modeling developmental hematopoiesis using pluripotent ...modeling developmental hematopoiesis using...
TRANSCRIPT
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