Modeling Developmental Hematopoiesis Using Pluripotent

Stem Cells

Christopher Sturgeon

February 14, 2017

hPSC

self-renewal

Pluripotent Stem CellsMesodermbloodcardiovascularmuscle

Endodermlungliverpancreas

Ectodermskinnervoussystem

PCS

ESC/iPSC

DevelopmentalBiology

DrugDiscoveryandTesting

CellReplacement

Therapy

DiseaseMechanism

LT-HSCPS cells

The goal of regenerative medicine

Hemogenic Endothelium

• Ventral side of the dorsal aorta

• “Aorta-Gonad-Mesonephros” region -AGM

• E10.5 in mouse• Endothelial characteristics

– CD34+CD45+/- KDR+ CD31+ CD144+ CD117low

– Sox17,Runx1, Gata2, Scl, Lmo2

– Endothelial-to-hematopoietic transition

– De novo HSC emergenceVecad+CD45-

Vecad+CD45-

Vecad+CD45+

Vecad+/-CD45+

Costa et al., 2012Clarke et al., 2013

What does a nascent HSC look like?

Ivanovs et al., 2011, 2014

CD34+ CD144+ CD45+ CD117+ CD90+ CD105+ CD38- CD45RA- RUNX1+

LT-HSCPS cellsRecapitulation of embryonic development

The goal of regenerative medicine

Stage-specific differentiation

hPSC endothelium hemogenicendothelium

hematopoietic progenitorsmesoderm

Yolk Sac P-Sp / AGM

Not all blood is created equal

PersistentTransient

Adult HSC

All lineages

Hemangioblast EMP

Nucleated erythroidMyeloidMegakaryocyte

Definitive erythroidMyeloidMegakaryocyte

E7.0 E8.5-9.5 E10.5 E12+

Hematopoietic development

Mk MkEryP EryDMyeloid Myeloid Lymphoid

HSC

Day24-33Day19Day14Day10

Hemangioblast

Primitive Definitive

HBE HBAHBG

Why T cell assays?

Mk MkEryP EryDMyeloid Myeloid Lymphoid

HSCHemangioblast

Primitive Definitive

HBE HBAHBG

No T cell potential ? = No HSC

Why T cell assays?No T cell potential ? = No HSC

HSC emergence requires transient Notch stimulation

Notch-dependent

Why T cell assays?No T cell potential ? = No HSC

HSC emergence requires transient Notch stimulation

T cell differentiation requires Notch signaling

CD43

CD

34

(pre?) HSC-like state

Notch-stimulation

posterior anterior

Location! Location! Location?

embryo extraembryonic Embryoid Body

Must find ways to distinguish between hematopoietic programs

PrimitiveHematopoiesis

Definitive / Primitive Hemogenic Endothelium

No anatomicalseparation

How do we specify a hESC-derived AGM-like hemogenic endothelium?

Lessons from the mouse

Primitive StreakE7.0

Modeling development in vitro

EmbryoidBody

Brachyury+Flk-1+

BlastColony

Vascular

HematopoieticHemangioblast

ES Cells

Late StreakEmbryo

BL-CFC

PrimitiveStreak

+BMP4+Wnt3a

VEGFR2Flk-1/KDR

+VEGF

+Activin/Nodal

Mouse ≠ Human

• Longer gestation

• Different early structure– Different signal

gradients

• Cell surface markers– CD34, CD43, CD45

hPSC Hematopoietic Differentiation

Kennedy* et al., 2012

10

12

9.2

61

7.9

0.7

1.3

86

CD43

CD

34

Activin

SB-431542

EryP

CD34+ Primitive and DefinitiveHematopoiesis

Kennedy* et al., 2012

10

12

9.2

61

7.9

0.7

1.3

86

CD43

CD

34

Activin

SB-431542

CD43

CD

34 Primitive

CD8

CD

4 Definitive

OP9-DL428+ days

hPSC Hematopoietic Differentiation

Kennedy* et al., 2012

Human primitive and definitive are separated simultaneously

Can we separate these mesoderm progenitors?

Glycophorin A (CD235a) is expressed on early mesoderm

KDR

KDR

CD235a CD235a

Program separation?SB Activin

Day 3

Day 9C

D34

CD

34

CD43 CD43

Primitive hematopoietic potential

SB Activin

Hemogenic Endothelium

KDR+CD235a-

KDR+CD235a+

Day9

CD43

CD34

CD43

CD34

KDR+CD235a-

KDR+CD235a+

Day9

CD43

CD34

CD43

CD34

3days

Hemogenic Endothelium

KDR+CD235a-

KDR+CD235a+

Day9

CD43

CD34

CD43

CD34

CD144

CD45

7days

Hemogenic Endothelium

Hematopoietic development

Mk MkEryP EryDMyeloid Myeloid Lymphoid

HSC

Day 33Day 19Day 14Day 10

Hemangioblast

Primitive Definitive

HBE HBAHBG

Definitive hematopoiesis?

OP9

• FACS Day 3

• FACS Day 9

• OP9DL4 coculture

Definitive erythroid potential

OP9DL1

7 daysMethylcellulose

Definitive erythroid potential

OP9DL1

7 daysMethylcellulose

EryP-CFC

EryP-CFC

Lymphoid - T cell

SCF + Flt3L + IL730+ days

OP9DL4

KDR+CD235a-

KDR+CD235a+

PrimitiveDefinitive

Human hematopoietic development in the dish

Day 19 Day 24+

EryP-CFCT/B cells

PrimitiveDefinitive

T-/B-lymphoid EryP-CFCEryD-CFC

EryP-CFC

EryD-CFCT-lymphoid

Tavian et al., 2001

Earliest identifiable progenitors- Selective differentiation strategy- Cell surface markers

Is WNT involved?

WNT regulation of hematopoietic specification

CHIR99021GSK3b inhibitorWNT agonist

IWP2Porcn inhibitorWNT antagonist

WNT WNT

No lymphopoiesis

T-lymphoid potential

OP9DL4

WNT WNT

8 88

15

CD8

CD

4

Program specification

Primitivehematopoiesis

Definitivehematopoiesis

Model of human hematopoietic development

Sturgeon et al., 2014

Palis Lab- University of RochesterJim PalisKathleen McGrathKate Fegan

FundingAmerican Cancer SocietyCenter of Regenerative MedicineNIH T32HL007088

AcknowledgementsSturgeon LaboratoryCarissa DegePhilip CreamerKendra SturgeonJolie Ho

Batista LaboratoryLuis BatistaWilson Fok

Fehniger LaboratoryTodd FehnigerMelissa Berrien-ElliottJulia WagnerTim Schappe