Takumi Era

Guided differentiation of ES cell into mesenchymal stem cell

Institute of Molecular Embryology and Genetics, Kumamoto University

Division of Molecular Neurobiology

AdipocyteOsteocyte

Chondrocyte

MesenchymalStem Cell

ParaxialMesoderm

Axial Mesoderm

Sox1+

PDGFRα-Sox1-

PDGFRα+Neuro-

epithelium

PDGFRα+

VEGFR2-

ImmatureMesoderm

PDGFRα-

VEGFR2+

Mesendoderm

PrimitiveEndoderm

LateralMesoderm

DefinitiveEndoderm

HepatocyteGut cell

Osteocyte

Blood cellEndothelial cell

GSC+

ECD+

CXCR4+

ECD+

CXCR4+

ECD-

PDGFRα+

VEGFR2+

PDGFRα+

VEGFR2+

PDGFRα+

VEGFR2-

PDGFRα-

VEGFR2+ES cell

Differentiation pathways of in vitro ES cell culture

AdipocyteOsteocyte

Chondrocyte

Nishikawa and Era, Nature Rev. Mol. Cell. Biol. , 2007

1. To visualize the intermediatesNew surface markers; PDGFRα and CXCR4

2. To establish the defined conditionsFactors; Activin and BMP4

3. To find the new molecules involved in development

Gene expressions in the intermediatesGene 1Gene 2

Sox17Eomeso

Brachyury

ES cell Activin addedMesoderm

Day 1 Day 2 Day 3

foregut Midgut

E8.5 mouse embryo

Day 4Endoderm

cell lineGsc+,E-cad+

Day 5

Expr

essi

on In

tens

ity

Endoderm differentiation

PDGFRα+VEGFR2-

PDGFRα-VEGFR2+

PDGFRα+VEGFR2+

ES cell-derived samplesTada and Era, Development, 2005Takebe and Era, Dev. Biol. 2006

Mixl1 expression restore the defect of endoderm by Eomeso KD

Izumi and Era, Stem Cells, 2007

Mesendoderm marker, Goosecoid-GFP100 101 102 103 104

100

101

102

103

104

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

2 15

34

1 21

33

3 4

31

Control Eomesodermin Knock-downVector only Vector only Mixl1-overexpressed

Endoderm

Mixl1

Sox17

Eomesodermin

Endo

derm

mar

ker,

Sox1

7-hC

D25

AdipocyteOsteocyte

Chondrocyte

MesenchymalStem Cell

ParaxialMesoderm

Axial Mesoderm

Sox1+

PDGFRα-Sox1-

PDGFRα+Neuro-

epithelium

PDGFRα+

VEGFR2-

ImmatureMesoderm

PDGFRα-

VEGFR2+

Mesendoderm

PrimitiveEndoderm

LateralMesoderm

DefinitiveEndoderm

HepatocyteGut cell

Osteocyte

Blood cellEndothelial cell

GSC+

ECD+

CXCR4+

ECD+

CXCR4+

ECD-

PDGFRα+

VEGFR2+

PDGFRα+

VEGFR2+

PDGFRα+

VEGFR2-

PDGFRα-

VEGFR2+ES cell

Differentiation pathways of in vitro ES cell culture

AdipocyteOsteocyte

Chondrocyte

Nishikawa and Era, Nature Rev. Mol. Cell. Biol. , 2007

4. To answer an important question in developmentalbiology

Marshak, D et al, Science, 1999

What is Mesenchymal stem cell (MSC) ?

Osteocytes

Chondrocytes

Adipocytes

Self-renewal

Fibroblastic morphology

Definition

Problems of MSCs ResearchUtilitiesMultipotency for differentiation Prevention from GVHD in transplantationSupport for regeneration of cardiac muscles

ProblemsDevelopemtal pathway is unclearSpecific surface markers are undefinedTransplantation methods are not established

ES cell study are useful for understanding what MSC is

Aims: To identify MSC progenitors and to define MSC differentiation pathway

Day 0 Day 5 Day 9-RA

MonolayerCulture

Day 0 Day 5 Day 9+RA

On day 2 and 3

MonolayerCulture

Condition A (-RA)

Condition B (+RA)

Two distinct conditions induce adipocytes in in vitro ES cell culture

ES cells

Dani C. and Austin S. et al, Development, 1997

Sakurai, Era and Nishikawa et al, Stem Cells, 2006

Adipogenesis

Oil red O staining

+ Insulin, dexamethasone,

IBMX and troglitazone

+ Insulin, dexamethasone,

IBMX and troglitazone

Day 18

100

101

102

103

104

0

10

20

30

21.9%

100

101

102

103

104

0

20

40

60

80

100

40.1%

100

101

102

103

104

0

20

40

60

80

20.4%

Day 5Day 4 Day 9

100

101

102

103

104

0

10

20

30

40

50

56.9 %

Mesoderm and Mesenchymal Marker, PDGFRα

100

101

102

103

104

0

20

40

60

80

15.5 %

100

101

102

103

104

0

30

60

90

120

3.47 %

Condition A (-RA)

Both condition induces the PDGFRα+ adipocytic progenitors

Condition B (+RA)

TG:26

TG: 327

Adipogenesis

Oil red O staining

100

101

102

103

104

0

10

20

30

21.9%

100

101

102

103

104

0

20

40

60

80

100

40.1%

100

101

102

103

104

0

20

40

60

80

20.4%

Day 5Day 4 Day 9

100

101

102

103

104

0

10

20

30

40

50

56.9 %

Mesoderm and Mesenchymal Marker, PDGFRα

100

101

102

103

104

0

20

40

60

80

15.5 %

100

101

102

103

104

0

30

60

90

120

3.47 %

Brachyury

Mesp2

βActin

OB-CAD

Vimentin

PDGFRβ

Mesogenin

RT-PCR

MesodermMarkers

MesenchymalMarkers

Condition A (-RA)

Condition B induces PDGFRα+ mesenchymal cells

Condition B (+RA)

Mesodermal cells

Mesenchymal cells

Cell Growth

104

109

1014

1019

0 10 20

Cel

l num

ber

30

Passage

PDGFRα+ mesoderm cells

PDGFRα+ mesenchymal cells

ES cell-derived PDGFRα+ Mesenchymal stem cells

Osteopontin

ChondrocytesAlucian blue staining

PPARγ

Adiponectin

Osteocalcin

x100

Col2a1

Sox9

Markers

AdipocytesOil red O staining

OsteocytesAlizalin red staining

Fibroblastic morphology

Cloning

Condition B-derivedCondition A-derived

ES cells ?? PDGFRα+ adipocyteschondrocytes

osteocytes

Mesenchymalstem cells

What kinds of cells are progenitors for MSCs ?

+RA

Mesogenin

Day 3 4 5 3 4 5-RA +RA

Brachyury

Foxa2

GATA4

Mesoderm

Endoderm

β-actin

Otx2

Sox1

Neurogenin1

Ectoderm

RA treatment induces neuro-epithelial cells

RT-PCR

Condition A Condition B

Visualization of neuroepithelium as Sox1-GFP+ cells

Pevny et al. Development, 1998

Sox1 Expression

ISH

8.5dpc 9.5dpc

J. Aubert et al. PNAS, 2003

Sox1 Promoter GFP

Sox1-GFP Knock-in ES cells

Neuroepithelium

Neural Tube

Neuro-epithelium is a origin of ES cell-derived MSC

Sox1-GFPNeuroepithelium marker

Mes

ench

yme

mar

ker

PDG

FRα

0.8 0.5

48

Day 4

Sox1+ Purification

30 4.5

17

Sox1-GFP

PDG

FRα

6 days after purification Adipogenesis

Condition B (+RA)

ES cells Sox1+PDGFRα-

Sox1-PDGFRα+

adipocyteschondrocytes

osteocytes

Mesenchymalstem cells

Differentiation pathway from ES cell to MSC

+RA

Neuro-epithelial cells

Tetraploid chimera

Review by Tam, PP. and Rossant, J. Development, 2003

4N tetraploid embryo

2N ES cell carrying GFP marker

Only ES cells can contribute to embryo.

E9.5 x100

Sox1/Nestin/TO-PRO

Sox1-GFPTetraploid

chimera

Sox1-GFP

PDG

FRα

41 4

31

Mesoderm

Neuroepithelium

E9.5 Mouse embryo

Sox1-GFP tetraploid chimera

Trunk region Osteogenesis

Chondrogenesis

Adipogenesis

Cloning

Visualization of Sox1+ cells in mouse embryos

Takashima, Era and Nishikawa Cell, 2007

Neuro-epithelium is the earliest origin of MSCs

FACS analysis

Gilbert, Developmental Biology

Development of Neural Crest CellsThey appear in the border between nuero-epithelium and epidermis and migrates from dorsal to ventral regions.

Bone, cartilage and neuron in head, and synpathetic neurons and pigment cells in trunk

Neural crest cells

Neuroepithelium

YFP marker are constitutively expressed during mouse development

Sox1-Cre Knock-in mouse

P0-Cre transgenic mouse

Tracing of Sox1+ neuro-epithelial and P0+ neural crest cells

Rosa26-STOP-YFP mouse

Rosa-p

STOP

loxP loxP

Crerecombinase

YFP

Rosa-p YFP

Cre gene is specifically expressed in Sox1+ neuro-epithelium cells

Cre gene is specifically expressed in P0+ neural crest cells

YFP expression in Sox1-Cre and P0-Cre embryos

Sox1-CreNeuroepithelium）

P0-Cre（Neural crest）

FACS Analysis

913

5

YFP

PDG

FRα

PDG

FRα

21 1

30

YFP

E14.5 embryos

Neuroepithelium-and neural crest-derived MSCs in mouse embryo

Sox1-Cre （neuroepithelium-derived）

Neuroepithelium- and neural crest-derived PDGFRα+ MSCsare present in the trunk of E14.5 mouse embryos

Colonies of MSCs, Colony Forming Unit-Fibroblast (CFU-F)

+Sox1/YFPPDGFRα -

+++-

-- +P0/YFP

PDGFRα -+

++-

--

P0-Cre（Neural crest-derived）

Neuroepithlium-derived

Neural crest-derived

100 101 102 103 104100

101

102

103

10421.1 1.27

29.847.9

Sox1-Cre, Rosa-YFP

PD

GFR

α Single cell sorting

Cell Morphology：Fibroblastic

Passage 18

PDG

FRα

Vim

entin

PDG

FRβ

OBC

AD

βAct

in

Slug

Snai

l

Gene expression

RT-PCR

E14.5 Sox1+-derived PDGFRα+ cells shows a multipotency and a sustained growth in vitro

Adipogenesis Osteogenesis Chondrogenesis

Differentiation Potential of clonal cell lines

Sox1-Cre YFP

PDG

FRα

FACS pattern of Femur bone(from P0 to P7)

3100 101 102 103 104

100

101

102

103

104

0.3

0.3

49 YFP+

PDGFRα+YFP-

PDGFRα+YFP-

PDGFRα-YFP+

PDGFRα-

1 2 3 4CFU-F assay

MSCs activity of neonate Sox1+-derived PDGFRα+ cells

Adipogenesis Osteogenesis Chondrogenesis

Differentiation activity of clonal cell linesFibroblastic Morphology

Passage 18

Single cellSorting

NE-derived MSC are reduced after birth

0.15

0.10

0.05

%

YFP+

PDGFRα+YFP+

PDGFRα-

P<0.01

0

Neonates

4-week old

Proportion of PDGFRα+ population

New differentiation pathway of MSCs

Takashima, Era and Nishikawa, Cell, 2007

MSCs

Sox1+

PDGFRα-

Neuro-epithelium

Sox1-

PDGFRα+P0+

Neural crest

Neuroepithelium origin

Unknown originMesoderm ?

Development

MSC activity

Embryo After Birth

Two developmental waves of MSCsNeuro-epithelium supplies the earliest and transient wave of MSCs

RIKEN CDB

Yasuhiro TakashimaShin-Ichi Nishikawa

Acknowledgements

Cambridge UniversityAustin Smith Stem Cell Biology Group

Kazuki Nakao

Animal Resources and Genetic Engineering