regulatory role of fibroblast growth factors on ... · university of groningen regulatory role of...
TRANSCRIPT
University of Groningen
Regulatory role of fibroblast growth factors on hematopoietic stem cellsYeoh, Joyce Siew Gaik
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.
Document VersionPublisher's PDF, also known as Version of record
Publication date:2007
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):Yeoh, J. S. G. (2007). Regulatory role of fibroblast growth factors on hematopoietic stem cells. s.n.
CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.
Download date: 09-04-2021
Regulatory Role of Fibroblast Growth Factors
on Hematopoietic Stem Cells
Joyce S. G. Yeoh
Regulatory Role of Fibroblast Growth Factors
on Hematopoietic Stem Cells
RIJKSUNIVERSITEIT GRONINGEN
Regulatory Role of Fibroblast Growth Factors on
Hematopoietic Stem Cells
Proefschrift
ter verkrijging van het doctoraat in de
Medische Wetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
woensdag 14 februari 2007
om 16.15 uur
door
Joyce Siew Gaik Yeoh
geboren op 26 november 1979
te Penang, Maleisië
Promotor Prof. dr. G. de Haan
Copromotor Dr. R. van Os
Beoordelingscommissie Prof. dr. E. Vellenga
Prof. dr. P. Coffer
Prof. dr. A. Mueller
In memory of my father
And for my dearest mother
The research described in this thesis was conducted in the Department of Cell
Biology, Section Stem Cell Biology, University Medical Center Groningen,
University of Groningen, the Netherlands.
This research project was financially supported by the National Institutes of Health
and Ubbo Emmius Foundation.
The printing of this thesis was financially supported by the Groningen University
Institute for Drug Exploration (GUIDE), Faculty of Medical Sciences, University of
Groningen, Amgen B.V. Breda, Harlan Nederland and Nederlandse Aardolie
Maatschappij B.V. (NAM).
© 2006 by J. S. G. Yeoh. All rights reserved. No part of this book may be reproduced
or transmitted in any forms or by any means without permission from the author.
ISBN: 90-367-2857-6
Page layout Joyce Yeoh and Chevy Yick
Cover design Josh Harrop and Adam Gourlay, Dept. of Medical Illustration,
University of Aberdeen
Cover illustration Cellular Dreamtime. Oil canvas painting of cells based on
aboriginal dreamtime paintings.
Printed by PrintPartners Ipskamp B.V., Enschede, The Netherlands
TABLE OF CONTENTS
SCOPE OF THESIS 11
CHAPTER 1 FIBROBLAST GROWTH FACTORS AS REGULATORS OF STEM CELL
SELF RENEWAL AND AGING 17
Submitted to Mechanisms of Ageing and Development, in press
CHAPTER 2 FIBROBLAST GROWTH FACTOR-1 AND 2 PRESERVE LONG-TERM
REPOPULATING ABILITY OF HEMATOPOIETIC STEM CELLS IN SERUM-
FREE CULTURES 39
Stem Cells, 2006, 24 (6) 1564 - 1572
CHAPTER 3 EFFECTS OF FIBROBLAST GROWTH FACTOR OVEREXPRESSION AND
CELLULAR LOCALIZATION ON HEMATOPOIETIC STEM CELL
FUNCTION 67
In preparation
CHAPTER 4 MOBILIZED PERIPHERAL BLOOD STEM CELLS PROVIDE RAPID
RECONSTITUTION BUT IMPAIRED LONG-TERM ENGRAFTMENT 105
Submitted
CHAPTER 5 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES 133
NEDERLANDSE SAMENVATTING 143
ACKNOWLEDGEMENTS 153
CURRICULUM VITAE 157
Scope of Thesis
Scope of Thesis
12
Scope of Thesis Hematopoietic stem cells (HSCs) are pluripotent cells with the ability to generate
progeny for at least eight major hematopoietic lineages; B lymphocytes, T
lymphocytes, erythrocytes, megakaryocytes/platelets, basophils, eosinophils,
granulocytes and monocytes/macrophages (Figure 1). Mature bloods cells have a
finite lifespan, ranging from a few (neutrophil) to more than 100 (erythrocytes) days
in humans. Thus, the ultimate function of HSCs is to ensure that billions of new blood
cells are produced each day over the lifespan of an organism. As a result of these
properties, HSCs are widely used to restore blood cell production in leukemia and
lymphoma patients that have received cytotoxic therapeutic agents.
HSC
Erythrocyte
Eosinophil
Basophil / Mast cell
T lymphocyte
B lymphocyte
Macrophage
Natural Killer cell
Megakaryocyte
Granulocyte
Neutrophil
Lymphoid progenitor cell
Multipotentprogenitor cell
HSC
Erythrocyte
Eosinophil
Basophil / Mast cell
T lymphocyte
B lymphocyte
Macrophage
Natural Killer cell
Megakaryocyte
Granulocyte
Neutrophil
Lymphoid progenitor cell
Multipotentprogenitor cell
Figure 1: Schematic representation of the HSC differentiation pathway. HSCs are able to self renew
and are responsible for generating a variety specialized blood cells such as erythrocytes, B
lymphocytes, T lymphocytes, granulocytes and basophils.
Scope of Thesis
13
The classical source of HSCs is bone marrow (BM). For more than 40 years clinicians
have been using HSCs isolated from BM for transplantations purposes. Following
migration from the BM, HSCs can also be isolated from the peripheral blood. Due to
its ease of collection and increased rate of engraftment, peripheral blood has become
the primary source of HSCs for medical treatments in the past 10 years. Despite its
prevalent use, few reports exist comparing the quality of blood stem cells and BM
stem cells. This knowledge may be relevant to already established clinical techniques.
The aim of many researchers is to culture and expand primitive, self renewing stem
cells isolated from multiple tissues (such as BM and brain) in order to generate
specific cell types so that they can be used to treat injury or disease. The maintenance
and expansion of stem cells in in vitro studies remains one of the greatest challenges.
Most culture conditions result in a net loss of stem cells indicating that differentiation
is favored over expansion. This is most evident in the hematopoietic system, where
stem cells are rare (< 1 per 105 BM cells) and attempts to expand numbers in culture
have been cumbersome. By using growth factors important during embryonic
development, a phase where stem cells were first generated, it may be possible to
generate and maintain stem cells in vitro. Commonalities may exist between distinct
stem cell systems functioning in different tissues so that all stem cells can be cultured
in a similar fashion with the same growth factors. Finally and most importantly, it
may be possible to genetically modify stem cells to improve their outcome, that is,
increase stem cell expansion whilst maintaining their self renewal property.
What is becoming increasingly evident is that the large family of fibroblast growth
factors (FGFs) plays an important role in regulating and maintaining stem cells. FGFs
belong to one of the largest families of polypeptide growth factors. During
embryogenesis, FGFs govern the development of organs such as the lung and limb. In
adult tissues they maintain tissue homeostasis by playing roles in mitogenesis, cellular
differentiation, cell migration and angiogenesis. This thesis focuses on the role of
FGFs in maintaining and regulating HSCs both in vitro and in vivo.
Chapter 1 provides an overview on the regulatory role of FGFs in maintaining stem
cell self renewal to counteract senescence/aging of HSCs, neural stem cells (NSCs)
and embryonic stem (ES) cells. Aging involves a slow deterioration of tissue function,
including an elimination of new growth and decreased capacity for repair. Aging is
also associated with increased cancer incidence in all tissues that contain stem cells.
In general, tissue homeostasis is lost during aging. These observations suggest a link
Scope of Thesis
14
between aging and stem cell self renewal function. We speculate that FGFs and their
receptors promote self renewal, maintenance and proliferation of HSCs, NSCs and ES
cells in order to sustain tissue homeostasis during aging.
Previous studies from our group demonstrated that the exogenous addition of FGF-1
to unfractionated bone marrow (BM) cultures in serum-free media resulted in a
sustained expansion of cells with lymphoid and myeloid repopulating capacity. FGF-1
was also shown to be involved in serially transplantable long-term repopulating
HSCs. In Chapter 2 we study the effects of exogenous addition of FGF-1, FGF-2 and
the combination of both to unfractionated C57BL/6.SJL (CD45.1) BM cells in serum-
free media. BM cells treated with FGFs for 1, 3 and 5 weeks were mixed with freshly
isolated BM cells from C57BL/6 (B6) mice and transplanted into lethally irradiated
B6 mice.
To further examine the roles of FGF in regulating and maintaining HSCs, in Chapter
3 we retrovirally overexpress FGF-1 and FGF-2 into CD45.1 5-Fluorouracil (5-FU)
BM cells. Additionally, we investigate the role of intracellular FGF localization and
its effects on hematopoietic cells. Site-directed mutagenesis was performed to create
two FGF-1 mutants. The first mutant, S130E, exchanges serine at phosphorylation site
130 for glutamic acid. This is expected to mimic the phosphorylated state of FGF-1
and is therefore constitutively exported into the cytoplasm. In the second mutant, the
serine is exchanged for alanine (S130A), thereby mimicking the unphosphorylated
state of FGF-1 and hypothesized to retain FGF-1 in the nucleus. In an attempt to
translocate FGF-2 from the cytoplasm into the nucleus, an artificial nuclear
localization signal (NLS) was inserted upstream of the FGF-2 gene (NLS/FGF-2).
Well-established in vitro assays exist for measuring the growth of precursors in the
hematopoietic system. However, currently the only conclusive assay for HSCs is to
assess their in vivo ability to give rise to the lymphoid and myeloid lineage in a
lethally irradiated host following transplantation. This assay, known as the
competitive repopulation assay, is thoroughly examined in Chapter 4. Mobilized
peripheral blood (MPB) has replaced BM cells as the primary source of hematopoietic
stem cells for clinical transplantations due to its ease of retrieval and faster
regeneration of neutrophils and platelets. Using the competitive transplantation assay,
in Chapter 4 we directly examine the quality and frequency of MPB stem cells
compared to normal BM stem cells.
Scope of Thesis
15
Finally, in Chapter 5 we briefly summarize and discuss the data obtained from
previous chapters followed by future perspectives.
16
17
CHAPTER 1
Fibroblast growth factors as regulators of stem cell
self renewal and aging
Joyce S. G. Yeoh and Gerald de Haan
Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands
Submitted in Mechanisms of Ageing and Development, in press
Fibroblast growth factors regulates stem cell self renewal and aging
18
Abstract Organ and tissue dysfunction which is readily observable during aging results from a
loss of cellular homeostasis and reduced stem cell self renewal. Over the past 10
years, studies have been aimed at delineating growth factors that will sustain and
promote the self renewal potential of stem cells and support the expansion of
primitive stem cells in vitro and in vivo. Recently, strong evidence is emerging
indicating that fibroblast growth factors (FGFs) play a crucial role in stem cell
maintenance. FGFs belong to a family of polypeptide growth factors that are involved
in multiple functions including cell proliferation, differentiation, survival and motility.
In this review, we discuss the regulatory role of FGFs on hematopoietic stem cells
(HSCs), neural stem cells (NSCs) and embryonic stem (ES) cells in maintaining stem
cell self renewal. These findings are useful and important to further our knowledge in
stem cell biology and for therapeutic approaches.
Fibroblast growth factors regulates stem cell self renewal and aging
19
Introduction FGFs are a family of polypeptide growth factors that are involved in embryonic
development and adult tissue homeostasis. Twenty-two FGF genes have been
identified in the genome of humans and mice1. During embryogenesis, FGFs (FGF-7,
-8 and -10) govern the development of parenchymal organs, such as the lung and
limb2-4. In adults, FGFs are important in maintaining general tissue homeostasis,
including functions such as tissue repair and regeneration, metabolism and
angiogenesis5;6.
Stem cells are active during embryonic development and in most adult tissues, playing
an important role in normal homeostasis and tissue integrity. In adult tissues, stem
cells have been identified or isolated from an ever increasing array of tissues, such as
bone marrow7;8, skin9-12, intestinal epithelium13, myocardium14;15 and brain16-18.The
unique ability of tissue specific stem cells to self renew, differentiate and proliferate is
critically important to an organism during development and to maintain tissue
homeostasis. Stem cell self renewal ensures that a potentially unlimited supply of
stem cells exists, irrespective of demands put on the system by repetitive cell turn-
over during aging or stress.
However, unlimited stem cell self renewal may not exist, as stem cells are exposed to
both intrinsic and extrinsic effectors of damage. For example, the skin will age more
rapidly for an individual who has more exposure to ultraviolet rays than one who does
not. The liver of an alcoholic would have aged more rapidly than a teetotaler and
chemotherapeutic drugs used to treat cancer will have deleterious effects on HSCs.
Evidently, exposure to environmental or genetic factors induces differentiation and
apoptosis or inhibits the asymmetrical self renewal division of stem cells. Tissues in
which stem cell self renewal is inhibited would not be able to replenish the
differentiated cells, thereby impending function and integrity. Thus, this suggests that
tissue aging results from stem cell aging which in turn results from a decrease in stem
cell self renewal capacity19-22. Consequently, it has become increasingly important to
study regulators which will enhance stem cell self renewal during aging. Here, we
review recent advances in our understanding of the effects of FGFs on enhanced self
renewal and maintenance of stem cells. Our focus will be on HSC, NSCs and ES cells
as FGFs are crucial growth factors for all three stem cell systems. In addition, these
three stem cell species are by far the best characterized stem cells in in vitro and in
Fibroblast growth factors regulates stem cell self renewal and aging
20
vivo models. Finally, these stem cells appear to be of vital relevance in a variety of
age-related diseases, ranging from hematopoietic disorders, cancer, cardiovascular
disease and neurodegenerative disorders.
Fibroblast Growth Factors To date, 22 FGFs have been identified. They range in molecular weight from 17 to 34
kDa and share 13-71% amino acid identity in vertebrates (reviewed by Ornitz and
Itoh, 20011). FGFs mediate their biological responses by binding with high affinity to
cell surface tyrosine kinase FGF receptors (FGFR). Four functional FGFR genes
(Fgfr1-Fgfr4) have been identified in vertebrates1. Following receptor binding, FGFs
induce dimerization and phosphorylation of specific cytoplasmic tyrosine residues.
The phosphorylation of FGFRs triggers the activation of downstream cytoplasmic
signal transduction pathways23. Furthermore, FGFs interact with low affinity to
heparan sulfate proteoglycans (HSPGs)24, which acts to stabilize FGFs and prevent
thermal denaturation and proteolysis. In addition HSPGs are required for FGFs to
activate FGFRs effectively (reviewed by Powers et al. 2000; Dailey et al. 2005 6;25).
The expression of all known FGFs in an array of mouse and human tissues and
organs, was analyzed by researchers at the Genomics Institute of the Novartis
Research Foundation (GNF) and is publicly available in the SymAtlas database
(http://symatlas.gnf.org/SymAtlas/)26. Figure 1.1 illustrates a selection of those FGFs
with the most variable expression levels across various tissues and organs. Most FGFs
are ubiquitously expressed. However, some FGFs are selectively expressed in tissues
and organs such as spleen (FGF-2), blastocysts (FGF-4), kidney (FGF-1), lung (FGF-
1), dorsal root ganglion (FGF-1 and -13), embryo (FGF-5 and -15), salivary gland
(FGF-23), pancreas (FGF-4 and -18) and pituitary (FGF-14). The complex expression
of FGFs suggests that these growth factors play an important role in maintaining
tissue homeostasis in many tissues and organs.
Fibroblast growth factors regulates stem cell self renewal and aging
21
Main olfactory epithelium
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Relative Expression
Fgf1 gnf1m11999_at Fgf2 gnf1m01118_at Fgf4 gnf1m27000_at
Main olfactory epithelium
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Fgf5 gnf1m11382_a_at
Fgf10 gnf1m12419_at Fgf13 gnf1m00725_at Fgf14 gnf1m24788_at Fgf15 gnf1m01116_a_at
Fgf18 gnf1m27457_at
Main olfactory epithelium
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Relative Expression
Relative Expression
Fg20 gnf1m27635_at Fgf23 gnf1m30275_atBrown fatAdipose tissueAdrenal glandBoneBone marrowAmygdalaFrontal cortexPreopticTrigeminalCerebellumCerebral cortexDorsal root gangliaDorsal striatumHippocampusHypothalamusMain olfactory epitheliumOlfactory bulbSpinal cord lowerSpinal cord upperSubstantia nigraBlastocystsEmbryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5Fertilized EggMammary gland (lact)OvaryPlacentaProstate
TestisUmbilical cordUterusOocyteHeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cellsLiverLungLymph nodeSkeletal muscleVomeronasal organSalivary glandTonguePancreasPituitaryDigitsEpidermisSnout epidermisSpleenStomachThymusThyroidTracheaBladderKidneyRetina
Legend
*900
*50,
000
*200
*85
*300
*450
*90
*9,0
00
*225
*150
*130
Main olfactory epithelium
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Relative Expression
Fgf1 gnf1m11999_at Fgf2 gnf1m01118_at Fgf4 gnf1m27000_at
Main olfactory epithelium
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Fgf5 gnf1m11382_a_at
Fgf10 gnf1m12419_at Fgf13 gnf1m00725_at Fgf14 gnf1m24788_at Fgf15 gnf1m01116_a_at
Fgf18 gnf1m27457_at
Main olfactory epithelium
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Brown fatAdipose tissueAdrenal gland
BoneBone marrow
AmygdalaFrontal cortex
PreopticTrigeminal
CerebellumCerebral cortex
Dorsal root gangliaDorsal striatumHippocampusHypothalamus
Olfactory bulbSpinal cord lowerSpinal cord upper
Substantia nigraBlastocysts
Embryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5
Fertilized eggMammary gland (lact)
OvaryPlacentaProstate
TestisUmbilical cord
UterusOocyte
HeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cells
LiverLung
Lymph nodeSkeletal muscle
Vomeronasal organSalivary gland
TonguePancreas
PituitaryDigits
EpidermisSnout epidermis
SpleenStomachThymusThyroid
TracheaBladderKidneyRetina
Relative Expression
Relative Expression
Fg20 gnf1m27635_at Fgf23 gnf1m30275_atBrown fatAdipose tissueAdrenal glandBoneBone marrowAmygdalaFrontal cortexPreopticTrigeminalCerebellumCerebral cortexDorsal root gangliaDorsal striatumHippocampusHypothalamusMain olfactory epitheliumOlfactory bulbSpinal cord lowerSpinal cord upperSubstantia nigraBlastocystsEmbryo day 10.5Embryo day 6.5Embryo day 7.5Embryo day 8.5Embryo day 9.5Fertilized EggMammary gland (lact)OvaryPlacentaProstate
TestisUmbilical cordUterusOocyteHeartLarge intestineSmall intestineB220+ B-cellsCD4+ T-cellsCD8+ T-cellsLiverLungLymph nodeSkeletal muscleVomeronasal organSalivary glandTonguePancreasPituitaryDigitsEpidermisSnout epidermisSpleenStomachThymusThyroidTracheaBladderKidneyRetina
Legend
*900
*50,
000
*200
*85
*300
*450
*90
*9,0
00
*225
*150
*130
Figure 1.1: Expression levels of FGFs in an array of tissues and organs obtained from SymAtlas
database. Only FGFs showing significant expression are shown in this figure. Further information
regarding the remaining FGFs can be found at http://symstlas.gnf.org/SymAtlas. Horizontal scales are
linear, ranging from zero to the final marked value.
Fibroblast growth factors regulates stem cell self renewal and aging
22
Effects of FGFs on stem cells Hematopoietic Stem Cells
HSCs are pluripotent cells, possessing high proliferative and self renewal potential.
They ultimately regenerate and maintain all blood cell types of both the lymphoid and
myeloid lineages, producing billions of new circulating but short-lived cells each day.
In the early 1960s, Till and McCulloch introduced the first quantitative in vivo assay
for stem cells in the bone marrow8. Ever since, researchers have focused on soluble or
cell-bound growth factors that promote HSC self-renewal, maintenance, survival and
proliferation.
Hematopoiesis involves interactions between HSCs and stroma to provide a favorable
microenvironment for the development of progenitors. Stromal cells are an important
source of cytokines that regulate proliferation, differentiation and maturation of
progenitor cells27-29. As hematopoietic regulatory factors, FGFs can potentially exert
their effects at two critical aspects of hematopoiesis: the HSC proper and/or
maintenance of stem cell supporting stromal cells30.
Initial studies demonstrated that FGF-1 induced granulopoiesis31 and
megakaryocytopoiesis32;33. Recent studies have shown that both FGF-1 and FGF-2
can sustain the proliferation of hematopoietic progenitor cells, maintaining their
primitive phenotype34-36. FGF-1 was shown to be involved in the expansion of multi-
lineage, serially transplantable long-term repopulating HSCs34. Most noticeably, we
reported that the culturing of unfractionated mouse bone marrow in serum-free media
supplemented only with FGF-1 resulted in a significant and sustained expansion of
cells with both lymphoid and myeloid repopulating capacity34. Additionally, we
recently reported that culturing bone marrow stem cells in the combination of FGF-1
and FGF-2 for at least five weeks, maintained long-term repopulation (LTR)35. Using
the FGF culture system as a tool, Crcareva et al. demonstrated that FGF-1 expanded
bone marrow cells could be utilized for gene delivery to promote radioprotection and
increase long-term BM reconstitution36. FGF-1 has also been used in combination
with stem cell factor (SCF), thrombopoietin (TPO) and insulin-like growth factor 2
(IGF2) to culture BM HSCs for 10 days. Competitive repopulation analysis revealed a
~20-fold increase in long-term HSCs37.
FGF-2 has been known to enhance the colony stimulating activity of IL-3,
erythropoietin (Epo) and granulocyte macrophage colony stimulating factor (GM-
Fibroblast growth factors regulates stem cell self renewal and aging
23
CSF) on hematopoietic progenitor cells derived from normal human adult peripheral
blood and murine bone marrow in vitro38;39. In addition, FGF-2 appears to have a
stimulatory role on murine pluripotent hematopoietic cells in a spleen colony-forming
unit (CFU-S) assay. Murine non-adherent bone marrow cells were infused into
lethally irradiated mice to reconstitute hematopoiesis in vivo. Preincubation of the
marrow cells with FGF-2 lead to an increase in the number of day 9 and day 12 CFU-
S39. Several reports exist demonstrating a role of other FGFs in maintaining
hematopoiesis and HSCs. For example, FGF-4 alters the hematopoietic potential of
human long-term bone marrow cultures by increasing the number of progenitors of
the cultures40.
The stimulation of both early and late hematopoiesis suggests the presence of FGFRs
on both pluripotent and lineage committed progenitors. Fgfr1 and Fgfr2 expression
was detected in murine unfractionated bone marrow cells, and in several purified
mature peripheral cell populations (megakaryocytes and platelets, macrophages,
granulocytes, T and B lymphocytes)32. Previous studies from our group have shown
that Fgfr1, -3 and -4 can be detected on a primitive mouse Lin-Sca-1+c-Kit+ HSC
population34.
Unlike systemically acting growth factors, such as Epo and granulocyte colony
stimulating factor (G-CSF), FGFs probably function locally in a paracrine or
autocrine manner. For example, it is clear that FGF-2 plays a regulatory role in early
and late hematopoiesis possibly by interacting directly with HSCs. However, as FGF-
2 lacks signal sequences it is a poorly secreted protein suggesting that it must be
produced locally by surrounding cells. Thus it is of importance to know which cells
produce the growth factor in situ as these cells may specify stem cell self renewal.
Hematopoietic stem cells are in intimate contact with the bone microenvironment
(niche) and cell-cell contact appears to be responsible for the proliferative capacity of
HSCs41.Within the niche, the bone marrow extracellular matrix is postulated to serve
as a reservoir of growth factors and hematopoietic cytokines42;43. Since FGF-2 was
found to be present in the bone marrow extracellular matrix, the most obvious source
of FGF-2 is the stromal fibroblasts44, which are able to both produce and respond to
FGF-2. FGF-2 was reported to be a potent mitogen for bone marrow stromal cells in
vitro38;45-48. Non-adherent hematopoietic progenitors can grow and differentiate over a
pre-established stromal cell layer in long-term bone marrow cultures. Under these
conditions, FGF-2 stimulated myelopoiesis by increasing the number of non-adherent
Fibroblast growth factors regulates stem cell self renewal and aging
24
myeloid precursors48. Clearly, FGFs, in particular FGF-2 affects both stroma and
hematopoietic progenitor cells, indicating that it is involved in both hematopoiesis and
maintenance of the microenvironment.
It is therefore highly probable that besides acting on the stem cell itself, FGFs may
interact with stromal niche cells (Figure 1.2)29;49;50. Both mechanisms may induce the
release of other molecules important for the regulation of cell proliferation. Taken
together, these results indicate that in vitro FGFs play a variety of regulatory roles
which prolong homeostatic tissue renewal. The relevance of these in vitro data for in
vivo stem cell behavior remains to be explored. Most notably, it would be highly
relevant to assess how activities of FGFs provide a buffer against age-related cell
stress that may occur.
Figure 1.2: Mechanism of action of FGFs on HSCs. FGFs may interact directly with the HSC
promoting self renewal and/or interact with progenitor cells, releasing autocrine growth factors or other
molecules important for cell proliferation. FGFs may also interact directly with stromal cells within the
stem cell niche, releasing secondary growth factors necessary for the expansion of HSCs. Both methods
are plausible as receptors for FGFs have been found on HSCs and stromal cells. Red diamonds
represent the FGF ligand, green rectangles represent FGFR and question marks indicate that the
existence of FGF/FGFR complex remains speculative.
Fibroblast growth factors regulates stem cell self renewal and aging
25
Neural stem cells
Neurogenesis occurs throughout vertebrate life in the subgranular zone of the
hippocampal dentate gyrus and in the telencephalic subventricular zone (SVZ).
Although neurogenesis persists in the adult, its rate declines with age in rats51, mice52,
monkeys53 and humans54;55. The functional consequence of age-associated reduction
in neurogenesis is not clear. However, restoring neurogenesis may be a strategy for
preventing neural stem cell aging.
Like many other stem cells, neural stem cells (NSCs) possess three cardinal
properties: self renewal, extensive proliferation and the ability to generate functional
end-stage cells such as neurons, glial cells and oligodendrocytes (see review by Gage,
2000; Alvarez-Buylla et al., 2001; Weiss et al., 1996; Seaberg et al., 2003 and Rao,
199956-60). NSCs appear to be present in the SVZ in all vertebrate species tested. They
can be selectively cultured from the central nervous system using only two growth
factors; epidermal growth factor (EGF) and FGF-218;61;62. For example, FGF-2 and
EGF have been used alone and in combination to isolate and maintain stem cells of
the adult SVZ, the spinal cord, adult striatum and hippocampus18;63-69.
In the presence of EGF and/or FGF-2, cultured NSCs in suspension give rise to
clonally expanded aggregates called neurospheres. Cultured neurospheres will
generate neurons and glia when plated without growth factor on adhesive substrates18.
Neurospheres have been derived from adult striatum, hippocampus, mesencephalon,
SVZ and spinal cord18;67;68;70;71. It is important to realize that not all the NSC progeny
in neurospheres are stem cells. Rather, a heterogenous population of cells exists in
which only 10% to 50% of the progeny retain stem cell features and the remaining
cells undergo spontaneous differentiation. Quite similar to the situation in
hematopoiesis, this brings up the concept of a stem cell niche. The cluster of a
heterogeneous population of cells may aid the survival of NSCs in vitro or enable
growth factors such as FGF-2 to act on differentiated/accessory cells, resulting in the
release of additional growth factors which regulate NSC self renewal and
proliferation. Following in vitro culturing differentiating/differentiated cells rapidly
die and only surviving NSCs that retain long-term, self-renewal capacity produce new
neurospheres72. Neurospheres may be cultured for extended periods of time,
representing a renewable source of NSCs that may facilitate neurogenesis. It is
therefore a promising cellular source for biotherapies of neurodegenerative diseases.
Fibroblast growth factors regulates stem cell self renewal and aging
26
There is also ample evidence that FGFs are relevant for in vivo neurogenesis. The
intraventricular delivery of FGF-2 increased cell proliferation within the adult
SVZ73;74, however it no longer promoted the generation of pyramidal neurons in the
cerebral cortex75. Further studies report that subcutaneous injections of FGF-2
enhanced dentate neurogenesis in both neonatal and adult brain76;77.
Intracerebroventricular infusion of FGF-2 has been shown to upregulate dentate
neurogenesis in the aged brain78. FGF-2 knock-out mice are viable, fertile and
phenotypically indistinguishable from wild-type. However they do display a reduction
in neuronal density in the motor cortex, neuronal deficiency in the cervical spinal cord
and ectopic neurons in the hippocampal commissure79;80. With these phenotypes, it is
not surprising that FGF-2 plays such a crucial role in neurogenesis and NSCs
regulation. Mice deficient for FGF-481, FGF-882-85, FGF-986 and FGF-1087 are lethal.
Taken together, it is evident that FGF-2 plays a key role in regulating the
proliferation, differentiation and survival of NSCs in vitro and in vivo. Despite a
reduction in basal neurogenesis in dentate gyrus and SVZ the aged mouse retains the
ability to respond to neurogenesis-stimulating effects of growth factors, such as FGF-
278. These observations suggest that a decrease in levels of stem/progenitor cell
proliferation factors, such as FGF-2, in the microenvironment of the subgranular zone
are one of the potential causes of age-related decreases in neurogenesis. Increasing the
concentration of FGF-2 and perhaps other growth factors may decrease the process of
stem cell aging and enhance neurogenesis.
Embryonic stem cells
Embryonic stem (ES) cells are derived from totipotent cells of the inner cell mass of
the early mammalian embryo and are capable of seemingly unlimited and
undifferentiated proliferation in vitro88;89. For unknown reasons they are refractory to
the senescence program that halts proliferation in adult cells. Potentially, the high
levels of telomerase in ES cells contribute to this characteristic indefinite self renewal
potential. As a result, ES cells have much greater developmental potential than adult
stem cells. ES cell lines have been derived from mouse (mES) cells and human (hES).
The factors and culture condition that regulate and mediate self-renewal of mouse and
hES cells appear to be different, although their isolation conditions are similar.
Similar to mES cells, hES cells were initially isolated by culturing inner cell mass
cells on fibroblast feeder layers in medium containing serum90;91. In contrast to mES
Fibroblast growth factors regulates stem cell self renewal and aging
27
cells92;93, leukemia inhibiting factor (LIF) does not sustain and support hES cells90;94.
It is now widely accepted that the exogenous addition of FGF-2 to hES cells promote
hES cell self-renewal and the capacity to differentiate into a large number of somatic
cell types95;96. The addition of FGF-2 to medium containing a commercially available
serum replacement enables the clonal culture of hES cells on fibroblasts95. Recently, it
was reported that high doses of FGF-2 are adequate to maintain hES cells over 30
passages under feeder-free and serum-free growth conditions97. Wang et al. showed
that incubation of conditioned media from feeders with a neutralizing antibody against
FGF-2 abrogates the capacity of conditioned media to support hES cells, suggesting
that indeed FGF-2 is an essential factor produced by feeder cells97. The recent
observation that high dose FGF-2 (40ng/ml) can sustain hES cells has been
independently reported, thereby corroborating this important insight98;99. It has
recently been demonstrated that elevated levels of FGF-2, FGF-11, FGF-13 and all
four FGFRs are expressed in undifferentiated hES cells100-105. Correlating with
reported data, SymAtlas database analysis (Figure 1) demonstrates that most FGFs
(15 out of 22) are expressed in blastocysts and fertilized egg of the mouse genome.
The mechanism of action for FGF-2 in maintaining self-renewal of hES cells is still
not understood. Because expression of all four FGFRs was observed in cultured hES
cells106;107, one can speculate that FGF-2 may stimulate undifferentiated hES cell
proliferation directly. Alternatively, FGF-2 may block the differentiation of hES cells,
as FGF-2 has been shown to inhibit maturation of oligodendrocyte precursors108;109.
Together, it is now clear that FGF signaling is unconditionally required for the
sustained self-renewal and pluripotency of hES cells. This knowledge may serve as a
base for future developments in using FGFs to culture undifferentiated hES cells for
cell-based therapies to counteract the process of age-related diseases.
Concluding Remarks and Future Perspectives The behavior of stem cells is carefully regulated to meet the demands of normal
homeostasis of the organism, ensuring that a balance between proliferation, survival
and differentiation exists. For many tissues, this balance is impeded during aging.
Data presented in this review describe the role of FGFs and their receptors in
promoting self-renewal, maintenance and proliferation of HSCs, ES cells and NSCs.
Many studies have shown striking similarities with respect to FGF biology shared
Fibroblast growth factors regulates stem cell self renewal and aging
28
between these three stem cell species. However, the molecular mechanisms by which
the effects of FGFs occur in all three types of stem cells remains unknown.
Although most stem cell studies that have been carried out were restricted to the use
of FGF-2. In total, 22 FGFs exist (not including spliced forms). Why has FGF-2
historically been the most commonly used growth factor? Given their pleiotropic
effects and sequence similarities it seems reasonable to argue that at least some other
members of the FGF family will possess interesting stem cell stimulating activity.
Clearly the next challenge will be to examine the effects of the remaining FGFs in
well characterized stem cell systems. Only then would we be able to begin to
understand the complete role of FGFs in regulating stem cell self renewal behavior
and its impact on cellular aging, tissue aging and indeed organismal aging.
Fibroblast growth factors regulates stem cell self renewal and aging
29
Acknowledgements This work was supported by the National Institutes of Health (R01HL073710) and by
the Ubbo Emmius Foundation
Fibroblast growth factors regulates stem cell self renewal and aging
30
References 1. Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biology
2001;2:3005.1-3005.12.
2. Martin G. Making a vertebrate limb: new players enter from the wings. Bioessays 2001;23:865-868.
3. Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev. 2002;16:1446-1465.
4. Ware LB, Matthay MA. Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair. Am.J.Physiol Lung Cell Mol.Physiol 2002;282:L924-L940.
5. Basilico C, Moscatelli D. The FGF family of growth factors and oncogenes. Adv.Cancer Res. 1992;59:115-165.
6. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr.Relat Cancer 2000;7:165-197.
7. Abramson S, Miller RG, Phillips RA. The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J Exp.Med. 1977;145:1567-1579.
8. Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat.Res. 1961;14:213-222.
9. Alonso L, Fuchs E. Stem cells of the skin epithelium. Proc.Natl.Acad.Sci.U.S.A 2003;100 Suppl 1:11830-11835.
10. Ito M, Liu Y, Yang Z et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat.Med. 2005;11:1351-1354.
11. Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990;61:1329-1337.
12. Lavker RM, Sun TT. Epidermal stem cells: properties, markers, and location. Proc.Natl.Acad.Sci.U.S.A 2000;97:13473-13475.
13. Bjerknes M, Cheng H. Clonal analysis of mouse intestinal epithelial progenitors. Gastroenterology 1999;116:7-14.
14. Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett. 2002;530:239-243.
15. Beltrami AP, Barlucchi L, Torella D et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763-776.
Fibroblast growth factors regulates stem cell self renewal and aging
31
16. Lois C, Alvarez-Buylla A. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc.Natl.Acad.Sci.U.S.A 1993;90:2074-2077.
17. Richards LJ, Kilpatrick TJ, Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc.Natl.Acad.Sci.U.S.A 1992;89:8591-8595.
18. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707-1710.
19. Juckett DA. Cellular aging (the Hayflick limit) and species longevity: a unification model based on clonal succession. Mech.Ageing Dev. 1987;38:49-71.
20. Rao MS, Mattson MP. Stem cells and aging: expanding the possibilities. Mech.Ageing Dev. 2001;122:713-734.
21. de Haan G. Hematopoietic stem cells: self-renewing or aging? Cells Tissues.Organs 2002;171:27-37.
22. Kamminga LM, de Haan G. Cellular memory and hematopoietic stem cell aging. Stem Cells 2006;24:1143-1149.
23. Itoh N, Ornitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet. 2004;20:563-569.
24. Boilly B, Vercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X. FGF signals for cell proliferation and migration through different pathways. Cytokine & Growth Factor Reviews 2002;11:295-302.
25. Dailey L, Ambrosetti D, Mansukhani A, Basilico C. Mechanisms underlying differential responses to FGF signaling. Cytokine & Growth Factor Reviews 2005;16:233-247.
26. Su AI, Cooke MP, Ching KA et al. Large-scale analysis of the human and mouse transcriptomes. Proc.Natl.Acad.Sci.U.S.A 2002;99:4465-4470.
27. Adams GB, Scadden DT. The hematopoietic stem cell in its place. Nat.Immunol. 2006;7:333-337.
28. Arai F, Hirao A, Suda T. Regulation of hematopoietic stem cells by the niche. Trends Cardiovasc Med. 2005;15:75-79.
29. Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149-161.
30. Kashiwakura I, Takahashi TA. Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells. Leukemia & Lymphoma 2005;46:329-333.
Fibroblast growth factors regulates stem cell self renewal and aging
32
31. Yang M, Li K, Lam AC et al. Platelet-derived growth factor enhances granulopoiesis via bone marrow stromal cells. Int.J.Hematol. 2001;73:327-334.
32. Bikfalvi A, Han ZC, Fuhrmann G. Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells. Blood 1992;80:1905-1913.
33. Chen QS, Wang ZY, Han ZC. Enhanced growth of megakaryocyte colonies in culture in the presence of heparin and fibroblast growth factor. Int.J.Hematol. 1999;70:155-162.
34. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental Cell 2003;4:241-251.
35. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures. Stem Cells 2006;24:1564-1572.
36. Crcareva A, Saito T, Kunisato A et al. Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery. Exp.Hematol. 2005;33:1459-1469.
37. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 2005;105:4314-4320.
38. Gabbianelli M, Sargiacomo M, Pelosi E et al. "Pure" human hematopoietic progenitors: permissive action of basic fibroblast growth factor. Science 1990;249:1561-1564.
39. Gallicchio VS, Hughes NK, Hulette BC, DellaPuca R, Noblitt L. Basic fibroblast growth factor (B-FGF) induces early- (CFU-s) and late-stage hematopoietic progenitor cell colony formation (CFU-gm, CFU-meg, and BFU-e) by synergizing with GM-CSF, Meg-CSF, and erythropoietin, and is a radioprotective agent in vitro. Int.J.Cell Cloning 1991;9:220-232.
40. Quito FL, Beh J, Bashayan O, Basilico C, Basch RS. Effects of fibroblast growth factor-4 (k-FGF) on long-term cultures of human bone marrow cells. Blood 1996;87:1282-1291.
41. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7-25.
42. Flaumenhaft R, Moscatelli D, Saksela O, Rifkin DB. Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis. J.Cell Physiol 1989;140:75-81.
Fibroblast growth factors regulates stem cell self renewal and aging
33
43. Roberts R, Gallagher J, Spooncer E et al. Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature 1988;332:376-378.
44. Brunner G, Nguyen H, Gabrilove J, Rifkin DB, Wilson EL. Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells. Blood 1993;81:631-638.
45. Oliver LJ, Rifkin DB, Gabrilove J, Hannocks MJ, Wilson EL. Long-term culture of human bone marrow stromal cells in the presence of basic fibroblast growth factor. Growth Factors 1990;3:231-236.
46. Dooley DC, Oppenlander BK, Spurgin P et al. Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures. J.Cell Physiol 1995;165:386-397.
47. Emami S, Merrill W, Cherington V et al. Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin. In Vitro Cell Dev.Biol.Anim 1997;33:503-511.
48. Wilson EL, Rifkin DB, Kelly F, Hannocks MJ, Gabrilove JL. Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures. Blood 1991;77:954-960.
49. Allouche M, Bikfalvi A. The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis. Prog.Growth Factor Res. 1995;6:35-48.
50. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-846.
51. Kuhn HG, ckinson-Anson H, Gage FH. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci. 1996;16:2027-2033.
52. Kempermann G, Kuhn HG, Gage FH. Experience-induced neurogenesis in the senescent dentate gyrus. J Neurosci. 1998;18:3206-3212.
53. Gould E, Reeves AJ, Fallah M et al. Hippocampal neurogenesis in adult Old World primates. Proc.Natl.Acad.Sci.U.S.A 1999;96:5263-5267.
54. Cameron HA, McKay RD. Restoring production of hippocampal neurons in old age. Nat.Neurosci. 1999;2:894-897.
55. Kukekov VG, Laywell ED, Suslov O et al. Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp.Neurol. 1999;156:333-344.
56. Gage FH. Mammalian neural stem cells. Science 2000;287:1433-1438.
57. Alvarez-Buylla A, Garcia-Verdugo JM, Tramontin AD. A unified hypothesis on the lineage of neural stem cells. Nat.Rev.Neurosci. 2001;2:287-293.
Fibroblast growth factors regulates stem cell self renewal and aging
34
58. Weiss S, Reynolds BA, Vescovi AL et al. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci. 1996;19:387-393.
59. Seaberg RM, van der KD. Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci. 2003;26:125-131.
60. Rao MS. Multipotent and restricted precursors in the central nervous system. Anat.Rec. 1999;257:137-148.
61. Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J.Neurosci. 1992;12:4565-4574.
62. Tropepe V, Sibilia M, Ciruna BG et al. Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev.Biol. 1999;208:166-188.
63. Gritti A, Cova L, Parati EA, Galli R, Vescovi AL. Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS. Neurosci.Lett. 1995;185:151-154.
64. Gritti A, Frolichsthal-Schoeller P, Galli R et al. Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci. 1999;19:3287-3297.
65. Morshead CM, Reynolds BA, Craig CG et al. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 1994;13:1071-1082.
66. Shihabuddin LS, Ray J, Gage FH. FGF-2 is sufficient to isolate progenitors found in the adult mammalian spinal cord. Exp.Neurol. 1997;148:577-586.
67. Weiss S, Dunne C, Hewson J et al. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci. 1996;16:7599-7609.
68. Gritti A, Parati EA, Cova L et al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J.Neurosci. 1996;16:1091-1100.
69. Gage FH, Coates PW, Palmer TD et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc.Natl.Acad.Sci.U.S.A 1995;92:11879-11883.
70. Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, varez-Buylla A. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 2002;36:1021-1034.
71. Zhao M, Momma S, Delfani K et al. Evidence for neurogenesis in the adult mammalian substantia nigra. Proc.Natl.Acad.Sci.U.S.A 2003;100:7925-7930.
Fibroblast growth factors regulates stem cell self renewal and aging
35
72. Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev.Biol. 1996;175:1-13.
73. Craig CG, Tropepe V, Morshead CM et al. In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J.Neurosci. 1996;16:2649-2658.
74. Kuhn HG, Winkler J, Kempermann G, Thal LJ, Gage FH. Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J.Neurosci. 1997;17:5820-5829.
75. Vaccarino FM, Schwartz ML, Raballo R et al. Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis. Nat.Neurosci. 1999;2:246-253.
76. Wagner JP, Black IB, Cicco-Bloom E. Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of basic fibroblast growth factor. J Neurosci. 1999;19:6006-6016.
77. Cheng Y, Black IB, Cicco-Bloom E. Hippocampal granule neuron production and population size are regulated by levels of bFGF. Eur.J Neurosci. 2002;15:3-12.
78. Jin K, Sun Y, Xie L et al. Neurogenesis and aging: FGF-2 and HB-EGF restore neurogenesis in hippocampus and subventricular zone of aged mice. Aging Cell 2003;2:175-183.
79. Ortega S, Ittmann M, Tsang SH, Ehrlich M, Basilico C. Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc Natl Acad Sci U S A 1998;95:5672-5677.
80. Miller DL, Ortega S, Bashayan O, Basch R, Basilico C. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol.Cell Biol. 2000;20:2260-2268.
81. Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Goldfarb M. Requirement of FGF-4 for postimplantation mouse development. Science 1995;267:246-249.
82. Meyers EN, Lewandoski M, Martin GR. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat.Genet. 1998;18:136-141.
83. Reifers F, Bohli H, Walsh EC et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 1998;125:2381-2395.
84. Shanmugalingam S, Houart C, Picker A et al. Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 2000;127:2549-2561.
Fibroblast growth factors regulates stem cell self renewal and aging
36
85. Sun X, Meyers EN, Lewandoski M, Martin GR. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 1999;13:1834-1846.
86. Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 2001;104:875-889.
87. Sekine K, Ohuchi H, Fujiwara M et al. Fgf10 is essential for limb and lung formation. Nature Genetics 1999;21:138-141.
88. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154-156.
89. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc.Natl.Acad.Sci.U.S.A 1981;78:7634-7638.
90. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-1147.
91. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat.Biotechnol. 2000;18:399-404.
92. Smith AG, Heath JK, Donaldson DD et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988;336:688-690.
93. Williams RL, Hilton DJ, Pease S et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-687.
94. Humphrey RK, Beattie GM, Lopez AD et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. Stem Cells 2004;22:522-530.
95. Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev.Biol. 2000;227:271-278.
96. Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat.Biotechnol. 2001;19:971-974.
97. Wang L, Li L, Menendez P, Cerdan C, Bhatia M. Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 2005;105:4598-4603.
98. Xu RH, Peck RM, Li DS et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nature Methods 2005;2:185-190.
Fibroblast growth factors regulates stem cell self renewal and aging
37
99. Xu C, Rosler E, Jiang J et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 2005;23:315-323.
100. Dvash T, Mayshar Y, Darr H et al. Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies. Hum.Reprod. 2004;19:2875-2883.
101. Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev.Biol. 2004;269:360-380.
102. Rao RR, Calhoun JD, Qin X et al. Comparative transcriptional profiling of two human embryonic stem cell lines. Biotechnol.Bioeng. 2004;88:273-286.
103. Sato N, Sanjuan-Ignacio M, Heke M et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Developmental.Biology. 2003;260:404-413.
104. Sperger JM, Chen X, Draper JS et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc.Natl.Acad.Sci.U.S.A 2003;100:13350-13355.
105. Skottman H, Mikkola M, Lundin K et al. Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells 2005;23:1343-1356.
106. Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev.Dyn. 2004;229:259-274.
107. Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev.Dyn. 2004;229:243-258.
108. McKinnon RD, Matsui T, Dubois-Dalcq M, Aaronson SA. FGF modulates the PDGF-driven pathway of oligodendrocyte development. Neuron 1990;5:603-614.
109. Bogler O, Wren D, Barnett SC, Land H, Noble M. Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. Proc.Natl.Acad.Sci.U.S.A 1990;87:6368-6372.
38
39
CHAPTER 2
Fibroblast growth factor-1 and 2 preserve long-term
repopulating ability of hematopoietic stem cells in
serum-free cultures
Joyce S. G. Yeoh1, Ronald van Os1, Ellen Weersing1,
Albertina Ausema1, Bert Dontje1, Edo Vellenga2, Gerald
de Haan1
1 Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands 2 Department of Hematology, University Medical Centre Groningen, The
Netherlands
Stem Cells 2006; 24 (6): 1564 – 1572
Fibroblast growth factors preserve stem cell functioning
40
Abstract In this study we demonstrate that extended culture of unfractionated mouse bone
marrow (BM) cells, in serum-free medium, supplemented only with Fibroblast
Growth Factor (FGF)-1, FGF-2 or FGF-1+2 preserves long-term repopulating
hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels
of stem cell activity were detectable at 1, 3 and 5 weeks after initiation of culture.
FGFs as single growth factors failed to support cultures of highly purified Lin-Sca-
1+c-Kit+ (LSK) cells. However, co-cultures of purified CD45.1 LSK cells with whole
BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing
that HSC activity originated from LSK cells. Subsequently, we tested reconstituting
potential of cells cultured in FGF-1+2 with the addition of early acting stimulatory
molecules, stem cell factor + interleukin-11 + Flt3 ligand. The addition of these
growth factors resulted in a strong mitogenic response, inducing rapid differentiation
and thereby completely overriding FGF-dependent stem cell conservation.
Importantly, although HSC activity is typically rapidly lost after short term culture in
vitro, our current protocol allows us to sustain stem cell repopulation potential for
periods up to 5 weeks.
Fibroblast growth factors preserve stem cell functioning
41
Introduction Hematopoietic stem cells (HSCs) play a vital role in establishing and maintaining
hematopoiesis throughout life. Key to all stem cell transplantation therapies is the
unique property of HSCs to undergo self-renewal and to functionally repopulate the
tissue of origin when transplanted into a myeloablated recipient.
It has been shown that HSCs can undergo a large number of self-renewing divisions
in vivo, where the actual number of HSCs can increase. For example, in mice, it has
been shown that a single stem cell can regenerate and maintain the entire
lymphohematopoietic system after transplantation into an irradiated or
immunocompromised host1-3.
Recent evidence suggests that signaling molecules involved in embryonic
development, when the hematopoietic system is first formed, play an important role in
regulating stem cell self renewal. These include Wnt, Bmp and Shh family members4-
9. In a recent study we showed that fibroblast growth factor (FGF)-1 is also a potent
stimulator of stem cell activity in vitro10. In addition, we showed that all long term
repopulating hematopoietic stem cell activity is contained in the lineage-depleted,
FGF receptor (FGFR)-positive cell population in mouse bone marrow (BM).
FGF-1 belongs to the family of FGFs of which to date, 22 FGFs and 4 FGF receptors
have been identified in vertebrate genomes11. All FGFs have a high affinity for
heparin and for cell surface heparan sulfate proteoglycan (HSPG)12. This complex
formation is crucial for high affinity binding of FGF to its receptors. Multiple
pleiotropic and overlapping activities of FGF family members have been reported. A
large body of evidence from human disorders and gene knockout studies shows that
FGF pathways are required for vertebrate and invertebrate development13-18. FGFs are
also prominent in the development of the limbs19-21, nervous system22;23, and
angiogenesis24;25. Additionally, several members of the FGF family are potent
inducers of mesodermal differentiation in early embryos26. Interestingly, FGF-2 has
been identified as a strong stimulator of human embryonic stem cell (hESC) self-
renewal. The addition of FGF-2 to serum-free medium allows the clonal culture of
hESCs27. Recently, it has been reported that hESC culture can be simplified by using
high doses of FGF-2 which are adequate to maintain hESCs long-term under feeder-
free and serum-free growth conditions28-30. Interestingly, neural stem cells grown in
Fibroblast growth factors preserve stem cell functioning
42
three dimensional sphere-like structures in vitro also require the presence of FGF-
231;32.
The role of FGFs for in vitro maintenance of hematopoietic stem cells has remained
largely unexplored. In the present study, we compared the growth of HSCs in serum-
free medium supplemented with FGF-1 and/or FGF-2. We show that long-term
repopulating stem cells can be conserved in vitro for periods up to 5 weeks.
Fibroblast growth factors preserve stem cell functioning
43
Materials and Methods Mice
Female C57BL/6 SJL CD45.1 mice, originally obtained from the Jackson Laboratory
(Bar Habor, ME, http://www.jax.org) and bred in our local animal facility were used
as a donor source of HSCs. C57BL/6-Tg(ACTB-EGFP)10sb/J transgenic green
fluorescent protein (GFP) mice originally purchased from The Jackson Laboratory
were bred in our local animal facility and also used in certain experiments. Wild type
female C57BL/6 mice were purchased from Harlan (Horst, The Netherlands,
www.harlaneurope.com) and maintained under clean conventional conditions in the
animal facilities of the Central Animal Facilities, University of Groningen (The
Netherlands). Mice were fed ad libitum with food pellets and acidified tap water (pH
= 2.8). All animal procedures were approved by the local animal ethics committee of
the University Medical Centre Groningen.
Hematopoietic Cells
Mice were sacrificed by cervical dislocation and BM cells were obtained by crushing
both femora. Marrow cells were resuspended in α-minimum essential medium (α-
MEM; Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with
2% fetal calf serum (FCS; Gibco-BRL). The cell suspensions were filtered through a
100μM cell strainer (BD Falcon, Two Oak Park, MA http://www.bdbioscience.com)
to remove debris. Cells were counted on a Coulter Counter Model Z2 (Coulter
Electronics, Hialeah, FL, http://www.beckmancoulter).
Stem Cell Expansion Culture System
Unfractionated C57BL/6.SJL CD45.1 BM cells were cultured at 5 x 106 cells per well
in a 6 well plate (Corning Incorporated, Corning, NY, http://www.corning.com) in
StemSpan serum free medium (Stem Cell Technologies; Vancouver, Canada,
http://www.stemcell.com) in the presence of 10ng/ml recombinant human FGF-1
(Gibco, Grand Island, NY, http://www.invitrogen.com), or with 10ng/ml FGF-2
(Sigma-Aldrich, St Louis, http://www.sigmaaldrich.com), or a combination of both
cytokines at 10ng/ml each. Culture media was also supplemented with 10μg/ml
heparin (H3149 Sigma-Aldrich). In some experiments, unfractionated C57BL/6.SJL
5.1 cells isolated and cultured with StemSpan serum-free medium, 10μg/ml heparin
Fibroblast growth factors preserve stem cell functioning
44
and FGF-1+2 were treated with a cocktail of hematopoietic growth factors (GFs). A
cocktail of SCF (300ng/ml) (Amgen, Thousand Oaks, CA), interleukin (IL)-11
(20ng/ml) (R&D Systems, Minneapolis, http://www.rndsystems.com) and Flt3 ligand
(Flt3L) (1ng/ml) (Immunex, Seattle, http://immunex.com) was added to the cultures
for 1, 3 and 5 weeks. Non-adherent cells were harvested weekly, counted to
determine growth kinetics and re-introduced into the expansion culture and fresh GFs
were added to the culture. At 1, 3 and 5 weeks of culture, non-adherent and adherent
cells were harvested and counted in preparation for cell analysis and in vivo
transplantation assay into lethally irradiated C57BL/6 mice.
Isolation of Lin-Sca-1+c-Kit+ Cells
Freshly isolated C57BL/6 BM cells were stained with biotinylated lineage-specific
antibodies (Mouse Lineage Panel, containing anti-CD45R, anti-CD11b, anti-TER119,
anti-Gr-1 and anti-CD3e (BD Pharmingen, San Diego,
http://www.bdbiosciences.com/pharmingen), FITC-anti-Sca-1 and APC-anti-c-kit
(BD Pharmingen). Lin-Sca-1+c-Kit+ cells were stained as described33. Cells were
either analyzed on the FACS Calibur (Becton, Dickinson and Company, San Jose,
CA, http:www.bd.com) or sorted by a MoFlow cell sorter (DakoCytomation, Fort
Collins, CO, http://www.dako.com).
Cell Analysis
FGF-expanded cells were spun for cytospin preparation. Cytospin preparations were
stained with May-Grünwald-Giemsa. Cytospots were washed with distilled water and
allowed to air dry before analysis under a microscope.
Levels of chimerism were determined by detecting the presence of GFP or CD45.1
and CD45.2 positive cells in transplanted mice. To detect CD45.1 and CD45.2
positive cells, cells were stained with anti-CD45.2 (FITC) and CD45.1
(phycoerythrin) antibodies (BD Pharmingen) for 30 minutes and analyzed on a flow
cytometer (FACS Calibur; Becton, Dickinson and Company).
Cobblestone Area Forming Cell Assays
Cobblestone area forming cell (CAFC) assays were performed as described34 to assess
the number of hematopoietic progenitor cells (day 7 CAFCs) or more primitive stem
cells (day 35 CAFCs) in the FGF expanded cultures. Adherent and non-adherent cells
Fibroblast growth factors preserve stem cell functioning
45
were collected and seeded in limiting dilution in 96-well plates (Corning Incoporated)
containing a pre-established fetal bone marrow-derived-1 stromal layer. The cells
were cultured in Iscove’s modified Dulbecco’s modified Eagle’s Medium (Gibco-
BRL, Paisley, Scotland, http://www.gibcobrl.com) supplemented with 20% horse
serum at 34oC in a 10% CO2 incubator. On a weekly basis, wells were scored and half
the volume of the medium was changed. Wells were scored positive if cobblestone
areas were present. P values were used to test the statistical significance of different
groups. The student’s t-test was used assuming unequal variances of the two
variables. The Poisson-based limiting dilution analysis calculation was used with a
95% confidence interval to determine significant differences at p < .05. As previously
reported, quantification of CAFCs at days 7 and 35 was performed by using
maximum likelihood ratio method35.
In Vivo Transplantation Assays
Female C57BL/6 mice were used to provide competitor cells and as recipient mice.
BM cells were obtained by flushing the femoral content 3 times with α-MEM
supplemented with 2% FCS. Recipient mice were irradiated with 9.5Gy γ-rays
(0.7026Gy/minute) in a IBL 637 Cesium 137 source (CIS bio-international, Gif-sur-
Yvette, France, http://www.cisbiointernational.fr), 24 hours prior to transplantation.
For competitive repopulation determination, varying doses of cultured BM cells were
mixed with a constant number of BM competitor cells. Thus, recipient mice were
intravenously transplanted with different dilutions of expanded stem cells, with or
without 2 x 105 life-sparing C57BL/6 BM competitor cells. Each transplant group
consisted of 6 recipients. After transplantation, blood samples (60μl) were taken
monthly from the retro-orbital sinus for flow cytometer analysis. At the time of
sacrifice, chimerism in BM samples was analyzed by fluorescence-activated cell
sorting (FACS) in the same manner. For each recipient, the competitive repopulating
index (CRI) was determined. CRI is a relative measure of the competitive ability of
cultured cells in comparison with that of fresh BM cells. The CRI was calculated by
using the following formula:
ted transplancells competitor tocells cultured of Rationcirculatio in the cells competitor tocells cultured of RatioCRI =
Fibroblast growth factors preserve stem cell functioning
46
A CRI value of 1 indicates by definition that cultured cells and competitor cells have
equal competitive ability. The repopulation ability of our cells can also be measured
in repopulation units (RU). The RU takes into account the total number of cells
generated. Each RU is equivalent to the repopulation function of 100 000 competitor
BM cells. The RU was calculated using the following formula:
( ) RU competitor of No. x ncirculatio in the cells competitor tocells cultured of Ratio RU =
( ) ( )( )cells ted transplanofNumber
per well cells cultured ofnumber Total x RU ofNumber RU/well =
Fibroblast growth factors preserve stem cell functioning
47
Results
Cell Growth Kinetics and Cell Morphology
We used FGF-1 and FGF-2 separately and the combination of both FGFs as the only
stimulus in serum-free media to culture CD45.1 BM cells for a period up to 5 weeks.
One and 3 weeks following initiation of culture, a decrease in the number of cells was
observed. The number of cells per well had dropped dramatically from 5 x 106 to an
average of approximately 5 x 105 cells for all FGF conditions (Figure 2.1A). Five
weeks after the initiation of culture, cells treated with FGF-1 and/or FGF-2 had
increased close to the input cell number, whereas cells cultured only in serum-free
media remained low throughout the 5-week culture period at 1 x 105 (Figure 2.1A).
As a first screen test, prior to in vivo assays, in vitro CAFC assays were setup. CAFC
subsets were quantified in cells harvested from the FGF-1+2 expansion cultures at 3
and 5 weeks. The absolute number of day 7 CAFCs that were harvested from 3-week
cultures had increased 1.5- fold (Figure 2.1B). Interestingly, a substantial 26-fold
expansion of day 7 CAFCs was observed at the 5 week culture time point. In contrast,
day 35 CAFCs at week 3 cultures, were slightly lower than input, whereas a 3.5-fold
expansion was apparent after 5 weeks of culture (Figure 2.1B).
May-Grunwald-Giemsa staining of the starting cell population (Figure 2.1C) and of
FGF-1+2-treated cells showed an accumulation of macrophages and blast-like cells
after 3 and 5 weeks of culture (Figure 2.1D and 2.1E). The presence of blast-like cells
and extensive CAFC activity indicated the possible existence of immature cells with
stem cell properties in FGF stimulated cultures.
Fibroblast growth factors preserve stem cell functioning
48
1
10
100
1000
10000
input Week 3 Week 51
10
100
1000
10000
100000
input Week 3 Week 5
0
1
2
3
4
5
6
7
8
0 1 3 5
Weeks in culture
A.
Day 0 Week 3 Week 5
C. D. E.
Num
ber o
f cel
ls/w
ell (
x106
)
B.
Num
ber o
f C
AFC
/wel
l
CAFC Day 35: FGF-1+2 culturesCAFC Day 7: FGF-1+2 cultures
No FGFs
FGF-1
FGF-2
FGF-1+2
P<0.05P<0.05
N.S.N.S.
1
10
100
1000
10000
input Week 3 Week 51
10
100
1000
10000
100000
input Week 3 Week 5
0
1
2
3
4
5
6
7
8
0 1 3 5
Weeks in culture
A.
Day 0 Week 3 Week 5
C. D. E.
Num
ber o
f cel
ls/w
ell (
x106
)
B.
Num
ber o
f C
AFC
/wel
l
CAFC Day 35: FGF-1+2 culturesCAFC Day 7: FGF-1+2 cultures
No FGFs
FGF-1
FGF-2
FGF-1+2
P<0.05P<0.05
N.S.N.S.
Figure 2.1: Cell growth, morphology and phenotype of cells. (A): Growth kinetics of unfractionated
bone marrow (BM) cells in culture for 5 weeks. These are 4 representative cultures out of 30 cultures.
Some were performed in 25cm2 flasks; the remaining in 6-well plates. Cells were cultured in serum-
free medium, supplemented with FGF-1, or FGF-2, or FGF-1+2 in the presence of 10µg/ml heparin.
(B): Unfractionated BM cells and cells cultured for 3 and 5 weeks with FGF-1+2, were placed in
limiting dilutions in a 96-well plate and absolute numbers of day 7 and 35 CAFCs were compared with
input cells. The input values refer to freshly isolated untreated bone marrow cells. Day 7 and 35 CAFC
activity was higher for cells after a culturing period of 5 weeks with FGF-1+2; p-values were calculated
using Student’s t test and Poisson-based limiting dilution analysis was used to determine the CAFC
frequency. (C): May-Grünwald-Giemsa staining was performed on BM cells cultured in the presence of
FGF-1+2 at day 0 of initiation of culture. (D): May-Grünwald-Giemsa staining was performed on BM
cells cultured in the presence of FGF-1+2 at 3 weeks after initiation of culture. (E): May-Grünwald-
Giemsa staining was performed on BM cells cultured in the presence of FGF-1+2 at 5 weeks after
initiation of culture; N.S., not significant.
Fibroblast growth factors preserve stem cell functioning
49
Long-Term Competitive In Vivo Reconstitution of FGF Expanded Cells
To assess whether cells cultured with FGF-1, FGF-2 or FGF-1+2 contained stem cell
activity, we competitively transplanted congenic B6.CD45.1 or transgenic B6.GFP+
FGF expanded cells with CD45.2 BM cells at 1, 3 and 5 weeks after the initiation of
culture. In each group of transplants, 6 recipient mice were transplanted. Animals
receiving 1 week cultured cells were transplanted with 2.5 x 105 cultured cells and 2.5
x 105 B6 CD45.2 BM cells. As shown in Figure 2.2A and Table 2.1, after 1 week of
culturing, the CRI of FGF-1+2 cultured cells, was approximately 50, 18 weeks after
transplant. Interestingly, CRI levels of FGF-1+2 cultured cells increased with time,
suggesting engraftment of relatively more cells with long-term repopulation ability.
Average chimerism levels after 18 weeks were 96% (Table 2.1). After 3 weeks of
culturing, recipient mice received 1.8 x 105, 2.2 x 105, and 3.5 x 105 FGF-1, FGF-2
and FGF-1+2 cultured cells, respectively, together with 2 x 105 B6 CD45.2
competitor cells. Chimerism levels of ~80% were achieved, corresponding to a CRI
level of approximately 5, 16 weeks post-transplant. No significant differences were
observed between the FGF groups (Figure 2.2B and Table 2.1). As expected, cell
cultured for 3 and 5 weeks in serum-free medium without any supplements had no
long-term reconstituting activity. Remarkably, 5 weeks after culturing 1.2 x 106 FGF-
2 and 1.1 x 106 FGF-1+2 cells still outcompeted 2 x 105 freshly isolated BM cells,
although CRI values dropped significantly compared to cells cultured for 1 or 3 weeks
(Figure 2.2C; Table 2.1). Low CRI values of 0.5 ± 0.2 for FGF-1 cultured cells were
obtained, suggesting that these cells provide little competitive repopulation ability
(Figure 2.2C) even though 1.62 x 106 cells were transplanted with 2 x 105 B6 CD45.2
BM cells. The reconstitution activity of FGF cultured cells as indicated by the total
number of repopulation units/well (RU) correlates with the CRI values (Figure 2.2D;
Table 2.1). A 5-fold increase in RU was observed with cells treated with FGF-1+2 for
1 week. After 5 weeks of culturing, both FGF-2 and FGF-1+2 cultured cells had a 1.5-
fold increase in RU compared to input cells (Figure 2.2D; Table 2.1).
Fibroblast growth factors preserve stem cell functioning
50
00.5
11.5
22.5
33.5
0 5 10 15 20
0
2
4
6
8
10
12
0 5 10 15 20
0102030405060708090
0 5 10 15 20
Weeks post-transplant
Ave
rage
CR
IA
vera
ge C
RI
Ave
rage
CR
I
A.
C.
B.
1 week
3 week
5 week
FGF-1FGF-2
No FGFsFGF-1+2
D.
FGF-1+2FGF-2
FGF-1No FGFs
1 3 5
RU
/wel
l
Weeks in culture
00.5
11.5
22.5
33.5
0 5 10 15 20
0
2
4
6
8
10
12
0 5 10 15 20
0102030405060708090
0 5 10 15 20
Weeks post-transplant
Ave
rage
CR
IA
vera
ge C
RI
Ave
rage
CR
I
A.
C.
B.
1 week
3 week
5 week
FGF-1FGF-2
No FGFsFGF-1+2
FGF-1FGF-2
No FGFsFGF-1+2
D.
FGF-1+2FGF-2
FGF-1No FGFs
1 3 5
RU
/wel
l
Weeks in culture
FGF-1+2FGF-2
FGF-1No FGFs
1 3 5
RU
/wel
l
Weeks in culture
Figure 2.2: In vivo competitive transplantation assay of FGF cultured cells. (A): B6 CD45.1 cells
cultured in serum-free medium alone, or in the presence of FGF-1, FGF-2 or both FGF-1+2 were
transplanted into lethally irradiated B6 CD45.2 recipient mice after 1 week of culture. (B): B6 CD45.1
cells cultured in serum-free medium alone, or in the presence of FGF-1, FGF-2 or both FGF-1+2 were
transplanted into lethally irradiated B6 CD45.2 recipient mice after 3 weeks of culture. (C): B6 CD45.1
cells cultured in serum-free medium alone, or in the presence of FGF-1, FGF-2 or both FGF-1+2 were
transplanted into lethally irradiated B6 CD45.2 recipient mice after 5 weeks of culture. For week 1
culture, mice were transplanted with 2.5 x 105 cultured cells and 2.5 x 105 B6 CD45.2 bone marrow
(BM) cells. Recipients receiving 3 weeks cultured cells were transplanted with 1.8 x 105 FGF-1, 2.2 x
105 FGF-2, 3.5 x 105 FGF-1+2 and 7.5 x 104 no FGF treated cells with 2 x 105 B6 CD45.2 BM cells. 5
weeks after culturing 1.6 x 106 FGF-1, 1.2 x 106 FGF-2, 1.1 x 106 FGF-1+2 and 4.8 x 105 no FGF
treated cells were transplanted into recipient mice with 2 x 105 B6 CD45.2 BM cells. Average CRI ±
Fibroblast growth factors preserve stem cell functioning
51
SD was calculated in each group consisting of 6 mice. (D): The absolute number of repopulating units
of cultured cells was compared to input cells. Each RU is equivalent to the repopulation function of
100,000 competitor bone marrow cells. Therefore, at initiation of culture, 5 x 106 whole BM cells
contain 50 RU. Following 1 week of culturing in FGF-1+2, 253 RU were generated. Both FGF-2 and
FGF-1+2 cultured cells after 5 weeks produced 75 RUs. Abbreviations: CRI, competitive repopulation
index; FGF, fibroblast growth factor; RU, repopulation unit.
Table 2.1 In vivo reconstitution of FGF expanded cells
The data presented in this table were used to generate Figures 2.1 and 2.2. All mice were transplanted
in competition with 2 x 105 B6 CD45.2 BM cells. N.D. indicates that the experiment was not
performed.
* Each RU is equivalent to the repopulation function of 100,000 competitor bone marrow cells.
Therefore, at day 0, 5 x 106 whole BM cells contain 50 RU
Abbreviations: CRI, competitive repopulation index; FGF, fibroblast growth factor; N.D., experiment
not performed; RU, repopulation unit.
Radioprotection and Long-Term In vivo Reconstitution of FGF-Expanded Cells
To test the ability of expanded cells in a more clinically relevant model, 1 x 104 and 5
x 104 FGF-1+2 expanded cells were transplanted in lethally irradiated C57BL/6 mice
without competitor cells. After 3 weeks of culture, 1 x 104 and 5 x 104 cells were able
to stably engraft into most recipient mice providing long-term repopulation.
Transplantation of 1 x 104 cells provided radioprotection to 60% of animals (Figure
2.3A). This value increased to 80% when 5 x 104 FGF-1+2 expanded cells were
transplanted (Figure 2.3A). The survival rate was higher when culture time was
extended to 5 weeks with 1 x 104 expanded cells providing radioprotection to 80% of
animals (Figure 2.3A). A further increase in survival was observed with 5 x 104
expanded cells providing radioprotection to 100% of the mice (Figure 2.3A). In all
cases a radioprotection endpoint of 4 weeks after transplant was used. Animals that
died of hematopoietic failure, died within 14 days post-transplant. Transplantation of
1 x 104 cells cultured for 3 weeks resulted in an average chimerism of 50%, whereas 5
x 104 cells engrafted with an average chimerism of 75%, 28 weeks after transplant
(Figure 2.3B). Chimerism results from transplants carried out with cells from 5 week
Fibroblast growth factors preserve stem cell functioning
52
cultures are shown in Figure 2.3C. With 1 x 104 FGF-1+2 expanded cells, engraftment
levels steadily increased, stabilized after 8 weeks and 26 weeks after transplant donor
contribution was more than 90%. Mice transplanted with 5 x 104 cells showed an
average level of chimerism of more than 95%.
0
20
40
60
80
100
0 5 10 15 20 25 30
0
20
40
60
80
100
0 5 10 15 20 25 30
0
20
40
60
80
100
3 5
A.
B.
C.
% C
D4 5
.1 C
h im
eris
m
Weeks after transplant
% S
urvi
val
Weeks in culture
Weeks after transplant
% C
D4 5
.1 C
h im
eris
m
5 x 104 cells
1 x 104 cells
5 x 104 cells
1 x 104 cells
3 week culture
5 week culture
5 x 104 cells
1 x 104 cells
0
20
40
60
80
100
0 5 10 15 20 25 30
0
20
40
60
80
100
0 5 10 15 20 25 30
0
20
40
60
80
100
3 5
A.
B.
C.
% C
D4 5
.1 C
h im
eris
m
Weeks after transplant
% S
urvi
val
Weeks in culture
Weeks after transplant
% C
D4 5
.1 C
h im
eris
m
5 x 104 cells
1 x 104 cells
5 x 104 cells
1 x 104 cells
3 week culture
5 week culture
5 x 104 cells
1 x 104 cells
5 x 104 cells
1 x 104 cells
Figure 2.3: Radioprotective potential of fibroblast growth factor (FGF)-expanded cells. (A): Survival
rate of mice transplanted only with expanded cells. (B): Unfractionated B6 CD45.1 bone marrow (BM)
cells were cultured in FGF-1+2 for 3 weeks. (C): Unfractionated B6 CD45.1 bone marrow (BM) cells
were cultured in FGF-1+2 for 5 weeks. Recipient CD45.2 C57BL/6 mice were lethally irradiated and
transplanted with 1 x 104 or 5 x 104 FGF-1+2 cultured cells without life sparing competitor cells.
Analysis of chimerism was performed and average results are shown.
Fibroblast growth factors preserve stem cell functioning
53
Growing of Lin-Sca-1+c-Kit+ Cells in Cocultures
In our 3 and 5 week FGF cultures described above, the percentage of LSK cells was
analyzed prior to transplantation. After 3 weeks of culturing LSK frequencies of FGF-
1 cultured cells was 0.7% whilst FGF-2 and FGF-1+2 cells were 1.3%. LSK
frequencies increased after 5 weeks of culturing to 0.9% for FGF-1 cells and 2.8% for
FGF-2 and FGF-1+2 cells (data not shown). Despite the high percentage of LSK cells
in these cultures compared to normal BM cells which have a LSK frequency of
∼0.2%, we were not successful at culturing purified LSK HSCs or bulk Lin+ and Lin-
cells in serum-free medium supplemented with FGF-1+2 (data not shown). Thus, we
speculated that either the stem cell growth in unfractionated BM cultures did not
originate from LSK cells, or alternatively, that an accessory population of cells
contained within the bone marrow was required. To test this hypothesis, we sorted
LSK cells from B6 CD45.1 mice and co-cultured 7 x 103 and 5 x 104 of these cells in
the presence of 5 x 106 CD45.2 unfractionated BM cells. FACS analysis showed that
5 x 106 unfractionated CD45.2 BM cells contained ~5,500 LSK cells. Purified stem
cells and whole BM were cocultured for 5 weeks. After culture, all cells were
harvested and 2 x 105 cells were transplanted into lethally irradiated recipients without
competitors. The percentage of white blood cells originating from the purified LSK
CD45.1+ fraction or from the CD45.2+ unfractionated cells was assessed in the
recipients. If only LSK cells were responsible for the FGF stimulated stem cell
activity in unfractionated BM, we would expect chimerism levels of the sorted 7,000
CD45.1 LSK cells to reach or come close to 60% (7,000 CD45.1 LSK cells + 5500
LSK CD45.2 cells: 7,000/(7,000+5,500) = 56%). Strikingly, 16 weeks post-transplant,
chimerism levels had increased to 51%, 52% and 64%, implying that indeed all FGF
induced stem cell activity is derived from the LSK population, (Figure 2.4A).
Chimerism levels in recipients transplanted with 5 x 104 CD45.1 LSK cultured in 5 x
106 CD45.2 unfractionated BM ranged from 70% to 99%, 16 weeks after transplant
(Figure 2.4B). Engraftment was seen in all mice transplanted. The estimated expected
level of chimerism in these animals was 50,000/(50,000+5,500) = 90%, clearly well in
range with the experimental findings. These results suggest that FGFs may be acting
both on LSK cells and on other cell types indirectly affecting LSK cells to induce
stem cell activity in vitro.
Fibroblast growth factors preserve stem cell functioning
54
0
20
40
60
80
100
0 5 10 15 20 25
0
20
40
60
80
100
0 5 10 15 20 25
A.
B.
Expected
Expected%
CD
45.1
Chi
mer
ism
Weeks after transplant
Weeks after transplant
% C
D45
.1 C
him
eris
m
0
20
40
60
80
100
0 5 10 15 20 25
0
20
40
60
80
100
0 5 10 15 20 25
A.
B.
Expected
Expected%
CD
45.1
Chi
mer
ism
Weeks after transplant
Weeks after transplant
% C
D45
.1 C
him
eris
m
Figure 2.4: Highly purified CD45.1 Lin-Sca-1+c-Kit+ (LSK) cells co-cultured with unfractionated
CD45.2 bone marrow (BM) cells. (A): Seven thousand B6 CD45.1 LSK cells were cocultured with 5 x
106 B6 CD45.2 whole bone marrow cells (estimated to contain 5500 CD45.2 LSK cells) for 5 weeks in
serum-free medium supplemented with fibroblast growth factor (FGF)-1+2. Subsequently, 2 x 105
cultured cells were transplanted into B6 CD45.2 mice without additional competitor cells. At start of
the culture, the percentage of CD45.1 LSK cells in the co-culture was 7,000/(7,000+5500) = 56%. 12
weeks after transplant, chimerism levels of all mice steadily increased to an average of 57%. (B): Five
week coculture of 50,000 B6 CD45.1 LSK and 5 x 106 B6 CD45.2 whole BM cells was set-up and
similarly transplanted. The percentage of CD45.1 LSK cells in this culture was 50,000/(50,000+5,500)
= 90%. Chimerism levels in transplanted recipients rapidly increased reaching 83%. Chimerism levels
for each transplanted recipient are denoted by different symbols.
Fibroblast growth factors preserve stem cell functioning
55
Effect of SCF, IL-11 and Flt3L on FGF-1+2 Induced Clonogenic Activity In Vitro
Although single LSK cells cultured in FGF-1+2 did not divide, they did remain
visible for 7 days (data not shown). Thus it appeared that the addition of FGF-1+2 to
cell cultures prolonged the lifespan of the cells, but did not induce a strong enough
mitogenic signal. More classical hematopoietic GFs such as SCF, IL-11 and Flt3L,
have a much stronger proliferating effect and are shown to maintain stem cells in
short-term cultures36. Therefore we cultured cells with a cocktail of SCF, IL-11 and
Flt3L with or without the addition of FGF-1+2. After 5 weeks of culturing, cell
numbers of GF treated cells had exponentially increased from 5 x 106 to 7 x 108,
similar to GF + FGF-1+2 cultured cells (Figure 2.5A; Table 2.2). We next tested
whether FGFs would be able to maintain stemness of cells when used in combination
with SCF, IL-11 and Flt3L, which provides stronger mitogenic signals.
CAFC assays were carried out to determine clonogenic activity of cells treated with a
cocktail of growth factors. We observed a significant (p < .05) increase in day 7
CAFC activity after 3 weeks of culturing in GFs (Figure 2.5B). CAFC day 7 numbers
for cells cultured in the presence of GFs and GF + FGF-1+2, increased 222- and 273-
fold, respectively, over the input value, whereas cells treated with FGF-1+2 had a
modest 1.5-fold increase over the input value. A contrasting pattern was observed
when primitive day 35 CAFC numbers were evaluated (Figure 2.5B). Day 35 CAFCs
of 3 week GFs alone, or GF + FGF-1+2 cultures were below the detection level (less
than 1 CAFC per 1x106). Nevertheless, FGF-1+2 cultured cells were able to maintain
day 35 CAFC activity.
Effect of GFs on FGF-1+2 Induced Stem Cell Activity In Vivo
Finally, we determined whether cells cultured in FGF-1+2 with or without SCF, IL-11
and Flt3L provided engraftment in vivo. To this end, varying cell doses ranging from
1.8 x 105 to 2 x 106 B6 CD45.1 cultured cells were transplanted in competition with 2
x 105 freshly isolated B6 CD45.2 BM cells and compared with results for transplanted
FGF-1+2 cells (Figure 2.2). Although highly elevated CRI levels were observed 16
weeks after transplant, when FGF-1+2 treated cells were cultured for 1 week, the CRI
had dropped to 1 for cells cultured with GFs alone or GF+FGF-1+2 treated cells
(Figure 2.5C; Table 2.2). Continued culturing of cells for 3 and 5 weeks decreased
CRI values for all conditions. All competitive repopulation ability was lost after 3
Fibroblast growth factors preserve stem cell functioning
56
weeks of culturing or cells treated with GF alone, or with the addition of GF + FGF-
1+2 (Figure 2.5C; Table 2.2).
1
10
100
1000
0 1 2 3 4 5 6
Num
ber o
f cel
ls/w
ell (
x106
)
1
10
100
1
10
100
1000
input FGF 1+2 GF only GF+FGF
1
10
100
1000
10000
100000
1000000
input FGF 1+2 GF only GF+FGF
CR
IA.
B.
Num
ber o
f C
AFC
/wel
l
CAFC Day 7: 3 week cultured cells CAFC Day 35: 3 week cultured cells
C.
Weeks in culture
N.D. N.D.
P<0.05
N.S.N.S.
GF (SCF+IL-11+Flt3L) treated cells
GF + FGF-1+2 treated cells
1 3 5GF
GF+FGF-1+2FGF-1+2
Weeks in culture
1
10
100
1000
0 1 2 3 4 5 6
Num
ber o
f cel
ls/w
ell (
x106
)
1
10
100
1
10
100
1000
input FGF 1+2 GF only GF+FGF
1
10
100
1000
10000
100000
1000000
input FGF 1+2 GF only GF+FGF
CR
IA.
B.
Num
ber o
f C
AFC
/wel
l
CAFC Day 7: 3 week cultured cells CAFC Day 35: 3 week cultured cells
C.
Weeks in culture
N.D. N.D.
P<0.05
N.S.N.S.
GF (SCF+IL-11+Flt3L) treated cells
GF + FGF-1+2 treated cells
GF (SCF+IL-11+Flt3L) treated cells
GF + FGF-1+2 treated cells
1 3 51 3 5GF
GF+FGF-1+2FGF-1+2
Weeks in culture Figure 2.5: Effects of GF treatment on FGF-cultured cells. (A): Growth kinetics of unfractionated bone
marrow (BM) cells cultured with a cocktail of GFs (SCF, IL-11 and Flt3L) in the presence or absence
of FGF-1+2. Cell growth exponentially increased from 5 x 106 to 7 x 108. (B): Day 7 and 35 CAFC
content of unfractionated BM cells and cells cultured for 3 weeks with FGF-1+2, GFs (SCF, IL-11 and
Flt3L) alone and GFs + FGF-1+2. No day 35 CAFC activity was observed for cells cultured with GFs
alone and GFs + FGF-1+2; p-values were calculated using Student’s t test, and Poisson-based limiting
dilution analysis was used to determine the CAFC frequency. (C): CRI ± SD values (shown in Table 2)
for mice transplanted with varying cell doses of FGF-1+2, GF and GF + FGF-1+2 cultured cells in
competition with 2 x 105 B6.CD45.2 cells. Data are the same as in Figure 2 for FGF-1+2 and are only
included for comparison. Abbreviations: CAFC, cobblestone area-forming cell; CRI, competitive
Fibroblast growth factors preserve stem cell functioning
57
repopulation index; FGF, fibroblast growth factor; Flt3L, Flt3 ligand; GF, growth factor; IL,
interleukin; N.D., not detected; N.S., not significant; SCF, stem cell factor.
Table 2.2: Effect of the addition of stem cell factor, interleukin-11 and Flt3 ligand on FGF-1+2
induced stem cell activity
The data on this table were used to generate Figure 2.5. Results shown for FGF-1+2 condition are also
presented in Table 1 and are added for comparison. All recipients were transplanted in competition
with 2 x 105 B6 CD45.2 bone marrow (BM) cells.
* Each RU is equivalent to the repopulation function of 100,000 competitor bone marrow cells.
Therefore, at day 0, 5 x 106 whole BM cells contain 50 RU
Abbreviations: CRI, competitive repopulation index; FGF, fibroblast growth factor; GF, growth factor;
RU, repopulation unit.
Fibroblast growth factors preserve stem cell functioning
58
Discussion In the present study, we tested the potential of different FGFs to support HSC growth
in serum-free medium. In all 3 FGF conditions, FGF-1, FGF-2 and FGF-1+2, similar
trends in overall cell growth and cell morphology were observed. Although not
evident from the growth and morphology of the cultured cells, the addition of FGF-1
and/or -2 to serum-free medium proved to be an effective culture condition to support
primitive HSCs. Our in vitro CAFC data and in vivo reconstitution results clearly
document that bona fide long-term repopulating stem cells can be preserved in vitro
for up to 5 weeks when FGFs are added to the medium (Figure 2.1 and 2.2).
To test the repopulating potential of FGF-treated stem cells in a clinically relevant
model, cultured cells were transplanted into lethally irradiated mice without
competitor cells. Importantly, BM cells cultured for 3 and 5 weeks in the presence of
only FGF-1+2 were able to provide radioprotection and reconstitution.
Thus, our data clearly document that FGFs (of which we tested 2 out of a family of
22) can be added to a growing list of signaling proteins that act on primitive stem
cells. Interestingly, whereas “classical” hematopoietic growth factors turn out to have
limited potential in sustaining and expanding HSCs37-40 the effect of growth factors
and morphogens that historically have been associated with embryonic development
(Wnt, BMP, FGF, Shh, and insulin-like GF [IGF]-2) may be more powerful4;7;41;42.
Recently it was reported that FGF-2 allows the clonal growth of hESCs in medium
containing serum replacement27. In addition, ESCs are known to express multiple
FGF receptors43. Xu et al, documented that FGF-2 synergizes with Noggin to suppress
BMP signaling and thus sustain undifferentiated proliferation of hESCs in the absence
of fibroblasts or conditioned medium29. It is also interesting to note that during
development TGF-β/BMP, IGF, Notch receptors and Wnts may be influenced and
dependent on heterologous factors such as FGFs44. Lately , it was reported that cross-
talk between the Notch and FGFR signaling pathways may be an important auto-
regulatory mechanism involved in the regulation of cell growth45. Also, FGFs and
Wnts have been shown to interact in a variety of developmental systems including
tracheal development in Drosophila, mesoderm induction in Xenopus, and brain,
tooth, and kidney development in other vertebrates46. FGF signaling maintains the
proliferation of multipotent neural stem cells and also affects subsequent lineage
commitment during neural differentiation. It was shown that these FGF effects are
Fibroblast growth factors preserve stem cell functioning
59
mediated by β-catenin47. Collectively, these data imply an important physiological
role for FGF in stem cell fate decisions. The specific biological response, such as
proliferation, apoptosis, and differentiation, that a cell will deliver in response to FGF
signals, will depend on the interaction with many other factors44.
We were unable to culture purified LSK cells with only FGFs, whilst recently Zhang
and Lodish were able to culture purified BM side population cells with a greater than
8-fold increase in repopulating HSCs when grown in low levels of SCF,
thrombopoietin (TPO), IGF-2 and FGF-1 in serum-free medium for 10 days48. In our
purified cell culture system, such cross-talk with other signaling networks was not
possible, suggesting that the combination of SCF, TPO, IGF-2 and FGF-1 are better
suited for the expansion of a purified population of HSCs. The results of Zhang and
Lodish and our results highlight the importance of FGF in an in vitro culture system to
maintain HSCs, however our findings also suggests that the effect of FGFs on stem
cells requires other stimuli. The addition of SCF, IL-11 and Flt3L increased the
proliferation of stem cells. We tested whether FGFs were able to maintain the
primitiveness of stem cells, thereby negating the potential differentiation effect of
these GFs when placed in combination. Thus, GF + FGF-1+2 cultured cells would
have been expected to provide competitive reconstitution whereas GF-only cultures
would have been expected to provide no or very little reconstitution. Unfortunately,
this was evidently not the case (Figure 2.5). The optimal combination of GFs required
for maintaining primitive HSC activity remains to be discovered.
Our studies were not aimed to delineate the molecular consequences of incubating
BM cells with FGFs. Consequently, we can only speculate on how FGFs maintain
stem cells in whole BM cultures. It has been shown that receptors for FGF-1 are
present on primitive hematopoietic cell subsets10. Therefore, we speculate that FGFs
maintain stem cells at least partially by acting directly on FGFRs expressed on the
stem cells. Additionally, other non-LSK cell types normally present in the stem cell
niche may carry FGFRs and therefore be responsive to FGFs and may play an
important role in the maintenance of stem cells. In the intact animal, stem cells are
found in close association with their cellular microenvironments49-51. These
observations suggest both the existence of stem cell niches6;52-54 and the notion that in
vivo stem cell regulatory mechanisms are likely to require cell-cell contact or short-
range interactions55, providing further evidence that additional stimuli may be
required for a desired effect. The niche is composed of stem cells and a diverse
Fibroblast growth factors preserve stem cell functioning
60
variety of neighboring hematopoietic and non-hematopoietic cell types such as
osteoblasts and endothelial cells. These act to secrete and organize a rich milieu of
extracellular matrix and other factors that allow stem cells to manifest their unique
intrinsic properties56. We speculate that for HSC to be properly maintained and
amplified in vitro, the whole BM in co-culture must act as a niche, facilitating stem
cells to expand.
It is tempting to postulate that many stem cell expansion studies have not been so
successful, because cultures almost invariably were initiated with purified cells. As
shown in our purification studies, disruption of HSC from their niche is likely to have
detrimental effects on their subsequent developmental potential. Our study represents
a clear example of an in vitro system using FGFs, capable of supporting primitive
HSCs for an extended period of time. Future studies will be aimed at creating a niche
for stem cells in vitro, while stimulating their proliferation.
Fibroblast growth factors preserve stem cell functioning
61
Acknowledgements We wish to thank Geert Mesander and Henk Moes for their assistance with cell
sorting and the animal facility staff for taking care of the mice. This work was
supported by grants from the Dutch Cancer Society (RUG 2000 – 2182) and (RUG
2000 – 2183), National Institutes of Health (R01-HL073710), European Union (EU-
LSHC-CT-2004-503436), and the Ubbo Emmius Foundation.
Fibroblast growth factors preserve stem cell functioning
62
References
1. Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369-377.
2. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc.Natl.Acad.Sci.U.S.A 1995;92:10302-10306.
3. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242-245.
4. Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448-452.
5. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003;423:409-414.
6. Zhang J, Niu C, Ye L et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836-841.
7. Bhatia M, Bonnet D, Wu D et al. Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. J.Exp.Med. 1999;189:1139-1148.
8. Ploemacher RE, Engels LJ, Mayer AE, Thies S, Neben S. Bone morphogenetic protein 9 is a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures. Leukemia 1999;13:428-437.
9. Bhardwaj G, Murdoch B, Wu D et al. Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat.Immunol. 2001;2:172-180.
10. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental Cell 2003;4:241-251.
11. Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biology 2001;2:3005.1-3005.12.
12. Ornitz DM. FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 2000;22:108-112.
13. Szebenyi G, Fallon JF. Fibroblast growth factors as multifunctional signaling factors. Int.Rev.Cytol. 1999;185:45-106.
14. Wilkie AO, Morriss-Kay GM, Jones EY, Heath JK. Functions of fibroblast growth factors and their receptors. Curr.Biol. 1995;5:500-507.
Fibroblast growth factors preserve stem cell functioning
63
15. Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Goldfarb M. Requirement of FGF-4 for postimplantation mouse development. Science 1995;267:246-249.
16. Sun X, Meyers EN, Lewandoski M, Martin GR. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 1999;13:1834-1846.
17. Sekine K, Ohuchi H, Fujiwara M et al. Fgf10 is essential for limb and lung formation. Nature Genetics 1999;21:138-141.
18. Ortega S, Ittmann M, Tsang SH, Ehrlich M, Basilico C. Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc Natl Acad Sci U S A 1998;95:5672-5677.
19. Niswander L, Tickle C, Vogel A, Booth I, Martin GR. FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 1993;75:579-587.
20. Fallon JF, Lopez A, Ros MA et al. FGF-2: apical ectodermal ridge growth signal for chick limb development. Science 1994;264:104-107.
21. Martin GR. The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 1998;12:1571-1586.
22. Crossley PH, Martinez S, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature 1996;380:66-68.
23. Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998;93:755-766.
24. Poole TJ, Finkelstein EB, Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev.Dyn. 2001;220:1-17.
25. Tsuboi R, Rifkin DB. Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice. Journal of Experimental.Medicine 1990;172:245-251.
26. Paterno GD, Gillespie LL, Dixon MS, Slack JM, Heath JK. Mesoderm-inducing properties of INT-2 and kFGF: two oncogene-encoded growth factors related to FGF. Development 1989;106:79-83.
27. Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev.Biol. 2000;227:271-278.
28. Wang L, Li L, Menendez P, Cerdan C, Bhatia M. Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 2005;105:4598-4603.
Fibroblast growth factors preserve stem cell functioning
64
29. Xu RH, Peck RM, Li DS et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nature Methods 2005;2:185-190.
30. Xu C, Rosler E, Jiang J et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 2005;23:315-323.
31. Gritti A, Parati EA, Cova L et al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J.Neurosci. 1996;16:1091-1100.
32. Gage FH. Mammalian neural stem cells. Science 2000;287:1433-1438.
33. de Haan G, Szilvassy SJ, Meyerrose TE et al. Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers. Blood 2000;96:1374-1379.
34. de Haan G, Van Zant G. Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene. J.Exp.Med. 1997;186:529-536.
35. Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 1991;78:2527-2533.
36. Uchida N, Dykstra B, Lyons KJ, Leung FY, Eaves CJ. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp.Hematol. 2003;31:1338-1347.
37. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 1997;94:13648-13653.
38. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. The Journal of Clinical Investigation 2000;105:1013-1021.
39. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J.Exp.Med. 1997;186:619-624.
40. Rebel VI, Dragowska W, Eaves CJ, Humphries RK, Lansdorp PM. Amplification of Sca-1+ Lin- WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential. Blood 1994;83:128-136.
41. Zhang CC, Lodish HF. Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. Blood 2003;103:2513-2521.
Fibroblast growth factors preserve stem cell functioning
65
42. Austin TW, Solar GP, Ziegler FC, Liem L, Matthews W. A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells. Blood 1997;89:3624-3635.
43. Sperger JM, Chen X, Draper JS et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc.Natl.Acad.Sci.U.S.A 2003;100:13350-13355.
44. Dailey L, Ambrosetti D, Mansukhani A, Basilico C. Mechanisms underlying differential responses to FGF signaling. Cytokine & Growth Factor Reviews 2005;16:233-247.
45. Small D, Kovalenko D, Soldi R et al. Notch activation suppresses fibroblast growth factor-dependent cellular transformation. J.Biol.Chem. 2003;278:16405-16413.
46. Moon RT, Brown JD, Torres M. WNTs modulate cell fate and behavior during vertebrate development. Trends Genet. 1997;13:157-162.
47. Israsena N, Hu M, Fu W, Kan L, Kessler JA. The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev.Biol. 2004;268:220-231.
48. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 2005;105:4314-4320.
49. Suda T, Arai F, Hirao A. Hematopoietic stem cells and their niche. Trends in immunology 2005;26:426-433.
50. Weiss L, Geduldig U. Barrier cells: stromal regulation of hematopoiesis and blood cell release in normal and stressed murine bone marrow. Blood 1991;78:975-990.
51. Wolf NS. The haemopoietic microenvironment. Clin.Haematol. 1979;8:469-500.
52. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7-25.
53. Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149-161.
54. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-846.
55. Dorshkind K. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu.Rev.Immunol. 1990;8:111-137.
56. Stier S, Ko Y, Forkert R et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. Journal of Experimental.Medicine 2005;201:1781-1791.
66
67
CHAPTER 3
Effects of fibroblast growth factor overexpression and
cellular localization on hematopoietic stem cell
function
Joyce S. G. Yeoh, Ellen Weersing, Bert Dontje, Leonid
Bystrykh, Ronald van Os, Gerald de Haan
Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands
In preparation
Fibroblast growth factor overexpression
68
Abstract Exogenous addition of Fibroblast Growth Factor (FGF)-1 and FGF-2 maintains and
expands long-term repopulating hematopoietic stem cells (HSCs) in vitro. These
proteins are also highly expressed by Lin-Sca-1+c-Kit+ (LSK) cells. In this study we
retrovirally transduced post 5-fluorouracil (5-FU) bone marrow (BM) cells with
retroviral vectors which express wild-type (WT) FGF-1 and WT FGF-2 to examine
their cell intrinsic role in hematopoietic cell expansion. In addition, we examine the
role of nucleocytoplasmic trafficking of FGFs in hematopoietic cells by
overexpressing two mutant isoforms of FGF-1 in which a serine residue at the
phosphorylation site is exchanged for glutamic acid (S130E) or alanine (S130A).
S130E mimics the phosphorylated state of FGF-1 and should hypothetically be
constitutively exported to the cytoplasm whilst S130A should remain in the nucleus.
A third mutant was created by inserting an artificial nuclear localization signal (NLS)
upstream of FGF-2. Using fluorescence microscopy, we demonstrate that FGF-1,
FGF-2 and S130E predominantly localizes to the cytoplasm whilst 2% of S130A and
11% of NLS/FGF-2 overexpressing cells shown nuclear localization of FGF-1 and
FGF-2 respectively. We present evidence demonstrating that in an in vivo competitive
repopulation assay, stem cells expressing S130A engrafted into secondary recipients
with superior kinetics compared to WT cells, suggesting that nuclear localization of
FGF-1 may improve hematopoietic stem cell functioning.
Fibroblast growth factor overexpression
69
Introduction A small population of HSCs plays a pivotal role in the lifelong maintenance of
hematopoiesis. A critical property of HSCs is their ability to undergo self-renewal.
The relative inability to expand HSCs ex vivo imposes substantial limitations on the
current use of HSC transplantation. While studies have shown that some self renewal
is clearly possible in vitro1-3, the magnitude of expansion obtained for human and
murine HSCs is modest4-7.
In studies aimed to maintain HSCs in vitro, recent attention has been focused on the
large family of FGFs which are involved in embryonic development and adult tissue
homeostasis. To date, there are 22 members of the FGF family and only four distinct
FGF receptors (FGFRs) (reviewed by Ornitz and Itoh 2001)8. There is a growing body
of evidence demonstrating the role of FGFs in hematopoiesis in general and HSCs in
particular. For example, previous in vitro studies showed impaired hematopoietic
development in FGFR-/- embryoid bodies (EB), such that the number of blast colonies,
primitive erythroid and myeloid progenitors were greatly reduced9. FGFs, in
particular FGF-2, have been shown to sustain the proliferation of hematopoietic
progenitor cells, maintaining their primitive phenotype10;11. FGF-1 induces
granulopoiesis12 and megakaryocytopoiesis13;14. Recently, our group showed that
Fgfr-1, -3 and -4 are expressed by mouse primitive hematopoietic cell subsets and that
FGF-1 was involved in the expansion of multi-lineage (lymphoid and myeloid), long-
term (LT) repopulating HSCs 15. Additionally, we recently reported that
unfractionated bone marrow cells could be cultured in the combination of FGF-1 and
FGF-2 for up to five weeks without loss of stem cell repopulation activity. We
showed that this originated from FGF cultured HSCs16.
Several lines of evidence exist indicating that nuclear localization of FGFs may be
required for the mitogenic effect in certain conditions, in different cells types. For
example, radiolabeled exogenous FGF-1 localized to the nuclear fraction and was
shown to stimulate DNA synthesis and cell proliferation in cells containing receptors
for FGF-117. In glioma cells and in primary cultures of human astrocytes, cell
proliferation rate and nuclear association of FGF-2 was reported to change in
parallel18. These observations support the notion that nuclear translocation of FGFs
could be related to mitogenesis in different cells.
Fibroblast growth factor overexpression
70
Studies have shown that the translocation of FGF-1 from the cytosol to the nucleus
requires tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) activity and that
phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC) at the only
functional phosphorylation site (Serine 130) 19-22. In contrast to FGF-1, FGF-2
contains both autocrine and intracrine effects resulting from the existence of different
isoforms. For example, human FGF-2 contains five different forms; a low molecular
mass form (18kDa), which acts as an autocrine/paracrine factor and four high
molecular mass forms (21-22, 22.5, 24 and 34kDa) which are intracrine effectors.
These four high molecular mass forms are generated by differential initiation of
translation sites from an upstream CUG codon. They contain at least two short N-
terminal extensions in which the NLS is located23;24. Only N-terminally extended
forms initiated at upstream CUG codons are translocated to the nucleus while the
normal 18 kDa AUG-initiated form is confined to the cytoplasm25-28. When the
intracrine FGF-2 forms were expressed in NIH 3T3 cells, high proliferation rates and
growth in soft agar were observed29 and stimulated cell growth under low-serum
conditions was evident30;31
In previous studies, our goal was to maintain and expand HSCs in vitro by
exogenously adding FGF-1 and FGF-2 to serum-free media15;16. In the current study
we focused on retroviral overexpression of FGF-1 and FGF-2 on HSCs to promote
expansion and maintenance of hematopoietic cells in both in vitro and in vivo studies.
Parallel to this, we examined the effects of nuclear localized FGFs and their ability to
provide long-term (LT) repopulation. To clarify the role of differential localization of
FGFs in maintaining hematopoietic cells, we created two FGF-1 mutants. Firstly, a
serine residue at the phosphorylation site (amino acid 130) was exchanged with
glutamic acid to mimic phosphorylated FGF-1, which is expected to be constitutively
transported to the cytosol (S130E)22. In the second mutant the same serine residue was
exchanged for alanine, which is expected to remain in the nucleus (S130A)22. A third
mutant for FGF-2 was also created whereby an upstream nuclear localization signal
(NLS) was inserted into the FGF-2 coding sequence.
Using retroviral overexpression, long-term in vivo repopulation and principles of
limiting dilution assays to quantitatively determine hematopoietic cell expansion for
both WT FGF-1 and FGF-2 and their mutant forms, we demonstrate that in in vitro
assay, endogenous FGFs confer increased mitogenic activity to BM cells. However, it
does not provide enhanced long-term repopulation in primary and secondary in vivo
Fibroblast growth factor overexpression
71
repopulating assays. Fluorescence microscopy images indicated that FGF-1, FGF-2
and S130E were preferentially located in the cytoplasm. In S130A and NLS/FGF-2
overexpressing cells, only 2% of FGF-1 and 11% of FGF-2 positive cells showed
nuclear FGF activity, respectively. S130A mutants transplanted competitively
engrafted into secondary recipients with improved efficiency compared to WT cells,
indicating that targeting FGF-1 to the nucleus may provide improved stem cell
quality.
Fibroblast growth factor overexpression
72
Materials and Methods Animals
Eight to twelve week old female B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice, bred at
our local animal facility, were used as a donor source of hematopoietic stem cells for
in vitro cultures and transplantations. Female C57BL/6 (CD45.2) (B6) mice were used
as recipients and were purchased from Harlan (Horst, The Netherlands). All animals
were maintained under clean conventional conditions in the animal facilities of the
Central Animal Facilities, University Medical Centre Groningen (The Netherlands).
Mice were fed ad libitum with food pellets and acidified tap water (pH = 2.8). All
animal procedures were approved by the local animal ethics committee of the
University Medical Centre Groningen.
DNA constructs, expression vectors and retroviral vectors
The MIEV vector (kindly provided by Prof. C. Jordan, University of Rochester)
contains an internal ribosomal entry site (IRES) sequence, ψ packaging signal and the
reporter gene for enhanced green fluorescent protein (eGFP). The empty MIEV
vector served as a control and was the backbone from which all other vectors were
made.
FGF-1 was initially cloned into pCR4 vector following SalI end modification by using
the following primers; 5’-CTACCACCGCTGCTTGC-3’ (forward), 5’-
GTCGACCAAAATAGAGAACACTCAG-3’ (reverse) (fragment size 529bp). The
FGF-1 coding sequence was cut with EcoRI/SalI and inserted into MunI/SalI site of
the MIEV vector in between the pPGK promoter and IRES. We refer to this vector as
WT FGF-1. A retroviral vector encoding for WT FGF-2 was created identically. The
primers used were; 5’-CCCCAAGAGCTGCCACAG-3’ (forward) and 5’-
TCAGTGACAGTGTCAAAAGTGAGTC-3’ (reverse) (fragment size 531bp).
Site directed mutagenesis was used to create two mutant isoforms of FGF-1. At the
phosphorylation site, the serine at amino acid 130 was exchanged for either glutamic
acid (S130E) or alanine (S130A). The initial FGF-1 cloned in pCR4 was used to
create the two mutants by amplifying the whole fragment with the following primers;
S130E, 5’- CAAGAAGAACGGGgagTGTAAGCGCGGTCC-3’ (forward) and 5’-
GGACCGCGCTTACACTCCCCGTTCTTCTTG-3’ (reverse); S130A, 5’-
CAAGAAGAACGGGgccTGTAAGCGCGGTCC-3’ (forward) and 5’-
Fibroblast growth factor overexpression
73
GGACCGCGCTTACAggcCCCGTTCTTCTTG-3’ (reverse). Lower case letters
indicates the place of mismatch. Both mutants were subcloned from pCR4 vector at
EcoRI/SalI site into MunI/SalI of MIEV vector.
Contrary to the human FGF-2 gene, in the mouse genome Fgf-2 does not contain an
upstream NLS. The 5’ UTR NLS was artificially created based on homology to the
human FGF-2 coding sequence using these primers; 5’-
ACGGACTGGGAGGCTGGCAG-3’ (forward) and 5’-
CGAGGTGATGCCGCTGGCAG-3’ (reverse) (fragment size 157bp). Following
successful sequencing results, an EcoRV and ATG site was inserted into the upstream
sequence with the following primers; 5’-
gatatcACGatggTACGGACTGGGAGGCTGGCAG-3’ (forward) and 5’-
CGAGGTGATGCCGCTGGCAG-3’ (reverse) (fragment size 140bp). Following
cloning into pCR4 vector, the 5’UTR NLS was excised from the vector at
EcoRV/NcoI site and ligated into the original FGF-2 pCR4 vector at SmaI/NcoI site.
The ligated 5’UTR NLS/FGF-2 was removed from pCR4 vector with EcoRI/SalI and
subcloned into MIEV at MunI/SalI site creating the NLS/FGF-2 retroviral vector.
Retroviral overexpression of FGFs in primary BM cells
Bone marrow cells were obtained from CD45.1 mice injected i.p. with 150mg/kg 5-
Fluorouracil (5-FU, Pharmachemie Haarlem, The Netherlands), 4 days prior to BM
isolation. Bone marrow cells were harvested by flushing the femoral content with
StemSpan (Stem Cell Technologies, Vancouver, BC, Canada). Cells were cultured for
48 hours in StemSpan supplemented with 10% fetal calf serum (FCS; GibcoBRL,
Invitrogen, CA), 300ng/ml Stem Cell Factor (SCF; Amgen, Thousand Oaks, CA,
USA), 20ng/ml Interleukin-11 (IL-11; R&D Systems, Minneapolis, MN, USA),
1ng/ml Flt3 Ligand (Flt3L; Amgen, Thousand Oaks, CA, USA), penicillin and
streptomycin (Invitrogen, Breda, The Netherlands).
Twenty-four hours prior to transfection, 3 x 105 ecotropic Phoenix packaging
cells/well (ATCC-LGC Promochem, Middlesex, United Kingdom) were seeded onto
6-well plates. Using Fugene 6 (Roche, Basel, Switzerland), plasmid DNA (1µg) was
transfected on to pre-seeded ecotropic phoenix packaging cells. Virus-containing
supernatants from transfected ecotropic Phoenix packaging cells were harvested 24
hours after transfection and seeded onto retronectin (Takara, Kyoto, Japan) coated 6-
well plates. Plates containing viral supernatant were centrifuged for 1 hour at
Fibroblast growth factor overexpression
74
2200rpm at room temperature and then incubated at 37oC with 5% CO2 for 4 hours.
The viral supernatant was then removed from the well prior to the incubation of 7.5 x
105 cultured BM cells with 4µg polybrene (Sigma, St Louis, MO, USA). The whole
procedure was repeated 48 hours after the initial transfection and 2µg of polybrene
was added. Four days after transduction, the transduction efficiency was determined
by flow cytometry (FACS Calibur, Becton Dickinson, Palo Alto, CA).
In vitro proliferation of BM cells
Four days following transduction, Green Fluorescent Protein (GFP+) cells were sorted
by a MoFlow cell sorter (DakoCytomation, Fort Collins, CO). 6 x 105 to 1 x 106 GFP+
cells were placed in 6-well plate in StemSpan supplemented with 10% fetal calf
serum, 300ng/ml SCF, 20ng/ml IL-11, 1ng/ml Flt3L, penicillin and streptomycin. On
a weekly basis, cells were counted, cultures were depopulated and passaged.
In vitro hematopoietic cell assays
Cobblestone Area Forming Cell Assays (CAFC) were performed as previously
described16;32 to assess the number of hematopoietic progenitor cells (CAFC day 7) or
more primitive stem cells (CAFC day 28-35) among the transduced BM cells.
Colony Forming Unit Granulocyte-Macrophage (CFU-GM) was determined using
standard methylcellulose cultures (0.8% methylcellulose, 30% FCS in α-MEM).
Transduced BM cells were added to methylcellulose cultures supplemented with
100ng/ml SCF and 10ng/ml recombinant mouse granulocyte-macrophage colony
stimulating factor (GM-CSF; Behringwerke, Marburg, Germany). Cultures were
cultured at 37oC with 5% CO2 and scored after 6-7 days.
In vitro proliferation of BM cells co-cultured on a stromal layer
Following retroviral transduction, 5,000 or 10,000 GFP+ cells were sorted and co-
cultured with Fetal Bone Marrow Derived (FBMD)-1 stromal layer in a culture flask.
Cells were cultured in Iscoves modified DMEM (IMDM; GibcoBRL, Paisley,
Scotland) supplemented with 20% horse serum (GibcoBRL, Paisley, Scotland),
pencillin/streptomycine (Gibco, Paisley, Scotland) with β-mercaptoethanol (Merck
Schuchardt, Hohenbrunn, Germany) and hydrocortisone (Sigma, St Louis, MO). Nine
Fibroblast growth factor overexpression
75
and 14 days after initiation of culture, all non-adherent cells were removed, counted
and placed in CFU-GM and CAFC assays.
Sorting of hematopoietic cell populations
Hematopoietic cells were stained and sorted for populations of cells that differentially
expressed Lin-, Sca-1 and c-Kit using the MoFlow cell sorter as previously
described16;33. BM cells were stained with biotinylated lineage-specific antibodies
Mouse Lineage Panel, containing anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1
and anti-CD3e (BD Pharmingen, San Diego, CA), FITC-anti-Sca-1 and APC-anti-c-
kit (BD Pharmingen, San Diego, CA). Biotinylated antibodies were visualized with
streptavidin-PE (Pharmingen, San Diego, CA). Four different populations were
sorted, Lin-Sca-1-c-Kit- (L-S-K-), Lin-Sca-1+c-Kit- (L-S+K-), Lin-Sca-1-c-Kit+ (L-S-K+)
and Lin-Sca-1+c-Kit+ (L-S+K+).
Reverse Transcriptase (RT-PCR) and Quantitative PCR (Q-PCR)
Total RNA was prepared from approximately 1 x 106 to 3 x 106 sorted GFP+ cells
using RNeasy Mini kit (Qiagen). M-MLV reverse transcriptase (Invitrogen, Breda,
The Netherlands) was used to synthesize cDNA. Expression of FGF-1 and FGF-2
transcripts was assessed by RT-PCR with the following primers; FGF-1, 5’-
CGGCTCGCAGACACCAAATGAGG-3’ (forward) and 5’-
GTCGACCAAAATAGAGAACACTCAG-3’ (reverse) (fragment size 238bp); FGF-
2, 5’–CCCCAAGAGCTGCCACAG-3’ (forward) and 5’-
TCAGTGACAGTGTCAAAAGTGAGTC-3’ (reverse) (fragment size 531bp). The
cDNA products were quantified using SYBR Green (Bio-rad) in a 96-well microtiter
plates in an iCycler thermal cycler (Bio-rad, Hercules, CA, USA). Primers used for
quantifying Fgf-1 and Fgf-2 expression were as follows; Fgf-1, 5’-
CGGCTCGCAGACACCAAATGAGG-3’ (forward), 5’-
CCATAGTGAGTCCGAGGACCGC-3’ (reverse) (fragment size 155bp) and Fgf-2,
5’- CGACCCACACGTCAAACTACAACTC-3’ (forward), 5’-
GAAGCCAGCAGCCGTCCATC-3’ (reverse) (fragment size 113bp).
FGF expression levels were compared with expression of housekeeping genes Gapdh
and Actin using relative quantification ΔΔCT technique34. This value was then
corrected for the initial number of cells used.
Fibroblast growth factor overexpression
76
Western blotting
Cell lysates were prepared using the acid-acetone precipitation method. 3 x 106
transduced cells were pelleted and washed with 0.1M hydrochloric acid (MERCK
KGaA, Darmstadt, Germany), followed by a 60 minute incubation at -20oC. Samples
were centrifuged for 30 minutes at maximum speed at 4oC, washed in 70% ethanol
and resuspended in chilled (-20oC) acetone. All acetone was removed and the pellets
were left to dry at 37oC for 1-5 minutes. The pellets were redissolved in sample buffer
at 60oC. Lysates were then boiled for 5 minutes. Proteins were separated by 12%
SDS-PAGE and transferred to nitrocellulose membrane. Following overnight
blocking in 5% skim milk, membranes were probed overnight with 1:1000 dilution of
rabbit anti-FGF-1 (Sigma, Saint Louis, MO, USA) and rabbit anti-FGF-2 (Santa Cruz
Biotechnology, Santa Cruz, California, USA). Membranes were washed and
incubated with anti-rabbit IgG Horseradish peroxidase secondary antibody
(Amersham Biosciences, Buckinghamshire, UK). ECL western blotting detection
reagents (Amersham, Biosciences, Buckinghamshire, UK) were used to develop the
membranes. Equal loading of membranes were verified with commassie staining of
SDS-PAGE gels.
Immunocytochemistry
Transduced GFP+ cells were spotted on to cytospin preparations following one week
of culturing in culture conditions described above. Cells were fixed with 4%
paraformaldehyde and permeabilized with 0.3% Triton X-100/PBS. FGF-1 expression
was detected using an anti-FGF-1 (C-19) goat polyclonal IgG antibody (1:20 dilution)
(Santa Cruz Biotechnology, Santa Cruz, California, USA) followed by a Donkey
Anti-Goat IgG-Cy3 (1:1500 dilution) (Jackson Immunoresearch Laboratories, West
Grove, PA, USA) secondary antibody. FGF-2 expression was detected using an anti-
FGF-2 (147) rabbit polyclonal IgG antibody (1:20 dilution) (Santa Cruz
Biotechnology, Santa Cruz, California, USA) followed by a Goat Anti-Rabbit IgG-
Cy3 (1:1500 dilution) (Santa Cruz Biotechnology, Santa Cruz, California, USA)
secondary antibody. DAPI (0.2µg/ml) (Sigma, St Louis, Missouri, USA) was used to
stain the nuclei. Images were taken with Leica DM6000B (Wetzlar, Germany,
www.leica-microsystems.com).
Fibroblast growth factor overexpression
77
Primary transplantations of transduced cells
Following transduction, 3 x 106 CD45.1 BM cells were transplanted into lethally
irradiated (9 Gy, IBL 637137Cs-source 0.7026Gy/min, CIS Biointernational, Gif-sur-
Yvette, France) CD45.2 recipients. GFP+ cells were not selected, thus transplants
consisted of a mixture of both transduced and non-transduced cells. Each transplant
group consisted of 10 recipients, and two independent experiments were performed.
Following transplantation, blood samples (60μl) were taken monthly to determine
donor chimerism. Levels of chimerism were determined by detecting the presence of
CD45.1+ and GFP+ cells in transplanted mice. To detect CD45.1+ cells, cells were
stained with anti-CD45.1 (PE) antibody (BD Pharmingen, San Diego, CA) for 30
minutes and analyzed on a flow cytometer (FACS Calibur; Becton Dickinson
Biosciences, San Jose, CA).
Secondary transplantations of transduced cells
Four months after primary transplantation, five recipients from each group were
randomly selected, sacrificed and BM cells were isolated from two hind legs.
Unfractionated BM cells were transplanted into lethally irradiated (9Gy) CD45.2
secondary recipients in three limiting dilutions (1:1, 1:2 and 1:4) with a fixed number
of 4 x 105 CD45.2 competitor cells. Blood analysis was carried out each month to
determine original donor CD45.1+GFP+ chimerism levels. Each transplanted group
consisted of 5 recipients and two independent experiments were performed.
Transplantation of cultured transduced cells
Ten days after cells were plated onto a FBMD-1 stromal layer, all cells (including
stromal cells) were removed and CD45.1+GFP+ cells were selected by FACS. Lethally
irradiated (9Gy) B6 recipients were transplanted with CD45.1+GFP+ cells in
competition with freshly isolated B6 BM cells. Recipients received; (i) 5 x 105 control
cells + 5 x 105 competitor cells, (ii) 2.8 x 105 WT FGF-1 cells + 8.5 x 105 competitor
cells, (iii) 1.5 x 105 S130E cells + 4.4 x 105 competitor cells and (iv) 2.5 x 105 S130A
cells + 7.5 x 105 competitor cells. On a monthly basis, blood was analyzed for
CD45.1+GFP+ chimerism.
Fibroblast growth factor overexpression
78
Statistical Analysis
P-values were calculated using the Mann-Whitney test or the student’s t-test
(assuming unequal variances of the two variables) to test the statistical significance (p
< 0.05) of different groups. Quantification of CAFC at day 7, 28 and 35 was
performed by using maximum likelihood ratio method35. The Poisson-based limiting
dilution analysis calculation was used with a 95% confidence interval (CI) to
determine significant differences at p < 0.05.
Fibroblast growth factor overexpression
79
Results Relative expression of FGF-1 and FGF-2 in HSCs
To quantify the normal expression levels of FGF-1 and FGF-2 in hematopoietic stem
cells, freshly isolated BM cells were stained with lineage (Lin) specific markers and
hematopoietic stem cell markers, Sca-1 and c-Kit. As shown in Figure 3.1A, 5% Lin-
cells were separated into four populations; Sca-1-c-Kit-, Sca-1+c-Kit-, Sca-1-c-Kit+ and
Sca-1+c-Kit+. The levels of expression of FGF-1 and FGF-2 relative to Sca-1-c-Kit- is
shown in Figure 3.1B and 3.1C. Both FGFs are expressed in all four populations.
Interestingly, the highest expression of FGF-1 and FGF-2 was in the Lin-Sca-1+c-Kit+
population, a population which is highly enriched for primitive hematopoietic stem
cells36;37. This further confirmed our hypothesis that FGFs may play an important role
in HSCs regulation.
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
40FGF1 FGF2
Sca- c
-Kit-
Sca
+ c-K
it-
Sca
- c-K
it+
Sca
+ c-K
it+
Rel
ativ
e ex
pres
sion
Sca
- c-K
it-
Sca
+ c-K
it-
Sca
- c-K
it+
Sca
+ c-K
it+
Rel
ativ
e ex
pres
sion
A.
B. C.
Line
age
c-K
it
Sca-1
5%
2.2%44%
38% 12%
FSC
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
40FGF1 FGF2
Sca- c
-Kit-
Sca
+ c-K
it-
Sca
- c-K
it+
Sca
+ c-K
it+
Rel
ativ
e ex
pres
sion
Sca
- c-K
it-
Sca
+ c-K
it-
Sca
- c-K
it+
Sca
+ c-K
it+
Rel
ativ
e ex
pres
sion
0
2
4
6
8
10
12
14
0
5
10
15
20
25
30
35
40FGF1 FGF2
Sca- c
-Kit-
Sca
+ c-K
it-
Sca
- c-K
it+
Sca
+ c-K
it+
Rel
ativ
e ex
pres
sion
Sca
- c-K
it-
Sca
+ c-K
it-
Sca
- c-K
it+
Sca
+ c-K
it+
Rel
ativ
e ex
pres
sion
A.
B. C.
Line
age
c-K
it
Sca-1
5%
2.2%44%
38% 12%
FSC
Line
age
c-K
it
Sca-1
5%
2.2%44%
38% 12%
FSC
Figure 3.1: Relative expression of FGF-1 and FGF-2 in Lin- Sca-1 and c-Kit populations. (A): FACs
plot of Lineage (Lin-), Sca-1 and c-Kit gates from B6 BM cells. Lineage- cells were sorted into four
populations, Sca-1-c-Kit-, Sca-1+c-Kit-, Sca-1-c-Kit+ and Sca-1+c-Kit+. (B): Relative expression of FGF-
1 by Q-PCR in the four populations of Sca-1 and c-Kit relative to Sca-1-c-Kit- BM cells. (C): Relative
expression of FGF-2 by Q-PCR in Sca-1 and c-Kit populations relative to Sca-1-c-Kit- BM cells.
Fibroblast growth factor overexpression
80
Expression of FGF-1 and FGF-2 in BM cells
A schematic representation of the control, WT FGF-1 and WT FGF-2 retroviral
vectors used to transduced 5-FU treated BM cells is shown in Figure 3.2A. The
control vector expressing only eGFP was used as a control in all assays. Bone marrow
cells harvested from 5-FU treated CD45.1 donor mice were transduced with control,
WT FGF-1 and WT FGF-2 retroviral vectors. Transduction efficiencies of 30-40%
were always achieved (data not shown). Following retroviral transduction, GFP+ cells
were selected and cultured for a total of 33 days in StemSpan supplemented with
10%FCS, IL-11, SCF and Flt3L. On a weekly basis, cells were counted for growth
kinetic analysis. Cell growth in control, FGF-1 and FGF-2 overexpressing cells was
comparable (Figure 3.2B). FGF-1 overexpressing cells maintained slightly higher but
not statistically significant cell numbers throughout the 33 days of culturing, peaking
at day 26 (Figure 3.2B). At day 26, a 2.6-fold and 16-fold increase (although not
statistically significant p > 0.05) in FGF-1 overexpressing BM cells was observed
when compared to FGF-2 and control cells respectively (Figure 3.2B). Twelve days
into the culture period the expression level of FGF-1 and FGF-2 in BM transduced
cells was assessed by RT-PCR (Figure 3.2C). The expression of FGF-1 was only
detectable in FGF-1 overexpressing BM cells and FGF-2 expression was detected
only in FGF-2 overexpressing BM cells (Figure 3.2C). Although both FGF-1 and
FGF-2 expression were detected by Q-PCR (Figure 3.1B), their expression in LSK
cells by RT-PCR were below detection levels, possibly due to the use of different
primers. Protein levels in FGF-1 and FGF-2 overexpressing BM cells correlated with
RT-PCR results (Figure 3.2D). The relative expression of both FGFs in transduced
BM cells was also examined by Q-PCR. As expected, high expression levels of FGF-
1 and FGF-2 were detected in FGF-1 and FGF-2 overexpressing BM cells
respectively (Figure 3.2E). It should be pointed out that expression of FGF-1 and
FGF-2 in control cells was extremely low with CT value of 33 whilst both FGF-1 and
FGF-2 displayed CT values of ~17.
Fibroblast growth factor overexpression
81
FGF1 17KDa
FGF2 18KDa
Control FGF
A.
B.
FGF-1238bp
GAPDH 451bp
FGF-2 531bp
WT
FGF1
Con
trol
LSK
WT
FGF2
WT
FGF2
Con
trol
LSK
WT
FGF1
C. D.
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 35106
107
108
109
1010
1011
Cum
ulat
ive
cell
grow
th
Days in culture
ControlWT FGF-1WT FGF-2
E.
Rel
ativ
e ex
pres
sion
0
10000
20000
30000
40000
50000
60000
70000
80000
Control FGF-1 FGF-2
500 1K 1.5K 2K 2.5K 3.5K3K 4K
Control
WT FGF-1
WT FGF-2
EGFPLTR Ψ pPGK IRES LTR
EGFPIRES LTRFGF1
EGFPIRES LTRFGF2
LTR Ψ pPGK
LTR Ψ pPGK
FGF1 17KDa
FGF2 18KDa
Control FGF
FGF1 17KDa
FGF2 18KDa
Control FGF
A.
B.
FGF-1238bp
GAPDH 451bp
FGF-2 531bp
WT
FGF1
Con
trol
LSK
WT
FGF2
WT
FGF2
Con
trol
LSK
WT
FGF1
FGF-1238bp
GAPDH 451bp
FGF-2 531bp
WT
FGF1
Con
trol
LSK
WT
FGF2
WT
FGF2
Con
trol
LSK
WT
FGF1
C. D.
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 35106
107
108
109
1010
1011
Cum
ulat
ive
cell
grow
th
Days in culture
ControlWT FGF-1WT FGF-2
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 351.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 35106
107
108
109
1010
1011
Cum
ulat
ive
cell
grow
th
Days in culture
ControlWT FGF-1WT FGF-2
ControlWT FGF-1WT FGF-2
E.
Rel
ativ
e ex
pres
sion
0
10000
20000
30000
40000
50000
60000
70000
80000
Control FGF-1 FGF-2
500 1K 1.5K 2K 2.5K 3.5K3K 4K
Control
WT FGF-1
WT FGF-2
EGFPLTR Ψ pPGK IRES LTR
EGFPIRES LTRFGF1
EGFPIRES LTRFGF2
LTR Ψ pPGK
LTR Ψ pPGK
500 1K 1.5K 2K 2.5K 3.5K3K 4K500 1K 1.5K 2K 2.5K 3.5K3K 4K
Control
WT FGF-1
WT FGF-2
EGFPLTR Ψ pPGK IRES LTR
EGFPIRES LTRFGF1
EGFPIRES LTRFGF2
LTR Ψ pPGK
LTR Ψ pPGK
Figure 3.2: Overexpression of WT FGF-1 and WT FGF-2 in 5-FU bone marrow cells. (A): Schematic
representation of control, WT FGF-1 and WT FGF-2 vectors used to retrovirally transduce 5-FU BM
cells. (B): Cumulative cell growth curve of 5-FU BM cells retrovirally transduced with control, WT
FGF-1 and WT FGF-2. Following retroviral transduction, GFP+ cells were selected and cultured for 33
days in StemSpan containing 10%FCS, IL-11, SCF and Flt3L. On a weekly basis cells were counted
and passaged. (C): RT-PCR analysis for FGF-1 and FGF-2 expression in cells transduced with control
vector, WT FGF-1 and WT FGF-2 after 12 days of culturing. As a further control RNA from non-
transduced LSK cells was included. (D): FGF-1 and FGF-2 protein expression in control, WT FGF-1
and WT FGF-2 transduced cells after 12 days of culturing. (E): Using Q-PCR, the relative expression
of FGF-1 and FGF-2 in WT FGF-1 and WT FGF-2 transduced cells relative to control transduced cells,
was determined five days after initiation of culture respectively. CT value of 33 was recorded for
control cells, whilst both FGF-1 and FGF-2 contained CT values of ~17.
Fibroblast growth factor overexpression
82
Subcellular localization of FGF-1 and FGF-2
A schematic representation of S130E, S130A and NLS/FGF-2 retroviral vectors is
shown in Figure 3.3A. Phosphorylation of FGF-1 by protein kinase C occurs at serine
13022. Phosphorylation at this site should initiate the translocation of FGF-1 to the
cytosol from the nucleus. Mutating the serine in the phosphorylation site to a
negatively charged glutamic acid (E) (S130E) should mimic the constitutively
phosphorylated state of FGF-1. Substituting serine to the uncharged alanine (A)
(S130A) should mimic the unphosphorylated state of FGF-1 and causing the growth
factor to remain in the nucleus21. Similar successful strategies had previously been
used with other proteins38-40. The artificial addition of NLS upstream of FGF-2 coding
sequence is expected to translocate FGF-2 from the cytosol into the nucleus.
The subcellular localization of FGF-1 and FGF-2 in transduced BM cells was
examined using fluorescence microscopy. We were not able to detect GFP and FGF
proteins in all spotted cells. Localization patterns of FGF-1 and FGF-2 were therefore
only quantified in GFP+ and FGF double positive cells. FGF-1 and FGF-2 were not
detected in control cells (Figure 3.3B and 3.3F respectively), correlating with RT-
PCR and western blot data in Figure 3.2C-E. Fluorescent images of BM cells
overexpressing WT FGF-1 and WT FGF-2 indicated that both FGF-1 and FGF-2 were
localized in the cytoplasm in all cells in which FGF expression could be detected
(Figure 3.3C and 3.3G respectively).
The S130E mutant mimics phosphorylated FGF-1. Thus, we hypothesize that it
should be constitutively transported to the cytosol. Overlay images of FGF-1 positive
cells indicate that the protein is indeed predominantly localized in the cytoplasm
(Figure 3.3D). The S130A mutant lacks a phosphorylation site. Therefore, FGF-1 was
expected to be localized in the nucleus. In FGF-1 positive cells (n = 100), 2% of cells
showed nuclear staining (Figure 3.3Ei) and the remaining 98% showed cytoplasmic
staining (Figure 3.3Eii). Similar to S130A mutant, in NLS/FGF-2 overexpressing
cells, FGF-2 was expected to be present in the nucleus of cells. In 11% (n = 100) of
FGF-2 positive cells, FGF-2 was indeed localized in the nucleus (Figure 3.3H). In
remaining cells, FGF-2 was found in the cytoplasm (results not shown). Although
only a small percentage of FGF-1 and FGF-2 were localized to the nucleus, the
localization pattern was distinct when compared to WT FGF-1, WT FGF-2 and
S130E, indicating that nuclear localization did occur.
Fibroblast growth factor overexpression
83
Our findings indicate that FGF-1 and FGF-2 are localized in the cytoplasm of BM
cells overexpressing WT FGFs. The localization distribution of FGF-1 and FGF-2 in
S130A and NLS/FGF-2 mutants respectively, suggests that the transport of FGF-1
and FGF-2 to and from the nucleus may be a dynamic process. FGFs enter the
cytoplasm following FGFR binding. Their NLS will transport FGFs into the nucleus
and upon phosphorylation, FGFs exit the nucleus into the cytoplasm. Cellular stress
will transport FGFs out of the cell41.
Growth kinetics of S130 and NLS/FGF2 mutants
Overexpression of both S130 mutants showed similar cell growth kinetics when
compared to the control vector in BM cells (Figure 3.4A). However, WT FGF-1
appears to have a slight increase in cell numbers suggesting that the change in
phosphorylation status of FGF-1 may not affect the mitogenic effect of BM cells. It
should be noted that growth kinetics of WT FGF-1 and control BM cells are the same
data as shown in Figure 3.2B.
It is often thought that nuclear localization of FGFs would increase its mitogenic
effect17;18;29. Bone marrow cells overexpressing NLS/FGF-2 displayed an increase in
cell growth during the culture period compared to WT FGF-2 and control BM cells.
Thirty-three days after initiation of culture there was a 7-fold increase in NLS/FGF-2
overexpressing cells when compared to WT FGF-2 overexpressing BM cells
respectively (Figure 3.4B). This suggests that NLS/FGF-2 increases the mitogenic
activity of BM cells. It should be noted that growth kinetics of WT FGF-2 and control
BM cells are the same data as shown in Figure 3.2B.
Fibroblast growth factor overexpression
84
A.S130E/S130A
NLS/FGF-2
500 1K 1.5K 2K 2.5K 3.5K3K 4K
EGFPIRES LTRFGF1
EGFPIRES LTRFGF2
S130
LTR Ψ pPGK
NLSLTR Ψ pPGK
Control
WT FGF-1
S130E
S130A
WT FGF-2
NLS/FGF-2
100%Cytoplasmic(n = 100)
2%Nuclear(n = 100)
98%Cytoplasmic(n = 100)
100%Cytoplasmic(n = 100)
Control
100%Cytoplasmic(n = 100)
11%Nuclear(n = 100)
B.
C.
D.
E. (i)
E. (ii)
F.
G.
H.
Anti FGF-1 and FGF-2Cy3 GFP Overlay
A.S130E/S130A
NLS/FGF-2
500 1K 1.5K 2K 2.5K 3.5K3K 4K
EGFPIRES LTRFGF1
EGFPIRES LTRFGF2
S130
LTR Ψ pPGK
NLSLTR Ψ pPGK
A.S130E/S130A
NLS/FGF-2
500 1K 1.5K 2K 2.5K 3.5K3K 4K500 1K 1.5K 2K 2.5K 3.5K3K 4K
EGFPIRES LTRFGF1
EGFPIRES LTRFGF2
S130
LTR Ψ pPGK
NLSLTR Ψ pPGK
Control
WT FGF-1
S130E
S130A
WT FGF-2
NLS/FGF-2
100%Cytoplasmic(n = 100)
2%Nuclear(n = 100)
98%Cytoplasmic(n = 100)
100%Cytoplasmic(n = 100)
Control
100%Cytoplasmic(n = 100)
11%Nuclear(n = 100)
B.
C.
D.
E. (i)
E. (ii)
F.
G.
H.
Anti FGF-1 and FGF-2Cy3 GFP Overlay
Control
WT FGF-1
S130E
S130A
WT FGF-2
NLS/FGF-2
100%Cytoplasmic(n = 100)
2%Nuclear(n = 100)
98%Cytoplasmic(n = 100)
100%Cytoplasmic(n = 100)
Control
100%Cytoplasmic(n = 100)
11%Nuclear(n = 100)
B.
C.
D.
E. (i)
E. (ii)
F.
G.
H.
Anti FGF-1 and FGF-2Cy3 GFP Overlay
Figure 3.3: Overexpression and subcellular localization of S130 mutants and NLS/FGF2 in 5-FU bone
marrow cells. (A): Schematic representation of S130 and NLS/FGF-2 retroviral vectors. At the
phosphorylation site of FGF-1 serine 130 is replaced with glutamic acid (E) (S130E) or alanine (A)
(S130A). Subcellular localization of FGF-1 and FGF-2 in transduced BM cells. Fluorescent microscope
images of BM cells transduced with (B): control vector stained with anti-FGF-1, (C): WT FGF-1, (D):
S130E, (Ei): S130A with cytoplasmic localization, (Eii): S130A with nuclear localization, (F): control
vector stained with anti-FGF-2, (G): WT FGF-2 and (H): NLS/FGF-2. Localization pattern was
quantified in 100 GFP+ cells expressing FGF-1 or FGF-2.
Fibroblast growth factor overexpression
85
A.
B.
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 35106
107
108
109
1010
1011
Cum
ulat
ive
cell
grow
th
Days in culture
ControlWT FGF-1
S130AS130E
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
0 5 10 15 20 25 30 35106
107
108
109
1010
Cum
ulat
ive
cell
grow
th
Days in culture
Control
WT FGF-2NLS/FGF-2
A.
B.
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 35106
107
108
109
1010
1011
Cum
ulat
ive
cell
grow
th
Days in culture
ControlWT FGF-1
S130AS130E
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 351.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
0 5 10 15 20 25 30 35106
107
108
109
1010
1011
Cum
ulat
ive
cell
grow
th
Days in culture
ControlWT FGF-1
S130AS130E
ControlWT FGF-1
S130AS130E
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
0 5 10 15 20 25 30 35106
107
108
109
1010
Cum
ulat
ive
cell
grow
th
Days in culture
Control
WT FGF-2NLS/FGF-2
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
0 5 10 15 20 25 30 351.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
0 5 10 15 20 25 30 35106
107
108
109
1010
Cum
ulat
ive
cell
grow
th
Days in culture
Control
WT FGF-2NLS/FGF-2
Control
WT FGF-2NLS/FGF-2
Figure 3.4: Cell growth of 5-FU BM cells overexpressing S130 mutants and NLS/FGF-2. (A):
Cumulative cell growth of 5-FU BM cells transduced with control, WT FGF-1, S130E and S130A
vectors. Following transduction GFP+ cells were selected and cultured for a total of 33 days in
StemSpan supplemented with 10% FCS, IL-11, SCF and Flt3L. Growth curves for control and WT
FGF-1 cells are the same as those in Figure 3.1 and were used for comparison purposes. (B):
Cumulative cell growth of 5-FU BM cells transduced with control, WT FGF-2 and NLS/FGF-2.
Growth curves for control and WT FGF-2 are the same as those in Figure 3.1 and were used for
comparison purposes.
Co-culturing of transduced cells on a stromal layer
Previous studies in our group demonstrated that we could culture purified LSK cells
in the presence of unfractionated BM16. Based upon these results we sorted 5,000 or
10,000 GFP+ cells transduced with control, WT FGF-1, S130E and S130A onto
FBMD-1 stromal layer (Figure 3.5A). Cultures were maintained for a maximum of 14
days. At each data point (day 9 and day 14), non-adherent cells were removed,
analyzed and discarded. Analysis of growth kinetics revealed that control and WT
Fibroblast growth factor overexpression
86
FGF-1 transduced BM cells displayed a slight increase (~ 1.3 fold) in cell numbers
compared to both S130 mutants (Figure 3.5B). The co-culturing of transduced BM
cells on a stromal layer results in the widespread presence of cobblestones, as can
easily be seen from images of co-cultures, taken eleven days after initiation (Figure
3.5C-F). More cobblestones were observed in co-cultures with WT FGF-1 and S130A
at day 11 (Figure 3.5D and 3.5F). S130E co-cultures contained more non-adherent
cells than the other co-cultures (Figure 3.5E).
5-FU treated Retroviral transduced cells
Sort GFP+ cells5000 or 10 000 GFP+ cells onFBMD stromal layer
A.
B.
Control d11 WT FGF-1 d11 S130E d11 S130A d11C. D. E. F.
0.00E+00
2.00E+06
4.00E+06
6.00E+06
8.00E+06
1.00E+07
1.20E+07
0 5 10 15Days after sort
Cum
ulat
ive
Cel
l Num
ber
0
2 x 106
4 x 106
6 x 106
8 x 106
10 x 106
12 x 106
SS
ControlWT FGF-1S130AS130E
5-FU treated Retroviral transduced cells
Sort GFP+ cells5000 or 10 000 GFP+ cells onFBMD stromal layer
A.
B.
Control d11 WT FGF-1 d11 S130E d11 S130A d11C. D. E. F.Control d11 WT FGF-1 d11 S130E d11 S130A d11C. D. E. F.
0.00E+00
2.00E+06
4.00E+06
6.00E+06
8.00E+06
1.00E+07
1.20E+07
0 5 10 15Days after sort
Cum
ulat
ive
Cel
l Num
ber
0
2 x 106
4 x 106
6 x 106
8 x 106
10 x 106
12 x 106
SS
ControlWT FGF-1S130AS130E
0.00E+00
2.00E+06
4.00E+06
6.00E+06
8.00E+06
1.00E+07
1.20E+07
0 5 10 150.00E+00
2.00E+06
4.00E+06
6.00E+06
8.00E+06
1.00E+07
1.20E+07
0 5 10 15Days after sort
Cum
ulat
ive
Cel
l Num
ber
0
2 x 106
4 x 106
6 x 106
8 x 106
10 x 106
12 x 106
SS
ControlWT FGF-1S130AS130ESS
ControlWT FGF-1S130AS130E
Figure 3.5: Co-culturing of control, WT FGF-1, S130E and S130A cells with FBMD-1 stromal cells.
(A): Schematic representation of co-culturing procedure. BM cells were retrovirally transduced with
control, WT FGF-1, S130E and S130A vectors. Following transduction, 5,000 or 10,000 GFP+ cells
were selected and co-culture in a culture flask containing pre-seeded FBMD-1 stromal cells. Cells were
cultured in IMDM supplemented with 20% horse serum, hydrocortisone and penicillin/streptomycin
containing β-mercaptoethanol. (B): Cumulative cell growth of co-cultured cells. Images of co-cultured
cells 11 days after initiation of culture transduced with (C): control, (D): WT FGF-1, (E): S130E and
(F): S130A.
Fibroblast growth factor overexpression
87
Co-cultured cell do not provide long-term repopulation
Nine and 14 days after initiation of co-cultures, non-adherent cells were placed in
CFU-GM assay. The number of CFU/GM per 105 cells is shown in Figure 3.6A.
CFU-GMs were not detected for control cells and no significant differences were
observed between WT FGF-1 and S130 mutants after nine days of culturing (Figure
3.6A). After 14 days of culturing, there was an increase in CFU-GM for S130A
transduced cells over the control, WT FGF-1 and S130E transduced cells (Figure
3.6A). In addition, 14 days after initiation of culture, non-adherent cells were placed
in a CAFC assay. S130A cells contained higher CAFC day 7 activity, correlating with
CFU-GM data (Figure 3.6B). S130E transduced cells were observed to have
extremely high CAFC day 28 activity (30/105, 17-61; 95% confidence interval) whilst
CAFC day 28 activity was not detected for the other groups (Figure 3.6B).
Ten days after co-culturing of GFP+ cells with the stromal layer, the whole culture
was harvested and CD45.1+GFP+ cells were sorted. CD45.1+GFP+ cells were
transplanted in competition with freshly isolated B6 BM cells. Disappointingly,
CD45.1+GFP+ cells co-cultured cells failed to provide engraftment 18 weeks after
transplant (Figure 3.6C). It should be noted that the calculated chimerism levels have
not been corrected for the different number of CD45.1+GFP+ cell transplanted. In
conclusion, these results demonstrate that co-culturing of retrovirally transduced BM
cells is ineffective.
Primary transplantation of WT FGF-1 and S130 mutants
Bone marrow cells from CD45.1 donor mice were isolated four days after 5-FU
administration and retrovirally transduced with control vector and vectors encoding
WT FGF-1, S130E and S130A. Following transduction, cells were assayed for CAFC
content and transplanted into primary recipients (Figure 3.7A). Transduction
efficiencies of ~50% were achieved (data not shown). Unsorted transduced BM cells
were placed in a 4-fold limiting dilution in in vitro CAFC assays. No significant
differences in CAFC day 7 activity were observed between the different cell sources
(Figure 3.7B). Low CAFC day 28 activity was observed for all cells, however control
cells contained higher CAFC day 28 activity compared to WT FGF-1, S130E and
S130A (Figure 3.7B). Following transduction, 3 x 106 non-fractionated BM cells were
transplanted into primary lethally irradiated recipients. Donor chimerism analysis of
CD45.1+GFP+ cells showed no differences in engraftment between control, S130E
Fibroblast growth factor overexpression
88
and S130A overexpressing cells (Figure 3.7C). Engraftment of FGF-1 overexpressing
cells throughout the transplant period was lower compared to control and S130 cells.
A ~2.5-fold decrease in engraftment was observed, 24 weeks after transplant (Figure
3.7C). Donor chimerism levels of S130E and S130A transplanted animals were
significantly higher than control and WT FGF-1 chimerism levels (p < 0.05) only at
20 weeks post transplant (Figure 3.7C).
A.
0
5
10
15
20
25
9 14
Days after sorting on to FBMDs
GM
/105
ControlWT FGF-1S130ES130A
B.
0
10
20
30
40
50
60
70
CA
FC/1
05 ce
lls
0
500
1000
1500
Day 7 Day 28
ControlWT FGF-1S130ES130A
N.D. N.D. N.D.CA
FC/1
05 ce
lls
0
1
MIEV FGF1 S130E S130A
Don
or c
him
eris
mC
D45
.1+ G
FP+
C.
A.
0
5
10
15
20
25
9 14
Days after sorting on to FBMDs
GM
/105
ControlWT FGF-1S130ES130A
ControlWT FGF-1S130ES130A
B.
0
10
20
30
40
50
60
70
CA
FC/1
05 ce
lls
0
500
1000
1500
Day 7 Day 28
ControlWT FGF-1S130ES130A
N.D. N.D. N.D.CA
FC/1
05 ce
lls
0
10
20
30
40
50
60
70
CA
FC/1
05 ce
lls
0
500
1000
1500
Day 7 Day 28
ControlWT FGF-1S130ES130A
ControlWT FGF-1S130ES130A
N.D. N.D. N.D.CA
FC/1
05 ce
lls
0
1
MIEV FGF1 S130E S130A
Don
or c
him
eris
mC
D45
.1+ G
FP+
C.
0
1
MIEV FGF1 S130E S130A
Don
or c
him
eris
mC
D45
.1+ G
FP+
C.
Figure 3.6: In vitro assays and in vivo repopulating assay of co-cultured cells. (A): CFU-GM analysis
of control, WT FGF-1, S130E and S130A overexpressing cells, 9 and 14 days after initiation of culture.
(B): Day 7 and day 35 CAFC activity of co-cultured cells 14 days after initiation of culture. (C):
Average CD45.1+GFP+ donor chimerism levels 18 weeks post transplant from recipients transplanted
with co-cultured control, WT FGF-1, S130E and S130A cells 10 days after initiation of culture.
Fibroblast growth factor overexpression
89
5-FU treated
Retroviral transducedcells
CD45.1 9Gy 3 x 106
cells
20 weeks
1:11:21:4
9Gy
CAFC
A.
B.
C. D.
CA
FC/1
05ce
lls
0
5000
10000
15000
20000
25000
30000
35000
Day 7 Day 280
2
4
6
8
10
12
14
16
ControlWT FGF-1
S130AS130E
0
0.1
0.2
0.3
0.4
%LS
K
Control WT FGF-1
S130E S130A B6 BM0
10
20
30
40
50
60
4 8 12 16 20 24Don
or c
him
eris
mG
FP+ Control
WT FGF-1 S130AS130E
Weeks post transplant
CAF
C/1
05ce
lls
5-FU treated
Retroviral transducedcells
CD45.1 9Gy 3 x 106
cells
20 weeks
1:11:21:4
9Gy1:11:21:4
9Gy
CAFC
A.
B.
C. D.
CA
FC/1
05ce
lls
0
5000
10000
15000
20000
25000
30000
35000
Day 7 Day 280
2
4
6
8
10
12
14
16
ControlWT FGF-1
S130AS130E
ControlWT FGF-1
S130AS130E
0
0.1
0.2
0.3
0.4
%LS
K
Control WT FGF-1
S130E S130A B6 BM0
0.1
0.2
0.3
0.4
%LS
K
Control WT FGF-1
S130E S130A B6 BM0
10
20
30
40
50
60
4 8 12 16 20 24Don
or c
him
eris
mG
FP+ Control
WT FGF-1 S130AS130E
Weeks post transplant
0
10
20
30
40
50
60
4 8 12 16 20 24Don
or c
him
eris
mG
FP+ Control
WT FGF-1 S130AS130EControl
WT FGF-1 S130AS130E
Weeks post transplant
CAF
C/1
05ce
lls
Figure 3.7: Transplantation of WT FGF-1, S130E and S130A overexpressing cells into primary and
secondary recipients. (A): Schematic representation of transplantation strategy. 5-FU treated BM cells
were retrovirally transduced with control, WT FGF-1, WT FGF-2, S130E, S130A and NLS/FGF-2
vectors. Following transduction, 3 x 106 unsorted BM cells were transplanted into lethally irradiated
(9Gy) primary B6 recipients. Parallel to this, transduced cells were analyzed for CAFC. Twenty weeks
after transplant, BM cells were harvested from primary recipients and transplanted in limiting dilutions
(1:1, 1:2 and 1:4) with 4 x 105 competitors into lethally irradiated secondary recipients. (B): Day 7 and
day 28 CAFC activity of control, WT FGF-1, S130E and S130A overexpressing cells. (C): Average
CD45.1+GFP+ donor chimerism levels of primary recipients (n=10 per group) transplanted with
control, WT FGF-1, S130E and S130A overexpressing cells. Chimerism in blood was analyzed on a
monthly basis. (D): LSK analysis of BM harvested from primary recipients 20 weeks after transplant.
Fibroblast growth factor overexpression
90
Secondary transplantation of WT FGF-1 and S130 mutants
Twenty weeks after transplant, BM was harvested from primary recipient mice and
analyzed for Lin-Sca-1+c-Kit+ (LSK) expression. In a normal C57BL/6 mouse, BM
cells contained 0.22 ± 0.05% LSK cells (Figure 3.7D). The percentages of LSK cells
in control, WT FGF-1 and S130A transplanted mice were comparable to that of B6
BM (Figure 3.7D). A small increase (1.45-fold) in the percentage of LSK cells was
observed in mice transplanted with S130E overexpressing BM cells (Figure 3.7D).
Secondary recipients were transplanted with 2 x 105 unfractionated donor BM cells in
competition with a fixed dose of 4 x 105 freshly isolated B6 CD45.2 BM cells (Figure
3.7A). Recipients were analyzed for the presence of CD45.1+GFP+ cells on a monthly
basis. As shown in Figure 3.8A, 24 weeks after transplant mice transplanted with
control, WT FGF-1 and S130E retrovirally transduced cells displayed CD45.1+GFP+
chimerism levels of ~ 8-10%. In contrast, S130A transduced cells displayed high
donor derived contribution in the peripheral blood. Chimerism levels of ~40% were
reached (Figure 3.8A). Individual donor chimerism levels of S130A secondary
recipients demonstrate that the effect brought about by S130A was delayed, with
chimerism levels only beginning to increase 12 weeks after transplant (Figure 3.8B).
FACS plots (inset) illustrate the increase in GFP+ cells from 4 weeks to 24 weeks after
transplant (Figure 3.8B). Comparison of S130A transduced cells (GFP+) over non-
transduced cells (GFP-), revealed a significant (p < 0.05) 10-fold increase in GFP+
cells compared to control, WT FGF-1 and S130E transduced cells, 24 weeks after
transplant (Figure 3.8C). These results suggest that transport of FGF-1 into the
nucleus may have an intracrine function maintaining stem cell quality.
Fibroblast growth factor overexpression
91
C.
0
2
4
6
8
10
12
0 5 10 15 20 25
ControlWT FGF-1
S130AS130E
CD
45.1
+ GFP
+vs
CD
45.1
+ GFP
-
Weeks post transplant
0
10
20
30
40
50
60
0 5 10 15 20 25
B.
Indi
vidu
al C
D45
.1+ G
FP+
chim
eris
m
Weeks post transplant
GFP
PE
GFP
PE
A.
Don
or c
him
eris
mC
D45
.1+ G
FP+
0
10
20
30
40
50
Control WT FGF-1 S130E S130A
2 x 105 donor cells + 4 x 105 competitor cells
C.
0
2
4
6
8
10
12
0 5 10 15 20 25
ControlWT FGF-1
S130AS130E
CD
45.1
+ GFP
+vs
CD
45.1
+ GFP
-
Weeks post transplant
C.
0
2
4
6
8
10
12
0 5 10 15 20 25
ControlWT FGF-1
S130AS130E
ControlWT FGF-1
S130AS130E
CD
45.1
+ GFP
+vs
CD
45.1
+ GFP
-
Weeks post transplant
0
10
20
30
40
50
60
0 5 10 15 20 25
B.
Indi
vidu
al C
D45
.1+ G
FP+
chim
eris
m
Weeks post transplant
GFP
PE
GFP
PE
0
10
20
30
40
50
60
0 5 10 15 20 25
B.
Indi
vidu
al C
D45
.1+ G
FP+
chim
eris
m
Weeks post transplant
GFP
PE
GFP
PE
GFP
PE
GFP
PE
A.
Don
or c
him
eris
mC
D45
.1+ G
FP+
0
10
20
30
40
50
Control WT FGF-1 S130E S130A
2 x 105 donor cells + 4 x 105 competitor cells
A.
Don
or c
him
eris
mC
D45
.1+ G
FP+
0
10
20
30
40
50
Control WT FGF-1 S130E S130A0
10
20
30
40
50
Control WT FGF-1 S130E S130A
2 x 105 donor cells + 4 x 105 competitor cells
Figure 3.8: S130A increases donor chimerism levels in secondary recipients transplanted with 2 x 105
S130A overexpressing cells and 4 x 105 competitor cells. (A): Average donor chimerism
(CD45.1+GFP+) levels 24 weeks post transplant of secondary transplant recipients. (n=5 per group).
(B): Individual donor chimerism of secondary recipients transplanted with S130A transduced cells.
FACS plots highlight the increase in GFP+ cells during the transplant period. (C): Ratio of transduced
GFP+ cells and non-transduced GFP- cells.
Primary transplantation of WT FGF-2 and NLS/FGF-2
Similar to WT FGF-1 and S130 mutants, 5-FU treated BM cells were retrovirally
transduced with control, WT FGF-2 and NLS/FGF-2. Transduction efficiencies of
Fibroblast growth factor overexpression
92
~50% were achieved (data not shown). Following transduction, unfractionated cells
were assayed for CAFC content in 3-fold limiting dilutions. CAFC day 7 activity for
control transduced cells was 3- and 2-fold higher than WT FGF-2 and NLS/FGF-2
transduced cells respectively (Figure 3.9A), indicating that control cells contain more
progenitors. Low CAFC day 35 activity was observed for all three groups (Figure
3.9A).
Lethally irradiated mice were transplanted with 3 x 106 unsorted transduced BM cells.
Monthly donor chimerism analysis (CD45.1+GFP+) showed that WT FGF-2 and
NLS/FGF-2 transduced cells had comparable chimerism levels (~16% ± 5, 20 weeks
after transplant) whilst control transduced cells maintain significantly higher
chimerism levels (29% ± 6, 20 weeks after transplant) during the 20 week
transplantation period (p < 0.05) (Figure 3.9B).
Secondary transplantation of WT FGF-2 and NLS/FGF-2
Unfractionated bone marrow from primary recipient mice 20 weeks post-transplant
were harvested and analyzed for the percentage of LSK cells. Similar to normal B6
BM cells (0.22 ± 0.05%), WT FGF-2 and NLS/FGF-2 BM cells contained 0.15 ±
0.02% and 0.27 ± 0.05% LSK cells, respectively (Figure 3.9C). Control transduced
cells contained 1.7-fold increase in LSK cells compared to B6 BM cells (Figure
3.9C).
As outlined above, secondary recipients were transplanted with harvested donor BM
and transplanted in limiting dilutions in competition with 4 x 105 freshly isolated B6
BM cells. Analysis of CD45.1+GFP+ chimerism levels 24 weeks after transplant
indicated that WT FGF-2 and NLS/FGF-2 provided little to no contribution in the
peripheral blood (Figure 3.9D). As expected, because of the higher levels of donor
chimerism levels in the primary transplant, slightly higher CD45.1+GFP+ engraftment
levels were also detected in secondary recipients of control transduced cells (~4 ± 8%)
(Figure 3.9D). Comparison of chimerism levels from the control with that of the
previous control group (Figure 3.8A) indicated that chimerism levels from both
groups were similar. This confirms that in this experiment the control cells do not
have an increase in engraftment, but that WT FGF-2 and NLS/FGF-2 transduced
donor cells have poorer stem cell quality and thus poorer engraftment ability.
Fibroblast growth factor overexpression
93
0
10
20
30
40
50
60
0 5 10 15 20 25
Don
or c
him
eris
mG
FP+
0
2
4
6
8
10
12
14
16
Don
or c
him
eris
mC
D45
.1+ G
FP+
Control WT FGF-2 NLS/FGF-2
A.
B. C.
D.
ControlWT FGF-2NLS/FGF-2
0
0.2
0.4
0.6
0.8
1
%LS
K
Control WT FGF-2
NLS/FGF-2
B6 BM
Weeks post transplant
0
1
2
CA
FC/1
05ce
lls
0
1000
2000
3000
4000
5000
Day 7 Day 35
Control
WT FGF-2NLS/FGF-2
CAF
C/1
05ce
lls
2 x 105 donor cells + 4 x 105 competitor cells
0
10
20
30
40
50
60
0 5 10 15 20 25
Don
or c
him
eris
mG
FP+
0
2
4
6
8
10
12
14
16
Don
or c
him
eris
mC
D45
.1+ G
FP+
Control WT FGF-2 NLS/FGF-20
2
4
6
8
10
12
14
16
Don
or c
him
eris
mC
D45
.1+ G
FP+
Control WT FGF-2 NLS/FGF-2
A.
B. C.
D.
ControlWT FGF-2NLS/FGF-2
ControlWT FGF-2NLS/FGF-2
0
0.2
0.4
0.6
0.8
1
%LS
K
Control WT FGF-2
NLS/FGF-2
B6 BM0
0.2
0.4
0.6
0.8
1
%LS
K
Control WT FGF-2
NLS/FGF-2
B6 BM
Weeks post transplant
0
1
2
CA
FC/1
05ce
lls
0
1000
2000
3000
4000
5000
Day 7 Day 35
Control
WT FGF-2NLS/FGF-2
CAF
C/1
05ce
lls
0
1
2
CA
FC/1
05ce
lls
0
1000
2000
3000
4000
5000
Day 7 Day 35
Control
WT FGF-2NLS/FGF-2
Control
WT FGF-2NLS/FGF-2
CAF
C/1
05ce
lls
2 x 105 donor cells + 4 x 105 competitor cells
Figure 3.9: Transplantation of WT FGF-2 and NLS/FGF-2 cells into primary and secondary recipients.
(A): Day 7 and day 35 CAFC activity of control, WT FGF-2 and NLS/FGF-2 overexpressing cells. (B):
Average CD45.1+GFP+ donor chimerism levels of primary recipients (n=10 per group) transplanted
with control, WT FGF-2 and NLS/FGF-2 overexpressing cells. Chimerism in blood was analyzed on a
monthly basis. (C): LSK analysis of BM harvested from primary recipients 20 weeks after transplant.
(D): Average donor chimerism (CD45.1+GFP+) levels 24 weeks post-transplant of secondary transplant
recipients transplanted (n=5 per group).
Fibroblast growth factor overexpression
94
Discussion In this study we undertook a detailed analysis of the ability of enforced
overexpression of FGF-1 and FGF-2 to maintain and expand hematopoietic cells in
vitro and in vivo. We demonstrate that overexpression of FGF-1 slightly increases the
mitogenic effect of 5-FU BM cells and that both FGF-1 and FGF-2 are almost
exclusively expressed in Lin-Sca-1+c-Kit+ cells, a primitive hematopoietic cell subset.
Finally, we show that secondary recipients transplanted with S130A mutant FGF-1
showed a delayed increase in donor chimerism levels and a 10-fold increase in GFP+
cells, implying that nuclear localized FGF-1 may play a role in maintaining stem cell
quality.
Unfortunately, in in vivo repopulation assays, low donor chimerism levels (~10% to
15%) were detected in primary recipients transplanted with WT FGF-1 and WT FGF-
2 and little to no reconstitution was observed in secondary recipients. This was
unexpected as previous studies had demonstrated that the exogenous addition of FGFs
could maintain whole BM cultures for up to five weeks and generate large numbers of
cells with lymphoid and myeloid long-term repopulating capacity15;16. Additionally,
the use of FGF-1 expanded BM cells as a source of stem cells for retroviral gene
delivery generated 15.5-fold increase in the number of bone marrow-derived
competitive repopulating units per mouse and provided radioprotection and long-term
BM reconstitution with average myeloid and lymphoid chimerisms of 70% and 50%
respectively42.
It is well known that FGFs are exogenous growth factors that activate the cell surface
receptors, thereby inducing activation of intracellular second messengers43. In
addition, the receptor-bound growth factor is endocytosed and translocated across the
vesicular membrane to reach the cytosol44;45. Several lines of evidence exist indicate
that after binding to receptors, FGF-1 and FGF-2 enter the nucleus and that this is
required for mitogenic response in certain cell types46-48. This led us to assume that
both WT FGF-1 and WT FGF-2 may not be entering the nucleus in order to exert its
full mitogenic activity. As a result, two S130 mutants and one NLS/FGF-2 were
created to further examine the effect of nuclear localized FGFs on BM cells.
Immunocytochemistry analysis of S130A and NLS/FGF-2 overexpressing cells
indicated that FGF-1 and FGF-2 were not exclusively localized in the nucleus in all
FGF positive cells. Considering that in only 2% of FGF-1 positive cells the protein
Fibroblast growth factor overexpression
95
was localized in the nucleus, the delayed but marked increase in CD45.1+GFP+
chimerism levels and the significant 10-fold increase in GFP+ cells of S130A
transduced cells in secondary recipients was exceptional. This suggests that the
localization of FGF-1 in the nucleus may play a role in maintaining stem cell quality.
The translocation of FGFs appears to be a dynamic process. For example, FGF-2 has
been shown to enter the nucleus, both when added exogenously46;49 and when
expressed endogenously26;50;51. In the former case, nuclear uptake is cell cycle-
dependent occurring only during G1 to S transition46. In the latter case, only the larger
(22-25kDa) isoforms are found in the nucleus while the 18kDa form remains
cytoplasmic25;26;50;51. Through cell fractionation studies Wiedlocha et al., clearly
showed that WT FGF-1 was found in all fractions (membrane, cytoplasmic and
nucleus) of the cell, S130A was found in the nuclear fraction whereas S130E was
mainly in the cytosolic fraction19. We wish to emphasize that since we have only
analyzed localization of FGF-1 and FGF-2 by immunocytochemistry, it is possible
that we are only able to detect small amounts of FGF-1 and FGF-2 in S130A and
NLS/FGF-2 in the nuclei. Thus, a more sensitive method such as cell fractionation
may be used in future studies.
Based upon previous studies from our group16, we hypothesized that a niche in the
form of stromal cells may increase the mitogenic activity of transduced cells and
maintain stem cell activity. A role is now emerging for the ‘stem-cell niche’ to affect
maintenance, differentiation and regulation of self-renewal of HSCs in vivo52-54. Since
stromal cells include a high proportion of fibroblast cells that respond to FGFs,
especially FGF-2 10;55, we co-cultured WT FGF-1, S130E and S130A transduced cells
with FBMD-1 stromal cells. Unfortunately, co-culturing of transduced cells with a
stromal layer was ineffective as these cells failed to reconstitute the peripheral blood
of recipients.
Heparin or heparin sulfate proteoglycans play a pivotal role in stimulating and
stabilizing the interaction of FGFs to FGFRs8;56;57. One possibility as to why no
dramatic repopulation was observed in FGF-1 and FGF-2 overexpressing cells may be
due to the lack of heparin in cultures. In previous studies involving exogenous FGFs,
heparin was also added exogenously. In our current study, heparin was not added.
Klingenberg et al., has shown that a correlation exists between mitogenic potency and
heparin affinity of FGF-1 phosphorylated mutants21. If the reason for the reduced
repopulation ability of FGF-1, FGF-2, S130 mutants and NLS/FGF-2 was due to the
Fibroblast growth factor overexpression
96
lack of heparin, future overexpression studies with FGFs should involve the addition
of heparin.
Fibroblast growth factor overexpression
97
Acknowledgements We thank Professor C. Jordan from the University of Rochester for providing us with
the MIEV vector. We also thank Geert Mesander and Henk Moes for their assistance
with cell sorting and the staff of the animal facility for taking care of the animals. This
work was supported by the National Institute of Health (R01-HL073710) and the
Ubbo Emmius Foundation.
Fibroblast growth factor overexpression
98
References 1. Ema H, Takano H, Sudo K, Nakauchi H. In vitro self-renewal division of
hematopoietic stem cells. J.Exp.Med. 2000;192:1281-1288.
2. Fraser CC, Eaves CJ, Szilvassy SJ, Humphries RK. Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells. Blood 1990;76:1071-1076.
3. Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood 1999;94:2161-2168.
4. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 1997;94:13648-13653.
5. Conneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc.Natl.Acad.Sci.U.S.A 1997;94:9836-9841.
6. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J.Exp.Med. 1997;186:619-624.
7. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. The Journal of Clinical Investigation 2000;105:1013-1021.
8. Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biology 2001;2:3005.1-3005.12.
9. Faloon P, Arentson E, Kazarov A et al. Basic fibroblast growth factor positively regulates hematopoietic development. Development 2000;127:1931-1941.
10. Allouche M, Bikfalvi A. The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis. Prog.Growth Factor Res. 1995;6:35-48.
11. Allouche M, Bayard F, Clamens S et al. Expression of basic fibroblast growth factor (bFGF) and FGF-receptors in human leukemic cells. Leukemia 1995;9:77-86.
12. Yang M, Li K, Lam AC et al. Platelet-derived growth factor enhances granulopoiesis via bone marrow stromal cells. Int.J.Hematol. 2001;73:327-334.
13. Bikfalvi A, Han ZC, Fuhrmann G. Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells. Blood 1992;80:1905-1913.
Fibroblast growth factor overexpression
99
14. Chen QS, Wang ZY, Han ZC. Enhanced growth of megakaryocyte colonies in culture in the presence of heparin and fibroblast growth factor. Int.J.Hematol. 1999;70:155-162.
15. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental Cell 2003;4:241-251.
16. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures. Stem Cells 2006;24:1564-1572.
17. Wiedlocha A, Falnes PO, Rapak A et al. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization. Mol.Cell Biol. 1996;16:270-280.
18. Joy A, Moffett J, Neary K et al. Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells. Oncogene 1997;14:171-183.
19. Wiedlocha A, Nilsen T, Wesche J et al. Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1. Mol.Biol.Cell 2005;16:794-810.
20. Wiedlocha A, Falnes PO, Rapak A et al. Translocation of cytosol of exogenous, CAAX-tagged acidic fibroblast growth factor. J.Biol.Chem. 1995;270:30680-30685.
21. Klingenberg O, Wiedlocha A, Olsnes S. Effects of mutations of a phosphorylation site in an exposed loop in acidic fibroblast growth factor. J.Biol.Chem. 1999;274:18081-18086.
22. Klingenberg O, Wiedlocha A, Rapak A et al. Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells. J.Biol.Chem. 1998;273:11164-11172.
23. Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol.Cell Biol. 1999;19:505-514.
24. Dono R, James D, Zeller R. A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling. Oncogene 1998;16:2151-2158.
25. Bugler B, Amalric F, Prats H. Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. Mol.Cell Biol. 1991;11:573-577.
26. Florkiewicz RZ, Baird A, Gonzalez AM. Multiple forms of bFGF: differential nuclear and cell surface localization. Growth Factors 1991;4:265-275.
Fibroblast growth factor overexpression
100
27. Davis MG, Zhou M, Ali S et al. Intracrine and autocrine effects of basic fibroblast growth factor in vascular smooth muscle cells. J.Mol.Cell Cardiol. 1997;29:1061-1072.
28. Quarto N, Finger FP, Rifkin DB. The NH2-terminal extension of high molecular weight bFGF is a nuclear targeting signal. J.Cell Physiol 1991;147:311-318.
29. Bikfalvi A, Klein S, Pintucci G et al. Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms. J Cell Biol. 1995;129:233-243.
30. Arese M, Chen Y, Florkiewicz RZ et al. Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals. Mol.Biol.Cell 1999;10:1429-1444.
31. Vagner S, Touriol C, Galy B et al. Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells. J Cell Biol. 1996;135:1391-1402.
32. de Haan G, Van Zant G. Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene. J.Exp.Med. 1997;186:529-536.
33. de Haan G, Szilvassy SJ, Meyerrose TE et al. Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers. Blood 2000;96:1374-1379.
34. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.
35. Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 1991;78:2527-2533.
36. Morrison SJ, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu.Rev.Cell Dev.Biol. 1995;11:35-71.
37. Okada S, Nakauchi H, Nagayoshi K et al. Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule. Blood 1991;78:1706-1712.
38. Alessi DR, Andjelkovic M, Caudwell B et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996;15:6541-6551.
39. Tagawa T, Kuroki T, Vogt PK, Chida K. The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal. J.Cell Biol. 1995;130:255-263.
Fibroblast growth factor overexpression
101
40. Engel K, Kotlyarov A, Gaestel M. Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J. 1998;17:3363-3371.
41. Prudovsky I, Bagala C, Tarantini F et al. The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export. J.Cell Biol. 2002;158:201-208.
42. Crcareva A, Saito T, Kunisato A et al. Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery. Exp.Hematol. 2005;33:1459-1469.
43. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr.Relat Cancer 2000;7:165-197.
44. Olsnes S, Klingenberg O, Wiedlocha A. Transport of exogenous growth factors and cytokines to the cytosol and to the nucleus. Physiol Rev. 2003;83:163-182.
45. Wiedlocha A, Sorensen V. Signaling, internalization, and intracellular activity of fibroblast growth factor. Curr.Top.Microbiol.Immunol. 2004;286:45-79.
46. Baldin V, Roman AM, Bosc-Bierne I, Amalric F, Bouche G. Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells. EMBO J 1990;9:1511-1517.
47. Imamura T, Engleka K, Zhan X et al. Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science 1990;249:1567-1570.
48. Wiedlocha A, Falnes PO, Madshus IH, Sandvig K, Olsnes S. Dual mode of signal transduction by externally added acidic fibroblast growth factor. Cell 1994;76:1039-1051.
49. Bouche G, Gas N, Prats H et al. Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing G0----G1 transition. Proc.Natl.Acad.Sci.U.S.A 1987;84:6770-6774.
50. Renko M, Quarto N, Morimoto T, Rifkin DB. Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J.Cell Physiol 1990;144:108-114.
51. Tessler S, Neufeld G. Basic fibroblast growth factor accumulates in the nuclei of various bFGF-producing cell types. J Cell Physiol 1990;145:310-317.
52. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-846.
53. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7-25.
Fibroblast growth factor overexpression
102
54. Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149-161.
55. Oliver LJ, Rifkin DB, Gabrilove J, Hannocks MJ, Wilson EL. Long-term culture of human bone marrow stromal cells in the presence of basic fibroblast growth factor. Growth Factors 1990;3:231-236.
56. Spivak-Kroizman T, Lemmon MA, Dikic I et al. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell 1994;79:1015-1024.
57. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 1991;64:841-848.
103
104
105
Chapter 4
Mobilized peripheral blood stem cells provide rapid
reconstitution but impaired long-term engraftment
Joyce S. G. Yeoh, Albertina Ausema, Piet Wierenga,
Gerald de Haan, Ronald van Os
Department of Cell Biology, Section Stem Cell Biology, University
Medical Centre Groningen, The Netherlands
Submitted
Mobilized peripheral blood stem cells have impaired long-term engraftment
106
Abstract In this study, we use competitive repopulation assay to compare the quality and
frequency of stem cells isolated from mobilized blood with stem cells isolated from
bone marrow (BM). Lin-Sca-1+c-Kit+ (LSK) cells were harvested from control BM
and peripheral blood of mice following Granulocyte Colony Stimulating Factor (G-
CSF) administration. LSK cells were used because of their resemblance with human
CD34+ cells. We confirmed that transplantation of phenotypically defined mobilized
peripheral blood (MPB) stem cells results in rapid recovery of blood counts.
However, in vitro results indicated that LSK cells purified from MPB had lower
cobblestone area forming cell (CAFC) day 35 activity compared to BM. Additionally,
evaluation of chimerism after co-transplantation of LSK cells purified from blood and
BM revealed that MPB stem cells contained 25-fold less repopulation potential
compared to BM stem cells. Competitive repopulating unit (CRU) frequency analysis
showed that freshly isolated MPB LSK cells have 8.8-fold fewer cells with long-term
repopulating ability compared to BM LSK cells. Secondary transplantation showed no
further decline in contribution of hematopoiesis relative to BM. We conclude that the
reduced frequency of stem cells within the LSK population of MPB, rather than
poorer quality, causes a reduced repopulation potential of MPB.
Mobilized peripheral blood stem cells have impaired long-term engraftment
107
Introduction The initial source of hematopoietic cells used for transplantations was bone marrow1.
However, due to the faster regeneration of both circulating neutrophils (9-11 days) 2;3
and platelets, peripheral blood stem cells have become the primary source of
hematopoietic stem cells for clinical transplantation over the past 10 years4.
Although multiple hematopoietic growth factors are capable of inducing mobilization
of hematopoietic progenitors, G-CSF is at present one of the most used mobilizing
molecule in clinical protocols5. The efficiency of G-CSF to mobilize bone marrow
precursors and long-term repopulating cells was initially shown in preclinical studies.
Molineux et al., observed a marked increase of the colony-forming unit spleen (CFU-
S) pool in the peripheral blood of mice treated with repeated doses of G-CSF6. In
addition, it was observed that G-CSF alone and in combination with SCF or IL-7
mobilizes hematopoietic precursors capable of both radioprotection and generating
sustained lympho-haematopoiesis in transplanted recipients7. In vitro data from
human patients appear consistent with the concept that the quality of human
mobilized peripheral blood progenitor cells is at least equivalent to that corresponding
to bone marrow grafts8;9.
Surprisingly, despite the prevalent use of hematopoietic stem cell mobilization in
clinical transplantation, few reports exist describing the competitive repopulating
quality of mobilized stem cells compared to bone marrow stem cells following G-CSF
treatment. Most available reports only outlined the differences in the kinetics and
efficiency of engraftment, in homing properties and in cell cycle profiles between
mobilized blood stem cells and those isolated from resting bone marrow5;10-13.
In view of the increased use of peripheral blood stem cells in clinical transplant
settings, it is of relevance to investigate long term functioning of stem cells isolated
from different sources. In this study, we directly assessed the function of mobilized
peripheral blood stem cells compared to control bone marrow stem cells when co-
transplanted in a single recipient mouse in an in vivo competitive repopulation assay.
We show a reduced frequency of repopulating stem cells in purified G-CSF-mobilized
peripheral blood LSK stem cells, which caused a 25-fold reduction in repopulation
potential compared with BM LSK cells.
Mobilized peripheral blood stem cells have impaired long-term engraftment
108
Materials and Methods Mice
Female C57BL/6 (B6), C57BL/6.SJL (CD45.1), (C57BL/6 x C57BL6.SJL) F1
(CD45.1/2) or C57BL/6-Tg(ACTB-EGFP)10sb/J transgenic GFP (GFP) mice were
used as donors, competitors or recipients of blood and marrow stem cells depending
on the experimental model. CD45.1 and transgenic GFP mice were originally
obtained from the Jackson Laboratory (Bar Harbor, Maine) and bred in our local
animal facility. Wild type female B6 mice were purchased from Harlan (Horst, The
Netherlands) and maintained under clean conventional conditions in the animal
facilities of University Medical Centre Groningen (The Netherlands). Mice were fed
ad libitum with food pellets and acidified tap water (pH = 2.8). All animal procedures
were approved by the local animal ethics committee of the University Medical Centre
Groningen.
Mobilization and harvesting of stem cells
Bone marrow cells were harvested by flushing the femoral content with α-MEM
(GibcoBRL, Invitrogen, CA) supplemented with 2% fetal calf serum (FCS;
GibcoBRL, Invitrogen, CA). Pegylated G-CSF (Neulasta) (250µg/kg/mouse)
(Amgen, Thousand Oaks, CA) was used to mobilize stem cells14. Two doses of G-
CSF were subcutaneously administered to donor mice at day -6 and day -3 before
stem cell harvest. Mobilized peripheral blood cells were harvested by cardiac
puncture. Approximately 1 ml of blood was collected and was diluted with 4 ml of
Iscove’s modified DMEM (IMDM; GibcoBRL, Paisley, Scotland) supplemented with
5% FCS (GibcoBRL, Invitrogen, CA) and heparin (25 IU) (Leo Pharma, Breda,
Netherlands). The collected blood cell suspension (5 ml) was centrifuged over an
equal volume of Lympholyte-M (Cedarlane Laboratories Ltd, Hornby, Canada) at 400
x g for 30 minutes at room temperature. After centrifugation, the mononuclear cells
within the opaque interface layer were isolated and washed in IMDM/5% FCS for 5
minutes at 2,000 rpm at 4oC. Alternatively, red blood cells were lysed using
ammonium chloride (NH4Cl) without prior density separation. Nucleated cells were
measured on a Coulter Counter Model Z2 (Coulter Electronics, Hialeah, FL).
Mobilized peripheral blood stem cells have impaired long-term engraftment
109
Isolation of Lin-Sca-1+c-Kit+ cells
Bone marrow and mobilized peripheral blood cells were stained as previously
described15 with biotinylated lineage-specific antibodies Mouse Lineage Panel,
containing anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1 and anti-CD3e (BD
Pharmingen, San Diego, CA), FITC-anti-Sca-1 and APC-anti-c-kit (BD Pharmingen,
San Diego, CA). Biotinylated antibodies were visualized with streptavidin-PE
(Pharmingen, San Diego, CA). After antibody staining, cells were sorted by a
MoFlow cell sorter (DakoCytomation, Fort Collins, CO). Lin-Sca-1+c-Kit+ (LSK) and
Lin- non-Sca-1+c-Kit+ cells were sorted and used in transplantation assays or in in
vitro CAFC assays.
Long term competitive repopulation ability
Female B6, CD45.1 or transgenic GFP mice were used as donors for competitor cells.
Female B6 mice were used as recipients in all experiments. Recipient mice were
irradiated with 9.5 Gy γ-rays (0.7026 Gy/min) in a CIS Biointernational IBL 637 137Cs-source, 20-24 hours prior to transplantation. For competitive repopulation
determination, unfractionated cells or mobilized peripheral blood LSK cells, were
mixed with competitor cells (unfractionated or LSK bone marrow cells) and
intravenously transplanted into recipient mice. Each transplant group consisted of 8-
10 recipients. Following transplantation, blood samples (60μl) were taken monthly to
determine donor chimerism. Levels of chimerism were determined by detecting the
presence of GFP+ or CD45.1+ and CD45.2+ cells in transplanted mice. To detect
CD45.1+ and CD45.2+ cells, cells were stained with anti-CD45.2 (FITC) and anti-
CD45.1 (PE) antibodies (BD Pharmingen, San Diego, CA) for 30 minutes and
analyzed on a flow cytometer (FACS Calibur; Becton Dickinson Biosciences, San
Jose, CA). In addition, the competitive repopulating index (CRI) was determined. CRI
is a relative measure of the competitive ability of test cells to that of fresh bone
marrow cells. The CRI was calculated by taking the ratio of WBC derived from
mobilized blood cells to bone marrow cells in the circulation and dividing it by the
ratio of mobilized blood cells to bone marrow cells transplanted. A CRI value of one
indicates by definition that mobilized peripheral blood cells and bone marrow cells
have equal competitive ability.
Mobilized peripheral blood stem cells have impaired long-term engraftment
110
Competitive Repopulation Unit assay
B6 recipient mice were transplanted with a series of diluted CD45.1 LSK cells (1,200,
600 and 300) from mobilized blood and control bone marrow and with a fixed number
of B6 competitor cells (5 x 105). Twelve weeks after transplantation, donor cell
contribution in the peripheral blood was determined. Recipients with a contribution of
≥ 5% in both myeloid and lymphoid lineages were considered to be positive.
To evaluate and quantify the repopulating potential of mobilized blood LSK cells and
control BM LSK cells, the frequency of competitive repopulation units (CRU) was
calculated. CRU frequencies per 1,000 LSK were calculated from the resultant
percentage positive recipients by limiting dilution analysis procedures which uses
Poisson statistics16.
Cobblestone area forming cell assays
Cobblestone area forming cell assays (CAFC) were performed as described17-19 to
assess the number of hematopoietic progenitor cells (CAFC day 7) or more primitive
stem cells (CAFC day 35) in mobilized peripheral blood stem cells.
Secondary Transplantations
In one of the competitive repopulation experiments, in which recipients were
transplanted in different ratios with CD45.1 mobilized peripheral blood LSK cells and
CD45.2 bone marrow LSK competitor cells, mice were sacrificed for secondary
transplantations. Bone marrow cells from B6 primary chimeric recipients were
isolated on basis of CD45 isoform. CD45.1 bone marrow cells represent cells derived
from mobilized peripheral blood LSK population (CD45.1 MPB LSK derived bone
marrow) and CD45.2 (B6) bone marrow cells represent cells derived from bone
marrow LSK cells (CD45.2 BM LSK derived bone marrow). Female CD45.1
recipient mice were transplanted with CD45.2 bone marrow LSK derived bone
marrow cells whilst B6 recipient mice were transplanted with CD45.1 mobilized
peripheral blood LSK derived bone marrow cells. Recipient mice were transplanted in
three ratios (1:1, 1:2 and 1:4) with a fixed number (4 x 105) of CD45.1/2 (F1)
competitors. Each transplant group consisted of five recipients and blood samples
(60µl) were taken on a monthly basis to determine donor chimerism and CRI.
Mobilized peripheral blood stem cells have impaired long-term engraftment
111
Statistical Analysis
P-values were calculated using the Mann-Whitney test or the student’s t-test
(assuming unequal variances of the two variables) and were employed to determine
the statistical significance between mobilized blood and normal bone marrow (p <
0.05). Quantification of CAFC at day 7 and 35 was performed by using maximum
likelihood ratio method20. The Poisson-based limiting dilution analysis calculation
was used with a 95% confidence interval to determine significant differences at p <
0.05.
Mobilized peripheral blood stem cells have impaired long-term engraftment
112
Results Unfractionated mobilized peripheral blood cells have reduced repopulation ability
compared to unfractionated bone marrow cells
To compare the repopulation ability of unfractionated bone marrow and
unfractionated mobilized peripheral blood cells, blood cells were extracted from
donor B6 mice following treatment with two doses of G-CSF. Untreated bone marrow
cells were obtained from GFP-Tg or CD45.1 mice. Clonogenic activity of the two cell
sources was measured by seeding in limiting dilutions in a CAFC assay. At day 7,
cells from each source displayed high CAFC activity, with average values of ~1,400
progenitors/106 cells [95% confidence intervals (CI): 908-2294]. No significant
differences were observed between bone marrow and peripheral blood (Figure 4.1A).
Whilst bone marrow cells isolated from either CD45.1 or GFP Tg mice displayed a
high CAFC day 35 activity of 10.5 (95% CI: 6.6-16.8)/106 and 16 (95% CI: 10-
24)/106 respectively, CAFC day 35 frequency in mobilized peripheral blood was only
1.8 (95% CI: 1-3.3)/106 (Figure 4.1A). These results indicate that unfractionated
mobilized peripheral blood cells had significantly lower primitive stem cell activity
compared to bone marrow cells.
To verify the in vitro results, three groups of lethally irradiated mice were
transplanted in a competitive repopulation assay. Donor chimerism levels of
mobilized peripheral blood cells were used to calculate CRI post transplant (Figure
4.1B) and were ~20% (data not shown). As expected, a CRI value of 1 was
maintained throughout the 7 month transplant period for recipient mice receiving
CD45.1 bone marrow mixed with GFP bone marrow, indicating equal competitive
repopulating ability of both sources (Figure 4.1B). However, mice transplanted with
mobilized peripheral blood in competition with either CD45.1 bone marrow or GFP
bone marrow exhibited significantly lower average CRI levels (0.33 ± 0.41 and 0.41 ±
0.29, respectively) at 5 months after transplant (Figure 4.1B).
Both in vitro CAFC results and in vivo competitive repopulation assays suggest that
unfractionated mobilized peripheral blood cells have lower repopulation ability
compared to unfractionated bone marrow cells. In addition, no apparent differences
were observed between different donor mouse strains. The fact that we used the
mononuclear cell fraction from mobilized blood (which is enriched for stem cells) and
Mobilized peripheral blood stem cells have impaired long-term engraftment
113
unseparated bone marrow cells may underestimate the difference in repopulating
ability.
0
0.20.4
0.6
0.8
11.2
1.4
1.61.8
2
0 5 10 15 20 25 30
Weeks after transplant
Com
petit
ive
Rep
opul
atio
n In
dex
MPB + CD45.1 control BM
MPB + GFP untreated BM
CD45.1 control BM + GFP control BM
1
10
100
1000
10000
7 35
CD45.1 control BM
GFP control BM
MPB
CAF
C fr
eque
ncy/
106
cells
Days
A.
B.
P < 0.05
0
0.20.4
0.6
0.8
11.2
1.4
1.61.8
2
0 5 10 15 20 25 30
Weeks after transplant
Com
petit
ive
Rep
opul
atio
n In
dex
MPB + CD45.1 control BM
MPB + GFP untreated BM
CD45.1 control BM + GFP control BM
MPB + CD45.1 control BM
MPB + GFP untreated BM
CD45.1 control BM + GFP control BM
1
10
100
1000
10000
7 35
CD45.1 control BM
GFP control BM
MPB
CD45.1 control BM
GFP control BM
MPB
CAF
C fr
eque
ncy/
106
cells
Days
A.
B.
P < 0.05
Figure 4.1: Unfractionated mobilized peripheral blood cells have reduced repopulation ability in
comparison with bone marrow cells. Unfractionated monunuclear cells from mobilized peripheral
blood (MPB) were obtained from B6 mice following treatment with 2 doses of Peg G-CSF 6 and 3
days before isolation. Control unfractionated bone marrow (BM) cells were collected from CD45.1 and
GFP-transgenic mice and transplanted in equal numbers. (A): Analysis of CAFC day 7 and day 35
activity for CD45.1 BM, GFP BM and MPB. (B): CRI analysis of mice transplanted with B6 MPB +
CD45.1 control BM, B6 MPB + GFP control BM and CD45.1 control BM + GFP control BM. All
mice were transplanted with 2 x 106 donor cells and 2 x 106 competitor cells. Average chimerism levels
at 20 weeks were 42% ± 18 (GFP BM + CD45.1 BM), 20% ±19 (B6 MPB + CD45.1 BM), and 16% ±
21 (B6 MPB + GFP BM). All chimerism levels were converted into CRI values as indicated in
materials and methods.
Mobilized peripheral blood stem cells have impaired long-term engraftment
114
Mobilized peripheral blood cells have a lower frequency of Lin-Sca-1+c-Kit+ cells
We next determined whether the poor repopulation ability of unfractionated mobilized
peripheral blood cells is reflected by the size of the phenotypically defined stem cell
population, LSK. To test this possibility we measured LSK frequencies in bone
marrow cells and mobilized peripheral blood. Typical FACS plots for isolating LSK
cells from bone marrow and mobilized peripheral blood cells are shown in Figure
4.2A and 4.2B. Mobilized peripheral blood differs from bone marrow cells, as they
contain fewer c-Kit+ cells and more Sca-1-c-Kit- cells (Figure 4.2B). This finding is
consistent with that of Levesque et al., who reported that mobilization with G-CSF
results in a down-regulation of c-Kit on mouse hematopoietic progenitor cells in
vivo21. On average, bone marrow cells have a LSK frequency of 0.17% ± 0.1,
significantly different to that of mobilized peripheral blood cells, which have a 3-4
fold lower frequency of 0.05% ± 0.04 (p < 0.05) (Table 4.1). The lower frequency in
mobilized blood may explain the lower repopulation potential of unfractionated
mobilized blood.
To compare mobilized blood and bone marrow LSK on a per cell basis, LSK cells
were isolated from bone marrow and mobilized peripheral blood and placed in
limiting dilution in a CAFC assay to assess clonogenic activity. CAFC day 7
frequency of 250/103 LSK cells (153-402; 95% CI) was recorded for control bone
marrow LSK cells, whilst mobilized peripheral blood LSK cells had a 2.4-fold higher
CAFC activity of (600/103 LSK cells [308-1160; 95% CI]) (Figure 4.2A and 4.2B).
CAFC day 35 frequency for mobilized peripheral blood LSK cells was 3.0/103 LSK
cells (1-8; 95% CI), similar to control bone marrow LSK cells with a CAFC activity
of 6.7/103 (0.9-48; 95% CI) (Figure 4.2A and 4.2B). Thus, these data suggest that,
compared to bone marrow, mobilized peripheral blood LSK cells contain somewhat
more progenitor cells, but slightly fewer stem cells.
It is well known that in normal bone marrow all stem cell activity is contained in the
LSK fraction22;23. To verify that in mobilized blood, stem cell activity is also
restricted to the LSK population, Lin- cells from mobilized peripheral blood were
further fractionated into non-Sca-1+c-Kit+ cells and placed in limiting dilution in a
CAFC assay (Figure 4.2C). As shown in Figure 4.2C, only some day 7 CAFC activity
was detected for Lin- non-Sca-1+c-Kit+ cells, but no CAFC day 35. This demonstrates
that cells outside the LSK gate do not have clonogenic activity. A summary of LSK
frequencies and CAFC day 35 frequencies and a calculation of their total pool size is
Mobilized peripheral blood stem cells have impaired long-term engraftment
115
shown in Table 4.1. Since we did not splenectomize our mice, a considerable number
of LSK cells may have been present in the spleen. However, we aimed at comparing
the phenotypically defined cell population (LSK) from mobilized blood with the same
population in steady state bone marrow.
0.1
1
10
100
1000
10000
7 35
C.
FSC
SSC
Sca-1
c-K
it
BL/6 Mobilized PB Lin- non Sca-1+c-Kit+
nd
CAF
C /1
03ce
lls
Days
87%
A.BL/6 control BM
Lin
5%
Sca-1
c-K
it 0.15%
B.BL/6 Mobilized PB
FSC
SSC
Sca-1
c-K
it
1
10
100
1000
10000
7 35CAF
C /1
03LS
K c
ells
Days
Control BM
MPB
0.04%
3.3%
*
0.1
1
10
100
1000
10000
7 35
C.
FSC
SSC
Sca-1
c-K
it
BL/6 Mobilized PB Lin- non Sca-1+c-Kit+
nd
CAF
C /1
03ce
lls
Days
87%0.1
1
10
100
1000
10000
7 35
C.
FSC
SSC
Sca-1
c-K
it
BL/6 Mobilized PB Lin- non Sca-1+c-Kit+
nd
CAF
C /1
03ce
lls
Days
87%
A.BL/6 control BM
Lin
5%
Sca-1
c-K
it 0.15%
B.BL/6 Mobilized PB
FSC
SSC
Sca-1
c-K
it
1
10
100
1000
10000
7 35CAF
C /1
03LS
K c
ells
Days
Control BM
MPB
0.04%
3.3%
*
A.BL/6 control BM
Lin
5%
Sca-1
c-K
it 0.15%
B.BL/6 Mobilized PB
FSC
SSC
Sca-1
c-K
it
1
10
100
1000
10000
7 35CAF
C /1
03LS
K c
ells
Days
Control BM
MPB
Control BM
MPB
0.04%
3.3%
*
Figure 4.2: Lin-Sca-1+c-Kit+ (LSK) contains the majority of cells with long-term repopulating ability
in both bone marrow and mobilized peripheral blood. Characteristic FACS plot of Lin- and Sca-1+c-
Kit+ sorts for (A): B6 bone marrow (BM) and (B): mobilized peripheral blood (MPB) cells and
comparison in limiting dilution analysis using the CAFC assay. (C): FACS plot of the Lin- non-Sca-
1+c-Kit+ sort for MPB cells placed in CAFC assay.
Mobilized peripheral blood stem cells have impaired long-term engraftment
116
Table 4.1: Average LSK frequencies in bone marrow and G-CSF mobilized blood
Frequency LSK (%) Total LSK cells/2
femurs or per ml blood
CAFC day
35/106
Control BM 0.17 ± 0.1 84837 ± 43361 12537 ± 9695
MPB 0.05 ± 0.04* 8968 ± 6377 2150 ± 1064
Average frequency, total LSK cells and CAFC day 35 frequency in control bone marrow (BM) (n=5)
and mobilized peripheral blood (MPB) (n=6).
* Indicates that LSK frequency is significantly (p < 0.05) lower compared to normal bone marrow
Mobilized peripheral blood stem cells promote accelerated hematological
reconstitution
To directly compare the rates of hematopoietic recovery of peripheral blood cell
values after transplant, 1,250 LSK cells were isolated from bone marrow or mobilized
peripheral blood and transplanted into two groups of lethally irradiated B6 mice.
Circulating blood cells were counted on day 7 and 10 and then once a week to 5
weeks after transplant. WBC counts of recipients transplanted with mobilized
peripheral blood stem cells increased at day 10 and day 14, reaching normal values of
~8 x 106/ml. At days 21 and 35, WBC counts were similar to those of animals
transplanted with bone marrow LSK cells (Figure 4.3). Consistent with the results in
Figure 4.2A and 4.2B (showing the presence of many progenitors/short-term
repopulating cells in blood), these data indicate that transplants with mobilized
peripheral blood stem cells results in faster recovery of WBC number compared to
control LSK bone marrow cells.
Mobilized peripheral blood stem cells have impaired long-term engraftment
117
0
2
4
6
8
10
12
14
0 7 14 21 28 35 42
1250 MPB LSK1250 control BM LSK
WB
C x
106 /m
l
Days after transplant
0
2
4
6
8
10
12
14
0 7 14 21 28 35 42
1250 MPB LSK1250 control BM LSK1250 MPB LSK1250 control BM LSK
WB
C x
106 /m
l
Days after transplant Figure 4.3: Mobilized peripheral blood LSK cells promote faster WBC recovery. Donor mice were
treated with 2 doses of Peg-G-CSF 6 days prior to donor cell isolation. Two groups of lethally
irradiated mice were transplanted with 1,250 LSK cells from control bone marrow (BM) (n=10) or
1,250 LSK cells from mobilized peripheral blood (MPB) (n=6). Following transplant, blood samples
were analyzed for WBC counts (WBC x 106/ml) from 7 days to 12 weeks after transplant.
Repopulation ability of peripheral blood stem cells
To further examine and directly compare the repopulation ability of mobilized
peripheral blood stem cells to bone marrow stem cells, LSK cells from bone marrow
and mobilized peripheral blood were tested in a competitive long-term repopulation
assay in different groups of mice. Recipients were transplanted with various ratios of
LSK cells from blood and LSK cells from bone marrow. A chimerism level of 50%
would be expected if both sources contained the same number of stem cells and
performed equally well. However, the contribution of cells derived from transplanted
mobilized peripheral blood was extremely low, with values of 2.6 ± 3% and 5.6 ± 4%
at 28 weeks when transplanted in a 1:1 ratio (Figure 4.4A). The CRI of peripheral
blood stem cells for each individual mouse is plotted in Figure 4.4B. The average CRI
(0.04 ± 0.04) for all transplanted mice indicate that purified mobilized peripheral
blood stem cells have a severely reduced repopulation potential (25-fold) compared to
bone marrow stem cells (Figure 4.4C). Recipient mice were either female B6 or
female CD45.1/2 mice.
Mobilized peripheral blood stem cells have impaired long-term engraftment
118
0
5
10
15
20
25
30
0 5 10 15 20 25 30
% D
onor
chi
mer
ism
A.
B.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15 20 25 30
Com
petit
ive
Rep
opul
atio
n In
dex
Weeks after transplant
C.
Weeks after transplant
Weeks after transplant
1000 MPB LSK + 1000 control BM LSK
750 MPB LSK + 750 control BM LSK
1000 MPB LSK + 1000 control BM LSK750 MPB LSK + 750 control BM LSK250 MPB LSK + 500 control BM LSK1600 MPB LSK + 400 control BM LSK
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30
Com
petit
ive
Rep
opul
atio
n In
dex
0
5
10
15
20
25
30
0 5 10 15 20 25 30
% D
onor
chi
mer
ism
A.
B.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15 20 25 30
Com
petit
ive
Rep
opul
atio
n In
dex
Weeks after transplant
C.
Weeks after transplant
Weeks after transplant
1000 MPB LSK + 1000 control BM LSK
750 MPB LSK + 750 control BM LSK
1000 MPB LSK + 1000 control BM LSK
750 MPB LSK + 750 control BM LSK
1000 MPB LSK + 1000 control BM LSK750 MPB LSK + 750 control BM LSK250 MPB LSK + 500 control BM LSK1600 MPB LSK + 400 control BM LSK
1000 MPB LSK + 1000 control BM LSK1000 MPB LSK + 1000 control BM LSK750 MPB LSK + 750 control BM LSK750 MPB LSK + 750 control BM LSK250 MPB LSK + 500 control BM LSK250 MPB LSK + 500 control BM LSK1600 MPB LSK + 400 control BM LSK1600 MPB LSK + 400 control BM LSK
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30
Com
petit
ive
Rep
opul
atio
n In
dex
Figure 4.4: Mobilized peripheral blood stem cells have reduced repopulation potential compared to
bone marrow stem cells. Mobilized peripheral blood (MPB) stem cells were obtained from donor mice
treated with 2 doses of Peg G-CSF. Both control bone marrow (BM) and MPB were sorted for LSK
cells. (A): Donor chimerism of MPB stem cells transplanted in a 1:1 ratio with competitor BM cells.
(B): Lethally irradiated recipients were transplanted with 1,600 LSK MPB + 400 LSK (n=5), 1,000
LSK MPB + 1,000 LSK control BM (n=6), 750 LSK MPB + 750 LSK control BM (n=10) and 250
LSK MPB + 500 LSK BM (n=4). Chimerism data was converted into a competitive repopulation index
(CRI). CRI values for each individual mouse is shown against weeks post transplant. (C): Average CRI
for all transplanted mice (n=25).
Mobilized peripheral blood stem cells have impaired long-term engraftment
119
Mobilized blood stem cells have a reduced frequency of long-term repopulating stem
cells
To determine whether the inefficiency of mobilized peripheral blood stem cells to
promote long-term repopulation was due to a decline in quality of blood stem cells or
that mobilized blood stem cells contained fewer long-term reconstituting stem cells,
we quantified the frequency of long-term repopulating stem cells in mobilized blood
and bone marrow stem cells. Mice were transplanted with CD45.1 mobilized blood or
control bone marrow LSK cells in limiting dilution (1,200, 600 and 300) with a fixed
number (5 x 105) of B6 competitors bone marrow cells. The repopulation ability of
both mobilized blood and control bone marrow LSK cells was assessed by calculating
the CRU per 1,000 LSK cells. Analysis of the frequency of CRU 12 weeks after
transplant revealed that control bone marrow LSK cells are more competitive in
reconstituting the recipient (17 of 18 = 94% contained ≥ 5% of engraftment) (data not
shown) resulting in a significantly (p < 0.01) higher frequency of CRU (6.6 CRU per
1,000 LSK; 1.6-15; 95% CI versus 0.75 CRU per 1,000 LSK; 0.35-2.8; 95% CI for
mobilized blood LSK) (Figure 4.5). This indicates that mobilized peripheral blood
stem cells have a reduced number of long-term repopulating cells compared to normal
bone marrow. Taken together, these results indicate that reduced repopulation
potential is due to lower frequency of stem cells rather than an impaired function of
mobilized blood LSK cells.
0
5
10
15
20
CR
U/1
000
LSK 6.6
0.75
MPB LSK BM LSK0
5
10
15
20
CR
U/1
000
LSK 6.6
0.75
MPB LSK BM LSK Figure 4.5: Reduced frequency of long-term repopulating stem cells in mobilized blood. Frequency of
competitive repopulation units (CRU) per 1,000 LSK cells with 95% confidence limits of recipient
mice transplanted in limiting dilution (1,200, 600, 300) with CD45.1 mobilized peripheral blood
(MPB) LSK cells and control bone marrow (BM) LSK cells with 5 x 105 B6 bone marrow cells. CRU
frequency of 0.75 CRU per 1,000 LSK (0.35-2.8; 95% confidence limit) was observed for mobilized
peripheral blood LSK group. Control bone marrow LSK group contained a significant (p < 0.01) 8.8-
fold higher CRU frequency (6.6 CRU per 1,000 LSK; 1.6-15; 95% confidence limit).
Mobilized peripheral blood stem cells have impaired long-term engraftment
120
Engrafted mobilized peripheral blood stem cells do not exhaust faster than bone
marrow stem cells
Recipient mice (CD45.1/2) previously transplanted with a mixture of mobilized blood
LSK cells and bone marrow LSK cells were sacrificed 37 weeks after transplant.
Donor CD45.1 mobilized blood LSK-derived bone marrow cells and CD45.2 bone
marrow LSK-derived bone marrow cells were selected, isolated by FACS sorting and
transplanted in limiting dilutions into secondary recipients with 4 x 105 CD45.1/2
competitor cells respectively (Figure 4.6A). Average CRI values of recipients
receiving CD45.1 blood LSK cells or CD45.2 bone marrow LSK cells are shown in
Figure 4.6B. Bone marrow cells originated from LSK blood cells displayed similarly
low CRI levels (~0.14 ± 0.1) as bone marrow derived from bone marrow LSK cells
(~0.1 ± 0.1). This indicates that the quality of stem cells from mobilized blood derived
donor did not decrease after transplantation and that exhaustion of initially engrafted
stem cells is similar for stem cells from both sources.
Reduced repopulation potential of LSK mobilized peripheral blood stem cells in a
non-competitive setting
To model a more clinically relevant protocol, recipient mice were transplanted with
1,250 LSK from pooled bone marrow or 1,250 LSK from pooled mobilized peripheral
blood cells without competitor cells (Figure 4.7). Donor chimerism for mice
transplanted with purified bone marrow stem cells displayed little variation, with
average donor chimerism of 87 ± 8.8% 20 weeks after transplant. In contrast, donor
chimerism of mice transplanted with mobilized peripheral blood LSK cells varied
widely (44 ± 39%), with half of the recipients exhibiting high chimerism levels (80 ±
4.2%) while the other half was below 10%. This suggests that the frequency of cells
that contribute to long-term repopulation is at limiting dilution; 1,250 MPB LSK cells
contain very few (0, 1, or 2) stem cells. This is consistent with our CRU data (0.75
CRU/100 LSK). Reducing the dose of bone marrow LSK to 625 led to more variation
in chimerism but overall levels were still higher (52 ± 34%) than those observed in
some recipients transplanted with 1,250 mobilized peripheral blood LSK.
Mobilized peripheral blood stem cells have impaired long-term engraftment
121
A.
B.
0
0.1
0.2
0.3
0.4
0 5 10 15 20
CR
I
Weeks after transplant
CD45.1 MPB LSK derived-BM cells CD45.2 BM LSK derived-BM cells
9Gy
CD45.1 MPB LSK derived-BM + 4x105 CD45.1/2
CD45.2 BM LSK derived-BM + 4x105 CD45.1/2
and
CD45.1 or B6 recipient
9Gy
1000 CD45.1 MPB LSK + 1000 CD45.2 control BM LSK
37 weeks, sacrifice Sort BM for CD45.1
and CD45.2
CD45.1 MPB LSK derived BM
CD45.2 BM LSK derived BM
FITC
PE 86%
3%
B6 recipient
A.
B.
0
0.1
0.2
0.3
0.4
0 5 10 15 20
CR
I
Weeks after transplant
CD45.1 MPB LSK derived-BM cells CD45.2 BM LSK derived-BM cells CD45.1 MPB LSK derived-BM cells CD45.2 BM LSK derived-BM cells
9Gy
CD45.1 MPB LSK derived-BM + 4x105 CD45.1/2
CD45.2 BM LSK derived-BM + 4x105 CD45.1/2
and9Gy9Gy
CD45.1 MPB LSK derived-BM + 4x105 CD45.1/2
CD45.2 BM LSK derived-BM + 4x105 CD45.1/2
and
CD45.1 or B6 recipient
9Gy
1000 CD45.1 MPB LSK + 1000 CD45.2 control BM LSK
9Gy9Gy
1000 CD45.1 MPB LSK + 1000 CD45.2 control BM LSK
37 weeks, sacrifice Sort BM for CD45.1
and CD45.2
CD45.1 MPB LSK derived BM
CD45.2 BM LSK derived BM
FITC
PE 86%
3% CD45.1 MPB LSK derived BM
CD45.2 BM LSK derived BM
FITC
PE 86%
3%
B6 recipient
Figure 4.6: Similar long-term functioning of engrafted mobilized blood and bone marrow stem cells.
(A): Primary recipients were transplanted with CD45.1 mobilized peripheral blood (MPB) LSK and
CD45.2 bone marrow (BM) LSK cells. Thirty-seven weeks after transplant, recipient mice were
sacrificed and their bone marrow was isolated and the CD45.1 and CD45.2 fractions were separated.
CD45.1 MPB LSK-derived BM and CD45.2 BM LSK-derived BM cells were isolated separated by
FACS sorting and transplanted in different ratios (1:1, 1:2 and 1:4) with 4 x 105 CD45.1/2 F1
competitor cells into lethally irradiated CD45.1 or B6 secondary recipients. (B): Average CRI values of
secondary recipients receiving CD45.1 mobilized blood LSK-derived bone marrow cells or CD45.2
bone marrow LSK-derived bone marrow cells.
Mobilized peripheral blood stem cells have impaired long-term engraftment
122
0
20
40
60
80
100
0 5 10 15 20 25
0
20
40
60
80
100
0 5 10 15 20 25
A.
B.
1250 LSK Control BM
1250 LSK MPB
% D
onor
chi
mer
ism
Weeks after transplant
% D
onor
chi
mer
ism
Weeks after transplant
0
20
40
60
80
100
0 5 10 15 20 25
0
20
40
60
80
100
0 5 10 15 20 25
A.
B.
1250 LSK Control BM
1250 LSK MPB
% D
onor
chi
mer
ism
Weeks after transplant
% D
onor
chi
mer
ism
Weeks after transplant
Figure 4.7: Mobilized peripheral blood stem cells have reduced long-term repopulation ability in a
non-competitive setting. (A): Lethally irradiated B6 recipients (n=7) were transplanted with 1,250 LSK
from CD45.1 bone marrow (BM). Donor chimerism levels are shown for each recipient. (B): Lethally
irradiated B6 recipients (n=6) transplanted with 1,250 LSK purified from CD45.1 mobilized peripheral
blood (MPB) cells. Donor chimerism levels are shown for individual recipients.
Mobilized peripheral blood stem cells have impaired long-term engraftment
123
Discussion In the present study, we exploited a murine model to evaluate the quality of mobilized
peripheral blood stem cells in in vitro and in vivo assays. Our data demonstrated that
unfractionated mobilized peripheral blood stem cells have a 5-fold reduction in CAFC
day 35 activity and a 5-fold reduction in stem cell repopulation potential compared to
unfractionated control bone marrow cells (Figure 4.1). Mobilized blood stem cells,
selected on basis of LSK expression, displayed an even greater fold reduction (25-
fold) in long-term repopulation potential when transplanted in competition with
normal bone marrow stem cells (Figure 4.4). Limiting dilution analysis showed that
this was a result of fewer stem cells rather than less potent stem cells as mobilized
blood stem cells have a 8.8-fold decrease in CRU compared to bone marrow stem
cells (Figure 4.5).
To compensate for the possible differences in stem cell frequency our experiments
were performed using a phenotypically defined stem cell population; Lin-Sca-1+c-Kit+
(LSK). Although LSK cells are not a pure stem cell population, in steady-state bone
marrow they contain all cells with long-term repopulation ability, very much like
CD34+ cells in humans22;23. The overall LSK cell number in mobilized peripheral
blood was markedly lower than in the bone marrow pool (Table 4.1). In mobilized
blood, fewer c-Kit+ cells were observed. These data are consistent with a down-
regulation of c-Kit on mouse hematopoietic progenitor cells in vivo resulting from
proteolytic cleavage as previously reported21. G-CSF administration also causes a
change in cell surface marker expression of CD3424. Importantly, we found no in vitro
stem cell activity in cells negative for either c-Kit or Sca-1, documenting that also in
mobilized blood all stem cells are contained in the LSK population.
Purified stem cells with the same phenotype do not always have the same functional
properties. One in five Thy-1lowSca-1+Lin- c-Kit+ cells isolated from young bone
marrow provides long-term reconstitution following transplantation into irradiated
mice25;26. Poorer repopulating efficiencies were reported for stem cells isolated from
other sources. Only 1 in 10 bone marrow stem cells from old mice engraft and 1 in 75
cyclophosphamide/G-CSF mobilized spleen Thy-1lowSca-1+Lin-c-Kit+ cells provided
long-term multilineage engraftment 27;28. Purification of mobilized spleen stem cells
using signalling lymphocytic activation molecule (SLAM) family markers,
CD150+CD48- resulted in a ~2-fold reduced long-term engrafting capacity when
Mobilized peripheral blood stem cells have impaired long-term engraftment
124
compared to the same population in young bone marrow stem cells29. This suggests
that fewer “true” stem cells are present in the Thy-1lowSca-1+Lin-c-Kit+ stem cell
population in mobilized blood. Our CRU data also indicate that LSK cells from blood
are inherently worse than LSK from bone marrow.
In our study, transplantation of purified stem cells from peripheral blood displayed a
rapid increase in recovery of WBC counts demonstrating an increase in short-term
engraftment, consistent with clinical and experimental data2;3;30;31. The observed
consistency between clinical and experimental data validates the use of LSK cells in
the murine system as a model to study the quality of mobilized peripheral blood stem
cells. Murine LSK, much like human CD34+ cells, are predominantly progenitor cells
but contain the vast majority of long-term repopulating stem cells.
The observed reduction in the frequency of stem cells rather than a faster deterioration
of blood stem cells suggest that recipients should receive higher cell dose of
mobilized blood stem cells to compensate for the decrease in repopulation potential.
In clinical studies, patients receiving mobilized peripheral blood typically receive a
much higher CD34+ cells dose than the bone marrow group30;32. Currently, 15-20 x
104 CFU-GM/kg or 2-2.5 x 106 CD34+ cells/kg is generally the agreed minimum
threshold below which rapid hematopoietic reconstitution may not occur4;33.
However, our data cautions the use of the frequency of a phenotypically defined
population such as CD34+ in human or LSK in mice, as the only parameter for
decisions about a minimum cell dose. The presence of fewer long-term repopulating
cells within this population, will increase the risk of re-growth of host (malignant)
haematopoiesis as shown in our non competitive transplants (Figure 4.7), increases.
Interestingly, a ~7-fold difference in CRI was observed between transplants with
unfractionated mobilized peripheral blood cells (CRI of 0.31 ± 0.1) (Figure 4.1) and
transplants with mobilized purified blood stem cells (CRI of 0.04 ± 0.04) (Figure 4.4).
This could be caused by the difference in cells used (mononuclear cells for MPB and
unseparated (>80% granuloid) cells from bone marrow, or it may suggest a potential
role of accessory cells to facilitate engraftment of mobilized peripheral blood cells. It
was reported that a CD8+/TCR- cell population from the donor bone marrow
facilitates engraftment of purified allogeneic bone marrow stem cells34. Mobilized
peripheral blood LSK cells may be more dependent on accessory cells than bone
marrow stem cells. Thus, it is possible that during the purification of LSK cells from
Mobilized peripheral blood stem cells have impaired long-term engraftment
125
mobilized peripheral blood, accessory cells facilitating engraftment were lost, leading
to a further decrease in repopulation of purified mobilized peripheral blood stem cells.
In summary, although mobilized peripheral blood stem cells promote faster
haematological recovery, our competitive and non-competitive transplant model
suggest that their lower stem cell frequency affects the long-term repopulation
potential of blood LSK stem cells.
Mobilized peripheral blood stem cells have impaired long-term engraftment
126
Acknowledgements We wish to thank Geert Mesander and Henk Moes for their assistance with cell
sorting and the staff of our animal facility for taking care of the mice. No conflicts of
interest exists between authors. This work was supported by grants from the European
Union (EU-LSHC-CT-2004-503436) and Ubbo Emmius Foundation.
Mobilized peripheral blood stem cells have impaired long-term engraftment
127
References
1. Mathe G, Thomas ED, Ferrebee JW. The restoration of marrow function after
lethal irradiation in man: a review. Transplant.Bull. 1959;6:407-409.
2. Goldman J. Peripheral blood stem cells for allografting. Blood 1995;85:1413-
1415.
3. Korbling M, Champlin R. Peripheral blood progenitor cell transplantation: a
replacement for marrow auto- or allografts. Stem Cells 1996;14:185-195.
4. To LB, Haylock DN, Simmons PJ, Juttner CA. The biology and clinical uses of
blood stem cells. Blood 1997;89:
5. Varas F, Bernad A, Bueren JA. Granulocyte colony-stimulating factor mobilizes
into peripheral blood the complete clonal repertoire of hematopoietic precursors
residing in the bone marrow of mice. Blood 1996;88:2495-2501.
6. Molineux G, Pojda Z, Dexter TM. A comparison of hematopoiesis in normal
and splenectomized mice treated with granulocyte colony-stimulating factor.
Blood 1990;75:563-569.
7. Yan XQ, Hartley C, McElroy P et al. Peripheral blood progenitor cells
mobilized by recombinant human granulocyte colony-stimulating factor plus
recombinant rat stem cell factor contain long-term engrafting cells capable of
cellular proliferation for more than two years as shown by serial transplantation
in mice. Blood 1995;85:2303-2307.
8. Demuynck H, Pettengell R, de Campos E, Dexter TM, Testa NG. The capacity
of peripheral blood stem cells mobilised with chemotherapy plus G-CSF to
repopulate irradiated marrow stroma in vitro is similar to that of bone marrow.
Eur.J.Cancer 1992;28:381-386.
9. Pettengell R, Luft T, Henschler R et al. Direct comparison by limiting dilution
analysis of long-term culture-initiating cells in human bone marrow, umbilical
cord blood, and blood stem cells. Blood 1994;84:3653-3659.
Mobilized peripheral blood stem cells have impaired long-term engraftment
128
10. Molineux G, McCrea C, Yan XQ, Kerzic P, McNiece I. Flt-3 ligand synergizes
with granulocyte colony-stimulating factor to increase neutrophil numbers and
to mobilize peripheral blood stem cells with long-term repopulating potential.
Blood 1997;89:3998-4004.
11. Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin expression are
associated with altered homing properties of Lin(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+)
hematopoietic stem cells following mobilization by
cyclophosphamide/granulocyte colony-stimulating factor. Exp.Hematol.
2002;30:176-185.
12. Szilvassy SJ, Meyerrose TE, Ragland PL, Grimes B. Differential homing and
engraftment properties of hematopoietic progenitor cells from murine bone
marrow, mobilized peripheral blood, and fetal liver. Blood 2001;98:2108-2115.
13. Uchida N, He D, Friera AM et al. The unexpected G0/G1 cell cycle status of
mobilized hematopoietic stem cells from peripheral blood. Blood 1997;89:-472.
14. de Haan G, Ausema A, Wilkens M, Molineux G, Dontje B. Efficient
mobilization of haematopoietic progenitors after a single injection of pegylated
recombinant human granulocyte colony-stimulating factor in mouse strains with
distinct marrow-cell pool sizes. Br.J.Haematol. 2000;110:638-646.
15. de Haan G, Szilvassy SJ, Meyerrose TE et al. Distinct functional properties of
highly purified hematopoietic stem cells from mouse strains differing in stem
cell numbers. Blood 2000;96:1374-1379.
16. Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ. Quantitative
assay for totipotent reconstituting hematopoietic stem cells by a competitive
repopulation strategy. Proc.Natl.Acad.Sci.U.S.A 1990;87:8736-8740.
17. Ploemacher RE, van der Sluijs JP, Voerman JS, Brons NH. An in vitro limiting-
dilution assay of long-term repopulating hematopoietic stem cells in the mouse.
Blood 1989;74:2755-2763.
Mobilized peripheral blood stem cells have impaired long-term engraftment
129
18. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and 2 preserve
long-term repopulating ability of hematopoietic stem cells in serum-free
cultures. Stem Cells 2006;24:1564-1572.
19. de Haan G, Van Zant G. Intrinsic and extrinsic control of hemopoietic stem cell
numbers: mapping of a stem cell gene. J.Exp.Med. 1997;186:529-536.
20. Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL. Use of
limiting-dilution type long-term marrow cultures in frequency analysis of
marrow-repopulating and spleen colony-forming hematopoietic stem cells in the
mouse. Blood 1991;78:2527-2533.
21. Levesque JP, Hendy J, Winkler IG, Takamatsu Y, Simmons PJ. Granulocyte
colony-stimulating factor induces the release in the bone marrow of proteases
that cleave c-KIT receptor (CD117) from the surface of hematopoietic
progenitor cells. Exp.Hematol. 2003;31:109-117.
22. Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express mouse c-
kit but do not depend on steel factor for their generation.
Proc.Natl.Acad.Sci.U.S.A 1992;89:1502-1506.
23. Okada S, Nakauchi H, Nagayoshi K et al. Enrichment and characterization of
murine hematopoietic stem cells that express c-kit molecule. Blood
1991;78:1706-1712.
24. Roberts MM, Swart BW, Simmons PJ et al. Prolonged release and c-kit
expression of haemopoietic precursor cells mobilized by stem cell factor and
granulocyte colony stimulating factor. Br.J Haematol. 1999;104:778-784.
25. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic
reconstitution by a single CD34-low/negative hematopoietic stem cell. Science
1996;273:242-245.
26. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and
characterization of fetal liver hematopoietic stem cells.
Proc.Natl.Acad.Sci.U.S.A 1995;92:10302-10306.
Mobilized peripheral blood stem cells have impaired long-term engraftment
130
27. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging
of hematopoietic stem cells. Nat.Med. 1996;2:1011-1016.
28. Morrison SJ, Wright DE, Weissman IL. Cyclophosphamide/granulocyte colony-
stimulating factor induces hematopoietic stem cells to proliferate prior to
mobilization. Proc.Natl.Acad.Sci.U.S.A 1997;94:1908-1913.
29. Yilmaz OH, Kiel MJ, Morrison SJ. SLAM family markers are conserved among
hematopoietic stem cells from old and reconstituted mice and markedly increase
their purity. Blood 2006;107:924-930.
30. To LB, Roberts MM, Haylock DN et al. Comparison of haematological recovery
times and supportive care requirements of autologous recovery phase peripheral
blood stem cell transplants, autologous bone marrow transplants and allogeneic
bone marrow transplants. Bone Marrow Transplant. 1992;9:277-284.
31. Wierenga PK, Setroikromo R, Kamps G, Kampinga HH, Vellenga E. Peripheral
blood stem cells differ from bone marrow stem cells in cell cycle status,
repopulating potential, and sensitivity toward hyperthermic purging in mice
mobilized with cyclophosphamide and granulocyte colony-stimulating factor. J
Hematother Stem Cell Res. 2002;11:523-532.
32. Korbling M, Anderlini P. Peripheral blood stem cell versus bone marrow
allotransplantation: Does the source of hematopoietic stem cells matter? Blood
2001;98:2900-2908.
33. Bender JG, To LB, Williams S, Schwartzberg LS. Defining a therapeutic dose of
peripheral blood stem cells. J.Hematother. 1992;1:329-341.
34. Kaufman CL, Colson YL, Wren SM et al. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994;84:2436-2446.
131
132
133
CHAPTER 5
Summarizing Discussion and Future Perspectives
Summarizing Discussion and Future Perspectives
134
Summarizing Discussion Expansion and maintenance of self renewing primitive hematopoietic stem cells
(HSCs) would have major implications in the areas of stem cell transplantation and
gene therapy. The ultimate goal of many scientists is to successfully expand and
maintain stem cells in their primary functional characteristic, namely their ability to
engraft and sustain long-term hematopoiesis. Establishing the ideal in vitro growth
conditions to expand and maintain HSCs has proven to be difficult. Recently, strong
evidence is emerging indicating that the large family of fibroblast growth factors
(FGFs) and FGF receptors (FGFRs) may play a key role in stem cell maintenance.
Chapter 1 provides a conceptual overview on FGFs and their receptors in
maintaining tissue homeostasis in HSCs, neural stem cells (NSCs) and embryonic
stem (ES) cells, in order to retain the self renewal capacity of stem cells.
Commonalities do exist between these three distinct stem cell systems. We summarize
evidence that FGFs are growth factors crucial for the regulation and culturing of all
three of these stem cell systems.
In Chapter 2 we studied the role of FGFs to maintain HSC function by culturing
unfractionated bone marrow (BM) cells in serum-free media supplemented only with
FGF-1, FGF-2 or the combination of both (FGF-1 + 2). Bone marrow cells were
cultured for a total of five weeks and then competitively transplanted into lethally
irradiated hosts. Cells cultured in FGF-1 + 2 showed a 5-fold and 1.5-fold increase in
repopulating units after culturing for one and five weeks respectively. In addition, we
co-cultured Lin-Sca-1+c-Kit+ (LSK) with unfractionated BM cells for a total of five
weeks in FGF-1 + 2 and transplanted without competitors into lethally irradiated
recipients. Overall, the data demonstrated that FGF cultured BM cells can be
maintained for up to five weeks while retaining long-term repopulating ability
(LTRA) and that all FGF-induced stem cell activity was derived from the LSK
population. Furthermore, the data signifies the importance of the stem cell niche, a
specialized microenvironment that houses, regulates and protects stem cells1. We
could not culture purified LSK cells in serum-free medium supplemented only with
FGF-1 and/or FGF-2. However in the presence of unfractionated BM cells, LSK cells
proliferated, suggesting that FGFs may be acting on other cell types to induce stem
cell activity in vitro. Receptors for FGF-1 have been shown to be present on primitive
hematopoietic cell subsets2. Therefore, it is also highly probable that FGFs maintain
Summarizing Discussion and Future Perspectives
135
stem cells by acting on FGFRs expressed on the stem cells. Additionally, non-LSK
cells present in the stem cell niche may carry FGFRs and therefore be responsive to
FGFs, playing an important role in the maintenance of stem cells. We speculate that
BM elements in the co-culture act as a pseudo niche facilitating the proliferation of
stem cells in vitro. These results demonstrate that we can maintain stem cell in culture
for up to five weeks with LTRA.
Quantitative PCR (QPCR) analysis in Chapter 3 demonstrated that FGF-1 and FGF-2
were predominantly expressed in LSK cells. The presence of both FGFRs2 and high
expression levels of FGFs on LSK cells strongly implies that FGFs may regulate
HSCs by autocrine signaling. To further examine the role of FGFs on HSCs, in
Chapter 3 we retrovirally overexpressed FGF-1 and FGF-2 in 5-Fluorouracil (5-FU)
treated BM cells. In addition, to assess the role of nucleocytoplasmic trafficking of
FGFs in hematopoietic cells, two mutant isoforms which altered the phosphorylation
status of FGF-1 were created and overexpressed. In the first mutant, the serine residue
at phosphorylation site 130 was exchanged for glutamic acid (S130E). This mutant
was expected to mimic the phosphorylated state of FGF-1, constitutively exporting
FGF-1 to the cytoplasm3. For the second mutant, the serine was exchanged for alanine
(S130A) which was hypothesized to prevent FGF-1 phosphorylation and remain
localized in the nucleus3. Higher molecular weight isoforms of human FGF-2 (21-22,
22.5, 24 and 34kDa) contain and upstream nuclear localization signal (NLS) which
translocates FGF-2 into the nucleus4;5. The smaller 18kDa isoform is confined to the
cytoplasm6-8. To create a nuclear localized isoform of mouse FGF-2, an artificial NLS
was inserted upstream of FGF-2 (NLS/FGF-2).
Fluorescent images of hematopoietic cells overexpressing wild-type (WT) FGF-1,
S130E or WT FGF-2 retroviral vectors showed that both FGF-1 and FGF-2 were
expressed predominantly in the cytoplasm. In contrast, overlay images of
hematopoietic cells overexpressing S130A and NLS/FGF-2 revealed that 2% of FGF-
1 positive cells and 11% of FGF-2 positive cells were localized in the nucleus
respectively. Although FGF-1 and FGF-2 mutant proteins were not predominantly
localized in the nucleus, compared to WT FGF-1, S130E and WT FGF-2 nuclear
localization did increase. More sensitive methods such as cell fractionation studies
should be performed to clarify these results. Recently, using cell fractionation studies,
Wiedlocha et al. showed that WT FGF-1 was found in all fractions (membrane,
cytoplasmic and nucleus) of the cell, S130A was found in the nuclear fraction
Summarizing Discussion and Future Perspectives
136
whereas S130E was mainly in the cytosolic fraction9. These data indicated that the
translocation of FGFs appears to be a dynamic process and each FGF can be regulated
differently.
Unfortunately, transplantation studies with WT FGF-1, WT FGF-2, S130E and
NLS/FGF-2 overexpressing cells resulted in low donor chimerism levels in primary
recipients and little to no reconstitution in secondary recipients. This was unexpected
as previous studies from our group (as described in Chapter 2) had shown that the
unfractionated BM cells treated with exogenously added FGF-1 and/or FGF-2 were
capable of long-term repopulation2;10. These results suggest that the constitutive
overexpression of FGF-1 and FGF-2 does not increase the repopulating potential of
hematopoietic cells. Interestingly, secondary recipients transplanted with S130A
overexpressing cells showed a delayed but marked increase in donor chimerism levels
and a significant increase in GFP+ cells. These results strongly suggest that nuclear
localized FGF-1 may play an important role in maintaining stem cell quality.
In vivo competitive transplantation assay is the ‘gold standard’ to test whether BM
derived cells are indeed HSCs with the potential for reconstituting all hematopoietic
lineages. The competitive repopulation assay11 has two key features. The first is that it
enables the detection of a very primitive class of hematopoietic stem cells and the
survival of lethally irradiated mice transplanted with very low numbers of such cells.
The second is the use of a limiting dilution experimental design to allow stem cell
quantitation.
To highlight the effectiveness of this assay, in Chapter 4 we use the competitive
transplantation assay to compare the functional qualities of mobilized peripheral
blood (MPB) stem cells to normal BM stem cells. Mobilized peripheral blood has
been used for the past 10 years in place of BM as a source of stem cells for
transplants. Their ease of collection and ability to promote faster regeneration of
neutrophils12;13 and platelets makes them a primary source for HSC transplantation.
Transplantation of MPB stem cells in competition with BM stem cells demonstrated
that MPB stem cells have a reduced long term repopulation potential. This impairment
in repopulation potential was due to the presence of fewer stem cells rather than a
decrease in stem cell quality. In actual fact, secondary transplantation of MPB stem
cells indicated that the quality of stem cells from MPB did not decrease after
transplantation and that exhaustion of initially engrafted stem cells is similar for both
Summarizing Discussion and Future Perspectives
137
BM and MPB stem cells. Clearly, in a clinical setting, more blood stem cells must be
transplanted to compensate for the decrease frequency of stem cells.
Future Perspectives In this thesis the exogenous and endogenous effects of FGF-1 and FGF-2 were
examined. We have shown that FGFs, in particular FGF-1 and FGF-2 play an
important role in regulating and maintaining stem cells.
In total, 22 FGFs exists (not including spliced forms) and we have shown that two out
of 22 FGFs are able to maintain HSCs. Most stem cell studies carried out are
restricted to FGF-1 or FGF-2. From mouse knock-out studies it has appeared that
other FGFs are more potent. For example, deletion of FGF-414, FGF-815-18, FGF-919
and FGF-1020 result in embryonic lethality whereas FGF-1 and FGF-2 knockout
mice21 are viable and fertile. Given their pleiotropic effects, similar receptor binding
properties, overlapping patterns of expression and sequence similarities, functionally
redundancy is likely to occur. It will be interesting to assess the effects of other FGFs,
in particular FGF-4, FGF-8, FGF-9 and FGF-10 on stem cells. Such studies would
increase our understanding on the biological role of FGFs on HSC maintenance and
regulation.
The mechanistic action of FGFs on stem cells remains unknown. Our results suggest
that the intracellular function of nuclear localized FGF-1 is biologically significant.
This indicates that the nuclear import/export trafficking pathway of FGFs may be key
to understanding the mechanistic action of FGFs. Future studies should be aimed at
assessing the trafficking pathway of FGFs in stem cells. Firstly, it will be interesting
to determine which FGFs bind to which FGFR and to what affinity. It should be noted
that a large number of splice variants of FGFR genes exist and must be taken into
account. Secondly, it would be appealing to determine the stage of the cell cycle
which enables the precise cueing of the nuclear localization of FGFs. This may
provide valuable information as to how nuclear localized FGF-1 (S130A) maintained
stem cell quality. Thirdly, it would be interesting to determine whether all FGFs,
which are highly homologous, have the same trafficking pathway.
The competitive transplantation assay serves as the only tool to detect long-term
repopulating stem cells with the potential for reconstituting all hematopoietic lineages.
We highlighted the effectiveness of this assay to study the qualities of MPB stem cells
Summarizing Discussion and Future Perspectives
138
compared to normal BM stem cells. Blood cells mobilized with Granulocyte-Colony
Stimulating Factor (G-CSF) have reduced repopulation ability due to a lower
frequency of stem cells. Many growth factors capable of migrating stem cells from the
BM to the blood exist. Each mobilizing agent, alone or in combination with
chemotherapeutic agents affects the stem cell differently. It will therefore be
interesting to examine the effects of different mobilization regimes and whether this
will improve the stem cell frequency and repopulating ability of blood stem cells. This
knowledge may be relevant and change techniques used in established clinical
application.
Summarizing Discussion and Future Perspectives
139
References 1. Schofield R. The relationship between the spleen colony-forming cell and the
haemopoietic stem cell. Blood Cells 1978;4:7-25.
2. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental Cell 2003;4:241-251.
3. Klingenberg O, Widlocha A, Rapak A et al. Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells. J.Biol.Chem. 1998;273:11164-11172.
4. Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol.Cell Biol. 1999;19:505-514.
5. Dono R, James D, Zeller R. A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling. Oncogene 1998;16:2151-2158.
6. Bugler B, Amalric F, Prats H. Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. Mol.Cell Biol. 1991;11:573-577.
7. Florkiewicz RZ, Baird A, Gonzalez AM. Multiple forms of bFGF: differential nuclear and cell surface localization. Growth Factors 1991;4:265-275.
8. Quarto N, Finger FP, Rifkin DB. The NH2-terminal extension of high molecular weight bFGF is a nuclear targeting signal. J.Cell Physiol 1991;147:311-318.
9. Wiedlocha A, Nilsen T, Wesche J et al. Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1. Mol.Biol.Cell 2005;16:794-810.
10. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures. Stem Cells 2006;24:1564-1572.
11. Harrison DE. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 1980;55:77-81.
12. Goldman J. Peripheral blood stem cells for allografting. Blood 1995;85:1413-1415.
13. Korbling M, Champlin R. Peripheral blood progenitor cell transplantation: a replacement for marrow auto- or allografts. Stem Cells 1996;14:185-195.
14. Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Goldfarb M. Requirement of FGF-4 for postimplantation mouse development. Science 1995;267:246-249.
Summarizing Discussion and Future Perspectives
140
15. Meyers EN, Lewandoski M, Martin GR. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat.Genet. 1998;18:136-141.
16. Reifers F, Bohli H, Walsh EC et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 1998;125:2381-2395.
17. Shanmugalingam S, Houart C, Picker A et al. Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 2000;127:2549-2561.
18. Sun X, Meyers EN, Lewandoski M, Martin GR. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 1999;13:1834-1846.
19. Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 2001;104:875-889.
20. Sekine K, Ohuchi H, Fujiwara M et al. Fgf10 is essential for limb and lung formation. Nature Genetics 1999;21:138-141.
21. Miller DL, Ortega S, Bashayan O, Basch R, Basilico C. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol.Cell Biol. 2000;20:2260-2268.
141
142
143
Nederlandse Samenvatting
Nederlandse Samenvatting
144
Nederlandse samenvatting Stamcellen zijn primitieve cellen met het vermogen zichzelf te vernieuwen en te
differentiëren in andere celtypen. Ze hebben het unieke vermogen schade in weefsels
te herstellen en/of andere cellen te activeren voor dit herstelproces. Stamcellen
worden onderverdeeld in embryonale of volwassen stamcellen. De studie beschreven
in dit proefschrift is gefocust op stamcellen uit het beenmerg (BM), die bloedcellen
produceren. Deze cellen worden hematopoietische stamcellen genoemd (HSCs).
HSC’s zijn in staat zich te vernieuwen en te differentiëren in een verscheidenheid van
gespecialiseerde bloedcellen zoals rode- (erytrocyten) en witte bloedcellen
(leukocyten). HSCs zijn verantwoordelijk voor het dagelijks aanmaken van miljarden
bloedcellen. Het is vanwege deze eigenschappen dat HSCs in de kliniek gebruikt
worden bij de behandeling van kanker, voornamelijk leukemie en lymfoma, kankers
onstaan uit bloedcellen. Ze worden na zware chemotherapie en/of radiotherapie
getransplanteerd om vernieuwe bloedcelaanmaak te garanderen.
HSCs kunnen geïsoleerd worden uit het BM en de bloedcirculatie. Het aantal
stamcellen in beenmerg en de bloedcirculatie is echter erg klein, daar er in BM slechts
minder dan 1 stamcel per 105 BM cellen aanwezig is. Het gevolg hiervan is dat
onderzoekers zijn gaan zoeken naar kweekmethodes met als doel meer primitieve
HSCs te genereren voor transplantatie. Om dit te verwezenlijken hebben onderzoekers
muizen-HSCs blootgesteld aan groeifactoren in een in vitro milieu.
Groeifactoren zijn kleine eiwitten die op cellulair niveau verschillende effecten
teweeg kunnen brengen. Er bestaan vele soorten groeifactoren. In dit proefschrift zijn
voornamelijk proeven beschreven die zich richten op de effecten van fibroblast
groeifactoren (FGFs) op de HSCs. FGFs zijn groeifactoren die specifiek reageren op
fibroblasten door het hele lichaam. Fibroblasten produceren de bouwstoffen van
fibreus weefsel, dat overal in het lichaam te vinden is. Tot op heden zijn 22 FGFs van
mens en muis bekend. FGFs zijn vooral bekend van wege hun functie tijdens
embryogenese wanneer de FGFs de ontwikkeling van organen zoals de longen en
ledematen besturen. Bij volwassenen spelen FGFs een belangrijke rol bij herstel van
weefsels, regeneratie, metabolisme en angiogenesis. FGFs verzorgen een biologische
respons door zich te binden aan gespecialiseerde eiwitten die receptoren genoemd
worden. Tot op heden zijn er in gewervelde dieren vier FGF-receptoren (FGFR)
geïdentificeerd, namelijk Fgfr1- Fgfr4.
Nederlandse Samenvatting
145
Dit proefschrift richt zich op handhaving en groei van jonge HSCs onder invloed van
FGFs in zowel in vitro als in vivo studies. Hoofdstuk 1 geeft een algemeen overzicht
van de regulerende rol van FGFs bij de handhaving van zich zelf vernieuwende
stamcellen om veroudering van HSCs, neurale stamcellen (NSCs) en embryonale
stamcellen (ES) tegen te gaan. Veroudering gaat gepaard met een langzaam
achteruitgaan van weefselfunctie, inclusief het afnemen van de groei van nieuwe
cellen en een afname van het herstelvermogen. Veroudering wordt ook geassocieerd
met de toename van het ontstaan van kanker in alle weefsels waarin stamcellen
aanwezig zijn. Dit geeft aan dat de balans tussen proliferatie, overleving en
differentiatie van stamcellen streng gereguleerd moet zijn. Deze waarnemingen
suggereren dat er een link bestaat tussen verouderingsprocessen en de rol van
stamcellen in het vernieuwingproces van stamcellen. We veronderstellen dat FGFs
met hun receptoren weefsel-homeostase tijdens veroudering ondersteunen door
vernieuwing, handhaving en proliferatie van HSCs, NSCs en ES te reguleren.
In hoofdstuk 2 bestudeerden we de effecten van de groeifactoren FGF1, FGF2, alleen
en in combinatie, op normale muizen BM cellen in kweken met serum-vrij medium.
De BM cellen die op deze manier, gedurende 1,3 en 5 weken met FGFs gekweekt zijn
werden onderzocht op hun vermogen gedurende lange tijd nieuwe bloedcellen te
vormen na transplantatie. Daartoe werden ze gemengd met vers geïsoleerde BM
cellen. Dit celmengsel werd geïnjecteerd in muizen die vooraf een hoge
bestralingsdosis toegediend kregen om er voor te zorgen dat de endogene cellen van
de muis niet meer functioneel zouden zijn. De gekweekte cellen en de vers
geisoleerde BM cellen verschillen op het Ly5 (CD45) locus, wat ervoor zorgt dat de
cellen aan de hand van hun fenotype nog te onderscheiden zijn na transplantatie. Deze
manier van transplantatie staat bekend als de competitieve transplantatie assay, en
stelt ons in staat te bewijzen dat met FGF gekweekte BM cellen inderdaad HSCs zijn.
Na transplantatie werd het bloed van de getransplanteerde muis geanalyseerd.
Wanneer getransplanteerde cellen, die behandeld waren met FGF-groeifactoren, na 24
weken terug gevonden worden in het bloed kan aangenomen worden dat er stamcellen
aanwezig waren. Er wordt in dat geval aangenomen dat deze stamcellen zorgen voor
kunnen lange termijn repopulatie, dus permanente bloedeelvorming. In hoofdstuk 2
laten we zien dat de stamcelactiviteit van BM cellen gekweekt met FGFs tot 5 weken
in stand gehouden kan worden, doordat deze cellen in de ontvanger muizen een lange
termijn repopulatie teweeg brengen. Vervolgens is gekeken of deze stamcelactiviteit
Nederlandse Samenvatting
146
afkomstig was van al bestaande HSCs of dat ze ontstaan zijn uit een andere populatie
cellen. Stamcellen uit BM kunnen gezuiverd worden door selectie op basis van hun
primitieve HSC-fenotypering. Stamcellen hebben geen lineage marker, maar wel Sca-
1 and c-Kit stamcel-markers (LSK). Deze cellen worden LSK-cellen genoemd. De
LSK cellen werden gezuiverd en gedurende 5 weken verder gekweekt en in
combinatie met normale BM cellen met alleen FGF-1+2 in serum vrij medium. Na 5
weken werden alle cellen getransplanteerd in muizen die een dodelijke
bestralingsdosis toegediend hadden gekregen. Analyse van het bloed liet zien dat de
geïsoleerde en gezuiverde stamcellen bijdragen aan de bloedcelvorming na
transplantatie. Dus de stamcelactiviteit in FGF gestimuleerde BM kweken is
afkomstig van LSK stamcellen.
In hoofdstuk 3 onderzoeken we de rol van FGFs bij de intrinsieke regulatie en
handhaving van HSCs. De hypothese was dat FGFs van belang zijn voor het
handhaven van de stamcelactiviteit van HSC. Als het niveau van FGFs in stamcellen
verhoogd zou worden door retrovirale overexpressie, zou de stamcelactiviteit
verhoogd worden of langer gewaarborgd kunnen zijn. Om dit te onderzoeken zijn BM
cellen van met 5-fluorouracil (5-FU) behandelde muizen getransduceerd met FGF-1
of FGF-2. 5-Fluorouracil is een middel dat dient om beenmergcellen te verrijken voor
primitieve cellen en deze te activeren. Dit verbetert de transductie van stamcellen.
Het doel van de transductie was om in stamcellen een extra FGF-1 of FGF-2 gen in te
brengen. FGFs komen dan tot overexpressie hetgeen een hogere niveau van deze
eiwitten in de cel oplevert. De met het FGF-1 of FGF-2 gen getransduceerde cellen
werden getest in het in vivo competitieve transplantatiemodel. De cellen die extra
FGF-1 of FGF-2 produceren, hadden echter geen voordeel boven cellen die geen extra
FGFs produceerden. Ook op de lange termijn, na doortransplantatie van
getransduceerde cellen in secundaire ontvanger muizen, leverde een extra FGF-1 of
FGF-2 geen voordeel op voor hematopoietische stamcellen.
Er wordt gesuggereerd dat de locatie van FGFs in de cel belangrijk is voor zijn
functie. FGFs aanwezig binnen in de cellen zouden verantwoordelijk zijn voor de
mitogene effecten. Om dit te onderzoeken hebben we verscheidene mutanten van FGF
gecreëerd om de invloed van localisatie van FGF op zijn functie in hematopoietische
stamcellen te onderzoeken. Twee mutaties werden aangebracht in het FGF-1 gen.
Voor de eerste mutant werd het eiwit S130E ontwikkeld, dat de gefosforyleerde fase
van FGF-1 zou moeten nabootsen, waardoor deze naar het cytoplasma van de nucleus
Nederlandse Samenvatting
147
getransporteerd kon worden. De tweede mutant, S130A, bootst de niet
gefosforyleerde fase van FGF-1 na, en veronderstelt dat FGF-1 in de kern gehouden
wordt. Om de FGF-2 van het cytoplasma over te brengen naar de kern werd een
kunstmatig nucleair localisatiesignaal (NLS) ingebracht vóór het FGF-2 gen
(NLS/FGF-2). Kwantitatieve analyse middels polymerase chain reaction (PCR) liet
zien dat zowel FGF-1 als FGF-2 duidelijk tot expressie komt in de LSK cellen. Dit
suggereert dat de FGFs een cruciale rol spelen bij de HSC regulatie en handhaving.
Fluorescentie beelden van hematopoietische cellen met overexpressie van FGF-1,
S130E en FGF-2 laten zien dat zowel FGF-1 als FGF-2 duidelijk voornamelijk
aanwezig zijn in het cytoplasma en nauwelijks in de celkern. Daarentegen laten
beelden van hematopoietische cellen met overexpressie van S130A en NLS/FGF-2
zien dat in respectievelijk 2% van de FGF-1 positieve cellen en 11% van de FGF-2
positieve het eiwit vooral in celkern aanwezig is. Deze bevindingen suggereren dat er
wel meer cellen zijn waar FGF zich in de kern bevindt maar dat de translocatie van
FGFs een dynamisch proces zou kunnen zijn waardoor dit toch in relatief weinig
cellen te zien was. Een schematische voorstelling van het transportmechanisme van
celkern naar cytoplasma (Figuur 1) laat zien dat FGF de cel binnenkomt door zich aan
FGFR te binden. Eenmaal binnen het cytoplasma gaan de FGFs de kern binnen met
behulp van hun NLS. Om de kern te verlaten moet het FGF-eiwit in gefosforyleerde
bv. (S130E) staat zijn. In een niet gefosforyleerde staat (S130A) blijft ermeer FGF-
eiwit in de kern.
De in vivo competitieve transplantatiestudies met S130E en NLS/FGF-2 dat de
endogene expressie van deze mutanten net als de normaal voorkomde FGF-1 en FGF-
2 eiwitten geen toename van het repopulatiepotentieel van hematopoietische cellen
bewerkstelligt. Stamcellen waar de niet gefosforyleerde vorm van FGF-1 (S130A) tot
overexpressie was gebracht, lieten na transplantatie ook geen verbeterd repopulatie
vermogen zien. Maar als deze cellen werden gezuiverd en doorgetransplanteerd in
secundaire ontvanger muizen werd een interessante bevinding gedaan. In eerste
instantie leken deze cellen het minder goed te doen, maar na verloop van tijd bleken
ze op de lange duur een verhoogd repopulerend vermogen te hebben. De niet
gefosforyleerde vorm van FGF-1 (localisatie in celkern) zou dus een belangrijke rol
kunnen spelen bij het handhaven van de kwaliteit van hematopoietische stamcellen.
Nederlandse Samenvatting
148
FGF
FGFR
FGF
Cytoplasma
Celkern
NLSP
FGF
FGF
Cellulairestress
S130E
FGF
FGFR
FGF
Cytoplasma
Celkern
NLSNLSPP
FGF
FGF
Cellulairestress
S130E
Figuur 1: Transportmechanisme van fibroblast groeifactoren (FGFs). FGF komt de cel binnen door
zich aan FGF-receptoren (FGFR) te binden. Eens binnen het cytoplasma, gaan de FGFs de kern binnen
met behulp van hun nucleair localisatiesignaal (NLS). Om de kern te verlaten moet het FGF-eiwit in
gefosforyleerde (bv. S130E) staat zijn. Het FGF-eiwit zal de cel verlaten tijdens cellulaire stress.
Hematopoietische stamcellen kunnen geïsoleerd worden uit bloed of BM. De
voornaamste bron van HSCs voor klinische transplantaties was BM. Door een
stamceldonor of een patient gedurende enkele dagen een hematopoietische groeifactor
toe te dienen (meestal Granulocyte Colony Stimulating Factor; G-CSF) migreren
stamcellen vanuit het beenmerg naar het bloed. Het is veel makkelijker en
aangenamer voor de stamceldonor of patient om stamcellen uit het bloed te halen dan
uit het beenmerg. Het proces van migratie van stamcellen van beenmerg naar bloed
wordt stamcelmobilisatie genoemd. Daarom zijn, mede als resultaat van de
toegenomen opbrengst, stamcellen uit het bloed de voornaamste bron van stamcellen
voor transplantaties geworden. In de meeste gevallen levert dit een versneld herstel
van het aantal rode en witte bloedcellen op. Er zijn echter weinig studies uitgevoerd
die de kwaliteit uit het bloed en BM op de lange termijn hebben vergeleken. In
hoofdstuk 4 passen we de competitieve transplantatie assay toe om de bloed
stamcellen met die van de normale BM stamcellen te vergelijken. Competitieve
transplantatie assay is de “gouden standaard” om te bewijzen dat de geteste cel
daadwerkelijk een HSC is. De test donor cel wordt gemengd met BM cellen en
geïnjecteerd in een muis, die eerder een hoge dosis bestraling heeft gekregen om zijn
Nederlandse Samenvatting
149
eigen bloed producerende cellen te doden. Wanneer de muis herstelt en alle typen
bloedcellen weer aanwezig zijn (die een genetische marker van de test donor dragen)
kan worden beoordeeld of de geïnjecteerde cellen stamcellen bevatten (Figuur 2).
Daarnaast kan de competitieve assay gebruikt worden om de frequentie van de
stamcellen te meten en om de kwaliteit van twee stamcelpopulaties te vergelijken. De
transplantatie van bloed stamcellen gemengd met normale BM stamcellen laat zien
dat bloed stamcellen minder vermogen tot repopulatie van de bestraalde muis
bezitten. Dit kan liggen aan een mindere kwaliteit of minder “echte” stamcellen in het
bloed; “echte” stamcellen bezitten de eigenschap om onbeperkt gedurende lange tijd
rijpe bloedcellen te produceren. Wij hebben gevonden dat het lagere repopulerend
vermogen van bloedstamcellen is te wijten aan een afname van frequentie van
stamcellen in het bloed.
Muis type BMuis type A
Bestraling
Muis type B
Bloed analyseerd
Type A
Type B
+Muis type BMuis type A
Bestraling
Muis type B
Bloed analyseerd
Type A
Type B
Type A
Type B
+
Figuur 2: Competitieve transplantatie assay. De testcellen van muis A worden gemengd met verse
beenmerg (BM) cellen van muis B. De celsuspensie wordt geïnjecteerd in lethaal bestraalde muizen.
Na transplantatie wordt het bloed van de getransplanteerde muizen geanalyseerd om de repopulatie
capaciteit van de testcellen van muis A te bepalen.
Nederlandse Samenvatting
150
In hoofdstuk 5 tenslotte, vatten we onze bevindingen samen en bediscussiëren we de
toekomstperspectieven. De FGF eiwit familie is groot en de eiwitten zijn zeer
homoloog, wat betekent dat ze erg op elkaar lijken. Het verwijderen van FGF-1 bij de
muis heeft bijvoorbeeld geen effect, waardoor we mogen concluderen dat de
functionele taak overbodig is. We hebben in onze studies slechts 2 van de 22 FGFs
getest. Het zal daarom erg interessant zijn de effecten van de overige 20 FGFs op
HSCs te onderzoeken zowel in vitro als in vivo. Verkregen resultaten hiervan zullen
waardevolle informatie verschaffen betreffende de biologische rol van FGFs in HSCs.
De mechanistische aspecten van FGFs zijn nog onbekend. De nucleaire localisatie van
FGF-1 blijkt waarschijnlijk een rol te spelen in de handhaving van de hoedanigheid
van de stamcellen. Toekomstige studies zouden gericht moeten zijn op het vraag
waarom er een vertraging optrad als gevolg van de effecten van de S130A mutant. Het
zal interessant zijn vast te stellen hoe de nucleaire gelocaliseerde FGF-1 de stamcel
hoedanigheid handhaaft omdat deze waardevolle informatie zou kunnen verschaffen
over de mechanistische aspecten van FGFs.
De bloedstamcellen die gemobiliseerd zijn met G-CSF hebben een beperkt
repopulatievermogen ten gevolge van een lage frequentie van stamcellen. Er bestaan
vele groeifactoren die in staat zijn stamcellen te doen migreren van BM naar bloed.
Elke gemobiliseerde groeifactor beïnvloedt de stamcel verschillend. Het zal daarom
interessant zijn de effecten van verschillende factoren van mobilisatie te onderzoeken
en of dit de stamcel-frequentie en het repopulatie-vermogen van de bloed stamcellen
zou kunnen verbeteren. Deze kennis zou van relevantie kunnen zijn en zou technieken
kunnen modificeren zodat die toegepast zouden kunnen worden in de kliniek om het
succes van stamceltransplantatie te verhogen.
151
152
153
Acknowledgements
As I sit back and reflect on the past four years and four months I can’t help but think
how quickly time has passed by. Naturally, these same thoughts and feelings never
went through my mind whilst in the crux of my PhD. I guess what they say is true –
sometimes it’s hard to see the bigger picture when you are standing in the middle of it.
The knowledge I have gained, the places I have seen and the many people involved
that have now become friends have all made this an incredible experience I will
forever remember. To these friends who have made it all possible, thank you.
Firstly, I would like to thank my supervisor Professor Gerald de Haan for accepting
me into the Department of Stem Cell Biology. Your steer and support was
instrumental towards the completion of this thesis. This opportunity has most
certainly helped further my scientific career. Special thanks to Ronald, my co-
promoter for your advice and assistance on experiments, proof reading my
manuscripts and for the constant repartee.
A big thank you goes to the members of the reading committee, Edo Vellenga, Paul
Coffer and Albrecht Mueller for reading and providing critical comments on my
thesis.
To my invaluable paranymphs, Bertien and Ellen you have provided the local support
and knowledge required to sort out the difficult intricacies towards my graduation.
Without both your help I would have never been able to complete all the experiments
that have formed the basis of this thesis. Bert, I really appreciated the many hours you
spent with me in the CDL. I will miss our chats and your great sense of humour. To
my roommates, Leonie, Alice and Lenya thank you for advice, guidance and, most
importantly, laughter. Leonie, I’m so pleased that you went through the graduation
procedures and formal documents with me. Lenya, I will miss your politically
controversial outlook on life and science. Your honesty is refreshing and sadly I will
never meet another Lenya. To my friends Sandra, Isabelle and Kyrjon, thanks for
being there when I needed to share a joke with you, when I had to let my frustrations
out or when I had some great gossip I badly needed to share. Many thanks to my
friends and colleagues in Radiation and Stress Cell Biology and Hematology for your
critical input. To my students Eelo and Alexandra, thank you for helping out with the
work. Your efforts were much appreciated.
154
Gerry, I can not thank you enough for everything you have done for me. Any
bureaucratic problems I had, whether it be my taxes, the IND or the university, you
solved it with a smile. If it wasn’t for your dream, I would not have been able to
graduate. Your kind-heartedness shines through in the many ways you selflessly help
others and I am delighted to have had the pleasure of meeting you.
My Mondays to Fridays would not have been complete without bumping into my
fellow international friends, Daniella, Larissa, Heni, Jola and Jarir. The overcooked
vegetables and fried food at the hospital was easier to take with the good company.
Special thanks to Babs, my fellow Aussie. Our lunches in the garden, frantically
chatting away, always put me in good spirits for the remainder of the day. Many
thanks for helping me with the Dutch summary. I don’t know what I would have done
without your help.
Groningen would have never been the same without my friends, Joe, Hermione,
Mensur, Jackie, Alex, Jim, Brad, Tim, Meagan, John, Susan, Tom, Anna, Leah, Ina,
Paul Hoban, Paul Hogarth, Greg, Karolien, Richard, Glen, Lucy, Jeanette, Josh, Tina,
Marcella, Thomas, Claire, and Karen. May our crazy skiing stunts, holidays and wild
parties continue wherever we are in this world. To the girls, the nights out at Da
Vinci’s will always form some of my fondest memories. There is nothing better than
pizza, pasta and lots of Rosé and whilst some of you had the luxury of Red Bull’s
wings to carry you through the Friday, I only had our canteen’s milk and cheese at my
disposal. The lack of culinary diversity never dampened my hungover spirit as I just
had to think back to the previous night’s fun and shenanigans to realise it was all
worth it. Alex and Jim, thank you for the chocolates, spring rolls, coffee sessions and,
most importantly, your perfectly timed phone calls. Josh and Tina, you deserve more
for your artistic talents than just food and booze but I’m glad you lowered your rates
for my cover. To my friends Michelle, Nathan, Rebecca, Zheng and Eu-Jin thank you
for all your continuous support from a distance. Always remember that “wherever you
are it is your friends who make your world” (William James).
Most importantly, a big thank you to my loving family. I am grateful for all the love
and support my family in Malaysia and Australia have given me all these years. To
my parents-in-law, Albert and Pauline, thank you for your care and continuous
prayers for me. We have been blessed in so many ways and will always try to remain
thankful. To my husband Chevy, thank you for your love and constant encouragement
especially when it came to solving mathematical problems. The laughter and fun we
155
shared in Groningen will always resonate in my memories as you made living so far
away from home seem not so far at times. You always made sure that I had everything
I needed to be happy and to achieve my goals, especially when it came to techno
wizardry. Without you I most definitely would not have travelled so much.
To my dearest mum and brother, I thank you from the very bottom of my heart for all
that you have done for me, not only during these past four years but throughout my
life. You were both always there for me whenever I needed to hear your voices or
have my own voice heard by a compassionate ear. I could always depend on either of
you to provide the motivation I needed, in the form of a supporting email or phone
call, wrapped in a bit of homely cosiness. I hope that I have made you both proud of
me and continue to do so in the next phase of my life.
With love
156
157
Curriculum vitae
Personal Details
Full Name: Joyce Siew Gaik Yeoh
Date of Birth: 26 November 1979
Country of Birth: Penang, Malaysia
Nationality: Australian
Education
PhD in Department of Cell Biology, Section Stem Cell Biology, University Medical
Centre of Groningen, May 2002 – August 2006
“Regulatory Role of Fibroblast Growth Factors on Hematopoietic Stem Cells”
Anticipated Graduation February 2007
Hons., BSc. in Pathology, University of Western Australia, Graduation, 2001
Work Experience
Research fellow in Department of Ophthalmology, University of Aberdeen, Scotland,
2006 – present
Production scientist in Verigen Transplantation Services International and Verigen
Australia Pty Ltd, 2001 to 2002
158
Papers published
Fibroblast growth factor-1 and 2 preserve long-term repopulating ability of
hematopoietic stem cells in serum-free cultures
Stem Cells 2006, 24 (6) 1564 – 1572
Fibroblast growth factors as regulators of stem cell self renewal and aging
Mechanism of Ageing and Development, in press
Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-
term engraftment
Bone Marrow Transplantation, submitted
Abstracts
In Vitro Generation of Hematopoietic Stem Cells
Joyce Yeoh, Ellen Weersing, Bert Dontje, Edo Vellenga, Ronald van Os, Gerald de
Haan
International Society of Experimental Hematology, Paris, France 2003
Fibroblast growth factors regulate stem cell functioning in vitro and in vivo.
Joyce Yeoh, Ronald van Os, Ellen Weersing, Bert Dontje, Edo Vellenga, Leonid
Bystrykh, Gerald de Haan
American Society of Hematology, San Diego, United States of America 2004
Fibroblast growth factors regulate stem cell functioning in vitro and in vivo.
Joyce Yeoh, Ronald van Os, Ellen Weersing, Bert Dontje, Edo Vellenga, Leonid
Bystrykh, Gerald de Haan
Dutch Program Tissue Engineering, Ede, The Netherlands 2005
Fibroblast growth factors regulate stem cell functioning in vitro and in vivo.
Joyce Yeoh, Ronald van Os, Ellen Weersing, Bert Dontje, Edo Vellenga, Leonid
Bystrykh, Gerald de Haan
International Society of Experimental Hematology, Glasgow, Scotland 2005
159
160