ips cell technology and disease modeling · inhibition of somatic regulators changes in histone...

ISTH Advanced Training Course

Dubai, UAE


iPS Cell Technology and

Disease Modeling

Presented by: Dr David Rabbolini

9th September 2016


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>


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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


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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


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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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Mayshar, Y., et al., Cell Stem Cell, 2010

Ben-David, U., et al., Cell Stem Cell, 2011


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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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- 38 -Antony-Debre I., et al., Blood, 2015.

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

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