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ISTH Advanced Training Course
Dubai, UAE
ISTH Advanced Training Course
iPS Cell Technology and
Disease Modeling
Presented by: Dr David Rabbolini
9th September 2016
ISTH Advanced Training Course
Dubai, UAE
Disclosures for David Rabbolini
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In compliance with COI policy, ISTH requires the following
disclosures to the session audience:
Research Support/P.I. No relevant conflicts of interest to declare
Employee No relevant conflicts of interest to declare
Consultant No relevant conflicts of interest to declare
Major Stockholder No relevant conflicts of interest to declare
Speakers Bureau No relevant conflicts of interest to declare
Honoraria No relevant conflicts of interest to declare
Scientific Advisory
BoardNo relevant conflicts of interest to declare
Presentation includes discussion of the following off-label use of a drug or medical device:
<N/A>
ISTH Advanced Training Course
Dubai, UAE
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Outline
iPS cell generation
Reprogramming strategies
Characterisation of iPS cells
iPS cell platforms for disease modeling abnormal megakaryopoiesis
GATA1
X +Y =
GFI1B
NFE2
FLI1
RUNX1
NFE2
Construct a design
Phenotypic analysis
Model production and maintenance
ISTH Advanced Training Course
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Potency
TotipotentAble to generate every cell type including extra-embryonic tissues
PluripotentAble to generate cells from all three embryonic germ layers
MultipotentAble to generate a variety of cells from a particular somatic structure
Unipotent
Only generate one cell type
ISTH Advanced Training Course
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Types of Stem Cells
Embryonic
From the inner cell mass of pre-implantation embryos, prior to formation of the 3 germ layers (ectoderm, mesoderm, endoderm)
Somatic
Undifferentiated cells found in specific locations in “mature” tissues
iPS cells
Induced pluripotent stem cells generated by reprogramming differentiated cells.
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Induction of Pluripotent Stem Cells
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Oct 3/4, Sox-2,
C-Myc, Klf-4
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10 candidates 4 candidates24 candidates
expressed in embryonic stem cells
Takahashi and Yamanaka, Cell, 2006
The 4 “Yamanaka Factors”
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Mechanisms underpinning iPSC
formation
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Somatic cells Intermediate cells “Immature” iPSCs “Mature” iPSCs
Mesenchymal genes
Epithelial genes
Pluripotency genes
Increased Proliferation
Metabolic changes
Inhibition of somatic regulators
Changes in histone marks
Activation of DNA repair
Activation of RNA processing
Activation of pluripotency genes
Inhibition of apoptosis pathways
Activation of glycolysis
Silencing of transgenes and factors
independence
Complete reprogramming
Epigenetic resetting
Huang, J., Cell Research, 2009.
Stadtfeld, M., et al., Genes and Development, 2010.
Bunganim, Y., Nature Reviews Genetics, 2013.
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Factor delivery
system
Viral
Non-integrating
Non-
viral
Integrating
But excisable
Non-integrating
Adenovirus, OSKM
~ 0.001
Transposon,
OSKM
~ 0.1
Integrating
Sendai, OSKM
~ 1
Retrovirus,
OSKM, OSK,
OSK+VPA,or OS
+VPA
~ 0.001-1Lentivirus,
OSKM or miR302/367
cluster + VPA
~0.1-1.1
Inducible Lentiviral,
OSKM or OSKMN
~0.1-2
Minicircle DNA,
OSNL
~ 0.0005
Modified mRNA,
OSKM or OSKML+
VPA ,~1-4.4
Protein, OS
~ 0.001
Plasmid, OSNL
~ 0.001
MicroRNA,
miR200c,miR302s or
miR-369, ~1-4.4
Small-molecule
compounds
Methods for reprogramming cells to iPS cells
Reprogramming Factors
Adapted from: Gonzalez F., et al., Nature Review Genetics, 2011.Robinton, D.A. and Daley, G.Q., Nature, 2013.Li., et al. Journal of Haematology and Oncology, 2014.Kumar, D., et al. World Journal of Stem cells, 2015.
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Modifiable factors to increase the efficiency
of reprogramming
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Safety
Eff
icie
ncy
Lentivirus
Retrovirus
Excisable lentivirus
Protein
Small molecule
Episomal vector
Transposon
RNA
Adenovirus
Availa
bil
ity
T cells
Ease of reprogramming
Keratinocytes
Adipose stem cells
Mesenchymal stem cells
Dental pulp cells
Cord blood cells
Hepatocytes
Amniotic fluid cells
Germline stem cells
Neural stem cells
Fibroblasts
Tissue Source Modes of Delivery
. Gonzalez F., et al., Nature Review Genetics, 2011.
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Apoptosis, cell cycle and
senescence p53, p16INK4A/ p19Arf,
microRNA, p21, p57, p38
Epigenetic regulators Histone deacetylase, Histone demethylase,
G9a, DNMT1
Signaling pathways TGFB, ERK-
MAPK, Aurora A kinase, MEK/ERK, Gsk3
To over-express:
Factors important in embryonic
Development OCT4, SOX2, NANOG,
UTF1, LIN28, SALL4, NR5A2, TBX3,
ESSRB, DPPA4
Proliferation and cell cycle MYC,
KLF4, SV40LT, MDM2, cyclin D1
Mutated reprogramming factors
Epigenetic regulators CHD1, PRC2
OthersWNT, Vitamin C, miR-294, TERT
To repress:
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Reprogramming enhancers and barriers
Modifiable factors to increase the efficiency of
reprogramming
Huang, J., Cell Research, 2009.
Stadtfeld, M., et al., Genes and Development, 2010.
Bunganim, Y., Nature Reviews Genetics, 2013.
Ebrahimi B, et al., Cell regeneration, 2015.
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IPSC characterisation and maintenance
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Colony morphology
Compact colonies with distinct
borders and well defined edges.
Cells have large nuclei, nucleoli and
scant cytoplasm.
Live staining with TRA1-60
This anti-human anti-body is specific
for stem cell-specific keratin sulphate
antigens expressed on the surface of
undifferentiated human embryonic stem
(ES) and iPS cells.
Characterising iPS cells
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Cells that are expected to behave like ES cells
acquire a number of molecular features.
The molecular features are acquired in a defined
sequence.
Changes include:
Silencing of the proviral transgenes (Oct 3/4, Sox-2, C-Myc,
Klf4).
Re-expression of pluripotency genes (Oct4 and Nanog).
Re-activation of the silenced X-chromosome in female cells.
Restoration of telomerase activity.
Epigenetic histone methylation changes.
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Characterisation: Molecular markers
Stadtfeld M., et al., Cell Stem Cell, 2008.
Payer BLJ., et al., Human Genet, 2011.
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Anti-SeVHoechst Nuclear
stain
Test
Control
Characterisation: Establishing vector free cells
and silencing transgenes
Reprogrammed iPSCs must be
idependent of transgene expression and
lack expression of the delivered factors.
Reprogramming factor indipendence is
marked by silencing of the proviral
transgenes.
Above left: RT-PCR is used to detect the SeVgenome and transgenes.Above right: Vector-free iPSC colonies
SeVC-Myc
-+Test
Klf-4
-+Test-+Test -+Test
KOS
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HiPSCs, like HESCs, may acquire genetic aberrations during culture.
Aberrations may affect the differentiation capacity
and increase tumorigenicity.
Causes of genomic instability include:
Aneuploidy in the parent somatic cells
Age
Tissue type
The reprogramming process
Selective pressure caused by dramatic changes in gene
expression and epigenetic modification
Culture adaptation
Time in culture (no. of passages)
Culture techniques
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Characterisation: Karyotype analysis
Ben-David, U. et al., Cell Cycle, 2010.
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Aneuploidy
Large scale aberrations may occur in upto 9% of iPSCs
The acquisition of mutations is not dependent on the type of
reprogramming vector used.
Specific aberrations predominate in iPSCs (trisomy 12)
Aberrations tend to occur stochastically and provide
selective advantage. Duplicated regions contain pluripotency genes – NANOG and PDF3
(duplicated and over-expressed)
Enrichment of other cell cycle genes
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Characterisation: Karyotype analysis
Mayshar, Y., et al., Cell Stem Cell, 2010
Ben-David, U., et al., Cell Stem Cell, 2011
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Characterisation: Karyotype analysis
JU14
JU16
JU3
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Copy Number Variation (CNVs)
Acquisition is not influenced by the reprogramming vectors
(retroviral or Piggybac transposon) or factors (absence of
MYC). Generated through the reprogramming process
More common in early passages
Occur at common fragile genomic sites.
For iPSCs, the reprogramming process is associated with
deletions of tumor suppressor genes, while time in culture is
associated with duplications of oncogenic genes.
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Characterisation: Subchromosomal copy number
variations in IPSCs
Laurent, L.C, et al., Cell Stem Cell, 2011
Hussein, S.M., et al., Nature, 2011
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Characterisation: CNVs
Inherent and acquired genetic differences may influence iPS
properties and haematopoietic potential.
SNP array analysis and/or genome sequencing should complement
Standard karyotype analysis.
(A)
(B)
(C)
Mills, J. A., et al., Blood, 2013
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Characterisation: Functional testing
iPSCs must be able to differentiate into lineages from
all three embryonic germ layers.
Test hierarchy: In vitro differentiation
Teratoma formation
Chimera contribution
Germline transmission
Tetraploid complementation (direct generation of entirely
ESC/iPSC-derived mice)
Mice
Maherali N.,Cell Stem Cell, 2008.
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Endoderm Mesoderm Ectoderm
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Endoderm Mesoderm Ectoderm
Characterisation: iPS in vitro differentiation
iPS cells are able to differentiate in vitro into cells of all 3 germ
cell layers
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“Scorecard” uses established iPSCline data to set a reference for the variation among iPSC lines.
Measures include:
DNA methylation
Gene-expression profiling
Quantitative differentiation assays
The data assists in predicting the functional consequences of these differences
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Characterisation: An iPS “Scorecard”
Embryoid bodies
Undifferentiated iPS cell colonies
Bock C., et al., Cell, 2011.
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“Scorecard” gene expression plot
Embryoid body
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“Scorecard” confirmation of pluripotency and
Prediction of differentiation potential
TaqMan® hPSC Scorecard™ Panel
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iPS cells – Disease Modeling
3 6
I
II
III
88
121
831 2
321185
121
9154
4249
229 250 215
(A)
(C)
(B)
654321
GFI1B
21-163 164-330 amino acids1-20
H294fsX307 C168F x2
C168F x2
(A)
(C)
(B)
Inherited thrombocytopenia caused by transcription factor mutation:
GFI1B – related thrombocytopenia
78
121 249
21525022991121185 DNA binding
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GFI1B C168F patients have:
Thrombocytopenia (78-121 x109/L)
But
No red cell anisopoikilocytosis
Normal platelet granule contents
No defect on aggregometry
And
Observed increased expression of
CD34 by megakaryocytes and
platelets (GFI1B Q287*)
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C168F H294fsX307
Stevenson, W.S., et al., J Thromb Haemost, 2013.
Monteferrario, D., et al., NEJM, 2013.
GFI1B Phenotype
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GFI1B is a transcriptional regulator of erythroid
and megakaryocyte development
GFI1B binds to the promoters of many genes repressing the activity of their promoters.
GFI1B binds to its own promoter auto-regulating its expression.
Zinc-finger 5 (H294fs) and Zinc finger 1 (C168F) mutations cause de-repression of transcription at GFI1B promoters.
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0.0
0.2
0.4
0.6
0.8
1.0
1.2
Empty WT T247fs C168F
Luciferase assay: CD34 promoter
C168FH294fsControlEmpty
Morel-Kopp MC., et al., J Thromb Haemost (Abstract), 2015.
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CD34 expression is altered by GFI1B
mutations
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Wild type platelets
GFI1B C168F platelets
GFIB H294fs platelets
CD34 surface expression is increased
on platelets with C168F and H294fs mutations
Controls ControlsH294fs C168F
CD34 is increased in platelet lysates byWestern blotting harbouring C168F andT247fs mutations
Morel-Kopp MC., et al., J Thromb Haemost (Abstract), 2015.
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CD34 expression is upregulated in
megakaryocytes from patient specific iPS cells
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Control
C168F
H294fs
H294fs C168F Control
P= 0.002
P=0.190
P= 0.003
iPSC derived megakaryocyte CD34 MFICD61 H&E
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CD34 expression may distinguish
thrombocytopenia caused by GFI1B mutations
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H294fs C168F GFI1B-WT RUNX1FLI1
P=0.046
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Understanding abnormal megakaryopoiesis
using iPS cell platforms
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Disorders of megakaryocyte differentiation
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The iPS model
iPS cells generated from skin
fibroblasts by retroviral transduction.
Reprogramming factors:
4(OCT3/4, SOX2, KLF4, and c-MYC)
or 3 (OCT3/4, SOX2, and KLF4)
1 x CAMT patient using multiple (x3
clones) vs. Normal iPSC clones.
Characterisation:
Molecular: SSEA- 4, TRA1-60, and
TRA1-81 , gene expression, silencing
of exogenous facotrs.
Functional: Teratoma formation in
NOD/SCID mice.
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iPS model recapitulates clinical
phenotype
Hirata S., et al. Journal of Clinical Investigation, 2013.
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CAMT iPSCs demonstrate that MPL is indespensible
for MPP maintenance and transition to MEPs
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MPP = CD34+CD43+CD41-GPA-
MEP = CD41+GPA+Hirata S., et al. Journal of Clinical Investigation, 2013.
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Disorders of megakaryocyte maturation – Familial Platelet
Disorder with predisposition to acute myeloid leukaemia
(FPD-AML)
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iPS cells generated from skin
fibroblasts by excisable lentivirus
transduction.
Reprogramming factors:
4( Oct4, Sox2, Klf4, c-Myc)
Test samples:
1x ipS clone from two family
members (R174Q mutation)
2x clones from one individual
(monoallelic deletion)
3x control lines
Characterisation:
Molecular: Flow cytometric
analysis of pluripotency markers
SSEA 3 and 4, TRA-1-60 and
TRA-1-80.
Confirmation of endogenous
pluripotency gene expression,
Oct4, Sox2, Klf4 and c-Myc.
Karyotype analysis and deep
sequencing and CNV analysis
using comparative genomic
hybridization assays
Functional: In vivo teratoma
formation
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The iPS model
Antony-Debre I., et al., Blood, 2015.
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RUNX1 mutated iPS clones produce reduced MK
progenitors and reduced MKs
MK = Megakaryocyte, MK-P = Megakaryocyte progenitor
Left: Number of MG-P generated by control
and mutant iPS clones
Above: MK populations derived from
mutant and control mutant iPS clones
Antony-Debre I., et al., Blood, 2015.
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RUNX1 alteration causes defective megakaryopoiesis
independent of the RUNX1 mutation
(A) and (B): Reduced pro-platelet
forming MKs from patient derived
iPS cells
(C): RUNX1 mutations alter
cytoskeletal components important
in proplatelet formation and factors
for ploidization
(A)
(B) (C)
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Summary
Reprogramming somatic cells into iPSCs provides investigators
with a unique source of patient-specific pluripotent cells from
which to study inherited diseases that may be otherwise difficult
to explore by conventional in vitro techniques.
The optimal protocol for deriving the most reliable and safest
iPSCs is still uncertain, however, many are being explored.
Appropriate characterisation is important to avoid artefactual or
inconsistent effects on iPSC differentiation.
Ongoing refinement of reprogramming, characterisation and
differentiation strategies is required.
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Acknowledgements:
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The Northern Blood Centre Research Team Prof. Christopher Ward
A/Prof. William Stevenson
Dr. Marie-Christine Morel Kopp
Dr. Giles Best
Walter Chen
Sara Gabrielli
Lucinda Beutler
Dr. Nicholas Blair (Neurogenetics, The Royal North Shore Hospital,
Sydney, Aus)
Dr. Nish Singh (Department of Cytogenetics, The Royal North
Shore Hospital, Sydney, Aus)
Referring clinicians A/Prof Lindsay Dunlop (Liverpool Hospital, Sydney, Aus)
Dr. Timothy Brighton (The Prince of Wales Hospital, Sydney, Aus)
Prof. Koji Eto (CiRA, Kyoto University, Japan)
Dr. Hideya Seo (CiRA, Kyoto University, Japan)
Royal North Shore Hospital