Modeling Developmental Hematopoiesis Using Pluripotent Stem Cells
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1 Modeling Developmental Hematopoiesis Using Pluripotent Stem Cells Christopher Sturgeon February 14, 2017
2 Pluripotent Stem Cells self-renewal hpsc Mesoderm blood cardiovascular muscle Endoderm lung liver pancreas Ectoderm skin nervous system
3 ESC/iPSC Developmental Biology Drug Discovery and TestingPCS Cell Replacement Therapy Disease Mechanism
4 The goal of regenerative medicine PS cells LT-HSC
5 Hemogenic Endothelium Ventral side of the dorsal aorta Aorta-Gonad- Mesonephros region - AGM E10.5 in mouse Endothelial characteristics CD34+CD45+/- KDR+ CD31+ CD144+ CD117 low Sox17,Runx1, Gata2, Scl, Lmo2 Endothelial-tohematopoietic transition De novo HSC emergence Vecad+ CD45+ Vecad+ CD45- Vecad+ CD45- Vecad+/- CD45+ Clarke Costa et al., et al.,
6 What does a nascent HSC look like? CD34+ CD144+ CD45+ CD117+ CD90+ CD105+ CD38- CD45RA- RUNX1+ Ivanovs et al., 2011, 2014
7 The goal of regenerative medicine hpsc mesoderm endothelium hemogenic endothelium hematopoietic progenitors Stage-specific differentiation PS cells LT-HSC Recapitulation of embryonic development
8 Not all blood is created equal E7.0 E E10.5 E12+ Yolk Sac Transient P-Sp / AGM Persistent Hemangioblast EMP Adult HSC Nucleated erythroid Myeloid Megakaryocyte Definitive erythroid Myeloid Megakaryocyte All lineages
9 Hematopoietic development Day 10 Day 14 Day 19 Day Primitive Definitive Hemangioblast HSC Mk Ery P HBE Myeloid Mk Ery D Myeloid Lymphoid HBA HBG
10 Why T cell assays? No T cell potential? = No HSC Primitive Definitive Hemangioblast HSC Mk Ery P HBE Myeloid Mk Ery D Myeloid Lymphoid HBA HBG
11 Why T cell assays? No T cell potential? = No HSC HSC emergence requires transient Notch stimulation Notch-dependent
12 Why T cell assays? No T cell potential? = No HSC HSC emergence requires transient Notch stimulation T cell differentiation requires Notch signaling (pre?) HSC-like state CD34 CD43 Notch-stimulation
13 Location! Location! Location? posterior anterior embryo extraembryonic Embryoid Body Primitive Hematopoiesis Definitive / Primitive Hemogenic Endothelium No anatomical separation Must find ways to distinguish between hematopoietic programs
14 How do we specify a hesc-derived AGM-like hemogenic endothelium?
15 Lessons from the mouse Primitive Streak E7.0
16 Modeling development in vitro ES Cells Brachyury+ Flk-1+ Embryoid Body +BMP4 +Wnt3a +Activin/Nodal BL-CFC Hemangioblast VEGFR2 Flk-1 / KDR Blast Colony Vascular +VEGF Hematopoietic Primitive Streak Late Streak Embryo
17 Mouse Human Longer gestation Different early structure Different signal gradients Cell surface markers CD34, CD43, CD45
18 hpsc Hematopoietic Differentiation 10 Activin CD SB CD43 Kennedy* et al., 2012
19 CD34+ Primitive and Definitive Hematopoiesis 10 Activin 9.2 CD34 Primitive 61 CD CD SB CD4 Definitive CD OP9-DL4 28+ days Ery P CD8 Kennedy* et al., 2012
20 hpsc Hematopoietic Differentiation Human primitive and definitive are separated simultaneously Can we separate these mesoderm progenitors? Kennedy* et al., 2012
21 Glycophorin A (CD235a) is expressed on early mesoderm
22 Program separation? SB Activin Day 3 KDR KDR CD235a CD235a Day 9 CD34 CD34 CD43 CD43
23 Primitive hematopoietic potential SB Activin
24 Hemogenic Endothelium Day 9 KDR+ CD235a- CD34 CD43 KDR+ CD235a+ CD34 CD43
25 Hemogenic Endothelium Day 9 3 days KDR+ CD235a- CD34 CD43 KDR+ CD235a+ CD34 CD43
26 Hemogenic Endothelium Day 9 7 days KDR+ CD235a- CD34 CD43 CD144 KDR+ CD235a+ CD34 CD43 CD45
27 Hematopoietic development Day 10 Day 14 Day 19 Day 33 Primitive Definitive Hemangioblast HSC Mk Ery P HBE Myeloid Mk Ery D Myeloid Lymphoid HBA HBG
28 Definitive hematopoiesis? FACS Day 3 FACS Day 9 OP9 OP9DL4 coculture
29 Definitive erythroid potential OP9 DL1 7 days Methylcellulose
30 Definitive erythroid potential OP9 DL1 7 days Methylcellulose EryP-CFC EryP-CFC
31 Lymphoid - T cell Definitive Primitive KDR+ CD235a- KDR+ CD235a+ OP9 DL4 SCF + Flt3L + IL7 30+ days
32 Human hematopoietic development in the dish Day 19 Day 24+ EryP-CFC T/B cells EryP-CFC T-lymphoid EryD-CFC Earliest identifiable progenitors - Selective differentiation strategy - Cell surface markers T-/B-lymphoid EryD-CFC Definitive EryP-CFC Primitive Tavian et al., 2001
33 Is WNT involved?
34 WNT regulation of hematopoietic specification WNT WNT CHIR99021 GSK3b inhibitor WNT agonist IWP2 Porcn inhibitor WNT antagonist
35 T-lymphoid potential WNT WNT OP9 DL4 CD CD8 15 No lymphopoiesis
36 Program specification
37 Model of human hematopoietic development Primitive hematopoiesis Definitive hematopoiesis Sturgeon et al., 2014
38 Acknowledgements Sturgeon Laboratory Carissa Dege Philip Creamer Kendra Sturgeon Jolie Ho Batista Laboratory Luis Batista Wilson Fok Palis Lab- University of Rochester Jim Palis Kathleen McGrath Kate Fegan Funding American Cancer Society Center of Regenerative Medicine NIH T32HL Fehniger Laboratory Todd Fehniger Melissa Berrien-Elliott Julia Wagner Tim Schappe
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