Name: directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells
Uploaded: 2017-11-01T12:30:00
Description: Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

This work has been supported by the Department of Internal Medicine, Division of Hematology, Washington University School of Medicine. CD was supported by T32HL007088 from the National Heart, Lung, and Blood Institute. CMS was supported by an American Society of Hematology Scholar Award.

1. Reagents
<ol>
	<li>Obtain cell lines; hESCs or hiPSCs1, mouse embryonic fibroblasts (MEFs)29, OP9-DL4 stroma30,31.</li>
	<li>Reagent preparation
	<ol>
		<li>Prepare a 0.1% w/v solution of gelatin in PBS. Sterilize by autoclaving and store at 4 &#176;C after aliquoting.
		<ol>
			<li>Prepare the gelatin-coated plasticware. Coat 6-well dishes with 1.5 mL of 0.1% gelatin solution. Incubate for 15 ...

<ol>
	<li>Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+to+clinically+relevant+populations:+lessons+from+embryonic+development.">Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.</a> Cell. 132 (4), 661-680 (2008).</li><li>Ditadi, A., Sturgeon, C. M., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+human+haematopoietic+development+from+the+Petri+dish.">A view of human haematopoietic development from the Petri dish.</a> Nat Rev Mol Cell Biol. 18 (1), 56-67 (2017).</li><li>Chen, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroid/myeloid+progenitors+and+hematopoietic+stem+cells+originate+from+distinct+populations+of+endothelial+cells.">Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.</a> Cell Stem Cell. 9 (6), 541-552 (2011).</li><li>McGrath, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Sources+of+Hematopoietic+Progenitors+Emerge+before+HSCs+and+Provide+Functional+Blood+Cells+in+the+Mammalian+Embryo.">Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.</a> Cell Rep. 11 (12), 1892-1904 (2015).</li><li>McGrath, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+definitive+erythroid+lineage+with+unique+regulation+of+the+beta-globin+locus+in+the+mammalian+embryo.">A transient definitive erythroid lineage with unique regulation of the beta-globin locus in the mammalian embryo.</a> Blood. 117 (17), 4600-4608 (2011).</li><li>Palis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+emergence+of+high+proliferative+potential+hematopoietic+precursors+during+murine+embryogenesis.">Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis.</a> Proc Natl Acad Sci U S A. 98 (8), 4528-4533 (2001).</li><li>Palis, J., Robertson, S., Kennedy, M., Wall, C., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+erythroid+and+myeloid+progenitors+in+the+yolk+sac+and+embryo+proper+of+the+mouse.">Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.</a> Development. 126 (22), 5073-5084 (1999).</li><li>Boiers, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphomyeloid+contribution+of+an+immune-restricted+progenitor+emerging+prior+to+definitive+hematopoietic+stem+cells.">Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells.</a> Cell Stem Cell. 13 (5), 535-548 (2013).</li><li>Bertrand, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+derive+directly+from+aortic+endothelium+during+development.">Haematopoietic stem cells derive directly from aortic endothelium during development.</a> Nature. 464 (7285), 108-111 (2010).</li><li>Hadland, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+requirement+for+Notch1+distinguishes+2+phases+of+definitive+hematopoiesis+during+development.">A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development.</a> Blood. 104 (10), 3097-3105 (2004).</li><li>Kumano, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+but+not+Notch2+is+essential+for+generating+hematopoietic+stem+cells+from+endothelial+cells.">Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells.</a> Immunity. 18 (5), 699-711 (2003).</li><li>Medvinsky, A., Rybtsov, S., Taoudi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+the+adult+hematopoietic+system:+advances+and+questions.">Embryonic origin of the adult hematopoietic system: advances and questions.</a> Development. 138 (6), 1017-1031 (2011).</li><li>Robert-Moreno, A., Espinosa, L., de la Pompa, J. L., Bigas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBPjkappa-dependent+Notch+function+regulates+Gata2+and+is+essential+for+the+formation+of+intra-embryonic+hematopoietic+cells.">RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells.</a> Development. 132 (5), 1117-1126 (2005).</li><li>Chadwick, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+BMP-4+promote+hematopoietic+differentiation+of+human+embryonic+stem+cells.">Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells.</a> Blood. 102 (3), 906-915 (2003).</li><li>Davis, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+a+GFP+reporter+gene+to+the+MIXL1+locus+of+human+embryonic+stem+cells+identifies+human+primitive+streak-like+cells+and+enables+isolation+of+primitive+hematopoietic+precursors.">Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors.</a> Blood. 111 (4), 1876-1884 (2008).</li><li>Kaufman, D. S., Hanson, E. T., Lewis, R. L., Auerbach, R., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cells+derived+from+human+embryonic+stem+cells.">Hematopoietic colony-forming cells derived from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 98 (19), 10716-10721 (2001).</li><li>Kennedy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+lymphocyte+potential+marks+the+emergence+of+definitive+hematopoietic+progenitors+in+human+pluripotent+stem+cell+differentiation+cultures.">T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2 (6), 1722-1735 (2012).</li><li>Ledran, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+hematopoietic+differentiation+of+human+embryonic+stem+cells+on+stromal+cells+derived+from+hematopoietic+niches.">Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches.</a> Cell Stem Cell. 3 (1), 85-98 (2008).</li><li>Ng, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+primitive+streak+gene+Mixl1+is+required+for+efficient+haematopoiesis+and+BMP4-induced+ventral+mesoderm+patterning+in+differentiating+ES+cells.">The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells.</a> Development. 132 (5), 873-884 (2005).</li><li>Pick, M., Azzola, L., Mossman, A., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+in+serum-free+medium+reveals+distinct+roles+for+bone+morphogenetic+protein+4,+vascular+endothelial+growth+factor,+stem+cell+factor,+and+fibroblast+growth+factor+2+in+hematopoiesis.">Differentiation of human embryonic stem cells in serum-free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis.</a> Stem Cells. 25 (9), 2206-2214 (2007).</li><li>Vodyanik, M. A., Bork, J. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+CD34++cells:+efficient+production+in+the+coculture+with+OP9+stromal+cells+and+analysis+of+lymphohematopoietic+potential.">Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.</a> Blood. 105 (2), 617-626 (2005).</li><li>Vodyanik, M. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukosialin+(CD43)+defines+hematopoietic+progenitors+in+human+embryonic+stem+cell+differentiation+cultures.">Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.</a> Blood. 108 (6), 2095-2105 (2006).</li><li>Yu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+the+generation+of+hematopoietic+progenitors+from+human+embryonic+stem+cell-derived+hemato-vascular+precursors.">Retinoic acid enhances the generation of hematopoietic progenitors from human embryonic stem cell-derived hemato-vascular precursors.</a> Blood. 116 (23), 4786-4794 (2010).</li><li>Zambidis, E. T., Peault, B., Park, T. S., Bunz, F., Civin, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+differentiation+of+human+embryonic+stem+cells+progresses+through+sequential+hematoendothelial,+primitive,+and+definitive+stages+resembling+human+yolk+sac+development.">Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development.</a> Blood. 106 (3), 860-870 (2005).</li><li>Sturgeon, C. M., Ditadi, A., Awong, G., Kennedy, M., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+Signaling+Controls+the+Specification+of+Definitive+and+Primitive+Hematopoiesis+From+Human+Pluripotent+Stem+Cells.">Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells.</a> Nat Biotechnol. 32 (6), 554-561 (2014).</li><li>Choi, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+hemogenic+endothelial+progenitor+and+its+direct+precursor+in+human+pluripotent+stem+cell+differentiation+cultures.">Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2 (3), 553-567 (2012).</li><li>Ditadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Definitive+Haemogenic+Endothelium+and+Arterial+Vascular+Endothelium+Represent+Distinct+Lineages.">Human Definitive Haemogenic Endothelium and Arterial Vascular Endothelium Represent Distinct Lineages.</a> Nat Cell Biol. 17 (5), 580-591 (2015).</li><li>Conner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryo+fibroblast+(MEF)+feeder+cell+preparation.">Mouse embryo fibroblast (MEF) feeder cell preparation.</a> Curr Protoc Mol Biol. Chapter 23, (2001).</li><li>La Motte-Mohs, R. N., Herer, E., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T-cell+development+from+human+cord+blood+hematopoietic+stem+cells+by+Delta-like+1+in+vitro.">Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro.</a> Blood. 105 (4), 1431-1439 (2005).</li><li>Schmitt, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+and+establishment+of+T+cell+competence+from+embryonic+stem+cells+differentiated+in+vitro.">Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.</a> Nat Immunol. 5 (4), 410-417 (2004).</li><li>Holmes, R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009 (2), (2009).</li><li>Polychronopoulos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+synthesis+of+indirubins+as+potent+and+selective+inhibitors+of+glycogen+synthase+kinase-3+and+cyclin-dependent+kinases.">Structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinases.</a> J Med Chem. 47 (4), 935-946 (2004).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat Chem Biol. 5 (2), 100-107 (2009).</li><li>Sugimura, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+and+progenitor+cells+from+human+pluripotent+stem+cells.">Haematopoietic stem and progenitor cells from human pluripotent stem cells.</a> Nature. , (2017).</li><li>Ohgushi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathway+and+cell+state+responsible+for+dissociation-induced+apoptosis+in+human+pluripotent+stem+cells.">Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.</a> Cell Stem Cell. 7 (2), 225-239 (2010).</li><li>Peschle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic----Fetal+Hb+switch+in+humans:+studies+on+erythroid+bursts+generated+by+embryonic+progenitors+from+yolk+sac+and+liver.">Embryonic----Fetal Hb switch in humans: studies on erythroid bursts generated by embryonic progenitors from yolk sac and liver.</a> Proc Natl Acad Sci U S A. 81 (8), 2416-2420 (1984).</li></ol>

This protocol describes a rapid, serum-free, stroma-free method for the differentiation of either primitive or definitive hematopoietic progenitors. Mesodermal specification of either primitive or definitive hematopoietic progenitors can be reliably achieved using our protocol, which uniquely exploits small molecule inhibitors of canonical WNT signaling. Stage-specific WNT activation by the GSK3&#946; inhibitor CHIR9902133 gives rise to definitive hematopoietic mesoderm, whereas WNT inhibition by ...

Human pluripotent stem cells (hPSCs) are defined as encompassing both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), and have the unique capability of not only undergoing self-renewal under appropriate growth conditions, but also, the capacity to differentiate into all cell types derived from the three germ layers: endoderm, mesoderm, and ectoderm1. Due to these unique abilities, hPSCs hold great promise for regenerative medicine, disease modeling, and cell-based therapies2. While multiple cell types have been successfully differentiated from hPSCs, one significant challenge is the in vitro specification of exclusively adult-like hPSC-derived hematopoietic stem cells (HSCs) and definitive hematopoietic progenitors.
One likely barrier to the development of human HSCs from hPSCs is the presence of multiple hematopoietic programs within the human embryo3. The first program which emerges, termed &#34;primitive hematopoiesis,&#34;&#160;originates within the extraembryonic yolk sac tissue and is best characterized by its transient production of erythroblast progenitors (EryP-CFC), macrophages, and megakaryocytes. Notably, this program does not give rise to HSCs, nor does it give rise to T and B lymphoid progenitors. However, the yolk sac does transiently give rise to restricted definitive hematopoietic progenitors, such as the erythro-myeloid progenitor (EMP4,5,6,7,8) and the erythroid-deficient lymphoid-primed multipotent progenitor (LMPP9). However, neither EMPs nor LMPPs are fully multipotent, or capable of HSC-like engraftment in adult recipients. In contrast, later in development, the classically defined &#34;definitive&#34; hematopoietic program is specified in the aorta-gonad-mesonephros region of the embryo proper, giving rise to all adult hematopoietic lineages, including the HSC. The specification of these intra-embryonic definitive hematopoietic cells occurs in a Notch-dependent fashion, via an endothelial-to-hematopoietic transition from hemogenic endothelium (HE)3,10,11,12,13,14. Aside from reconstitution capacity, the multilineage potential and Notch-dependence of these cells can be used to distinguish these definitive hematopoietic progenitors from the EMP and the LMPP (reviewed in references3,13).
Understanding the mechanism(s) governing primitive and definitive hematopoietic specification from hPSCs is likely critical to the reproducible production of definitive hematopoietic progenitors across a variety of hPSC lines. Until recently, hPSC differentiation protocols that could separate multipotent primitive and definitive hematopoietic progenitors did not exist15,16,17,18,19,20,21,22,23,24,25. Many approaches using fetal bovine serum (FBS) and/or stromal co-culture first outlined the hematopoietic potential of hPSC differentiation, with mixtures of primitive and definitive hematopoietic potential15,16,17,19,22,23,25. Further, many serum-free hematopoietic protocols have described the signal requirements for the specification of mesoderm from hPSCs that harbors hematopoietic potential18,20,21,24. However, as these methods still gave rise to heterogeneous mixtures of both programs, their use in clinical applications and understanding developmental mechanisms may be limited.
We have recently built on these studies, having outlined the stage-specific signal requirements for ACTIVIN/NODAL and WNT signaling in primitive and definitive hematopoietic specification from hPSC-derived mesoderm18,26. The latter was particularly unique, as its use of stage-specific WNT signal manipulation allows for the specification of either exclusively primitive or exclusively definitive hematopoietic progenitors26. During mesoderm specification, the inhibition of canonical WNT signaling with the PORCN inhibitor IWP2 results in the specification of CD43+ EryP-CFC and myeloid progenitors, with no detectable lymphoid potential. In sharp contrast, stimulation of canonical WNT signaling with the GSK3&#946; inhibitor, CHIR99021, during the same stage of differentiation resulted in the complete absence of detectable CD43+ EryP-CFC, while simultaneously leading to the specification of CD34+CD43&#8722; HE. This population possessed myeloid, HBG-expressing erythroid, and T-lymphoid potential. Subsequent analyses identified this HE as lacking the expression of CD7327,28 and CD18428, and its hematopoietic potential was NOTCH-dependent28. Further, single-cell clonal analyses demonstrated that these definitive hematopoietic lineages could be derived from multipotent single cells28. Taken together, these studies indicate that stage-specific WNT signaling manipulation can specify either pure primitive hematopoietic progenitors, or multipotent NOTCH-dependent definitive hematopoietic progenitors.
Here, we outline our differentiation strategy that yields exclusively primitive or definitive hematopoietic progenitors, via manipulation of canonical WNT signaling during mesodermal patterning, and their downstream hematopoietic lineage assays. This protocol is of great value to investigators who are interested in the production of either primitive or definitive hematopoietic progenitors from hPSCs for regenerative medicine applications.

<table>
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMD)</td>
			<td>Corning</td>
			<td>10-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), ES cell rated</td>
			<td>Gemini Bioproducts</td>
			<td>100-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine, 200 mM solution</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, porcine skin, Type A</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-MEM</td>
			<td>Life Technologies</td>
			<td>12000-022</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Corning</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Knock-out serum replacement</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td>&#34;KOSR&#34;</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA)</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mercaptoethanol, 55 mM solution</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction V, Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Corning</td>
			<td>10-080</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement, no vitamin A</td>
			<td>Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stempro-34 (SP34)</td>
			<td>Life Technologies</td>
			<td>10639011</td>
			<td>&#34;SP34&#34;</td>
		</tr>
		<tr>
			<td>Growth factor reduced Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>&#34;MAT&#34;</td>
		</tr>
		<tr>
			<td>L-absorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>10652202001</td>
			<td></td>
		</tr>
		<tr>
			<td>Monothioglycerol (MTG)</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Roche</td>
			<td>11088831001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Life Technologies</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Calbiochem</td>
			<td>260913</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D Systems</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D Systems</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF</td>
			<td>R&#38;D Systems</td>
			<td>293-VE</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>R&#38;D Systems</td>
			<td>255-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF-1</td>
			<td>R&#38;D Systems</td>
			<td>291-G1</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>R&#38;D Systems</td>
			<td>203-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>R&#38;D Systems</td>
			<td>206-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-7</td>
			<td>R&#38;D Systems</td>
			<td>207-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-11</td>
			<td>R&#38;D Systems</td>
			<td>218-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>TPO</td>
			<td>R&#38;D Systems</td>
			<td>288-TP</td>
			<td></td>
		</tr>
		<tr>
			<td>EPO</td>
			<td>Peprotech</td>
			<td>100-64</td>
			<td></td>
		</tr>
		<tr>
			<td>Flt-3 ligand (FLT3-L)</td>
			<td>R&#38;D Systems</td>
			<td>308-FK</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>IWP2</td>
			<td>Tocris</td>
			<td>3533</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiotensin II</td>
			<td>Sigma-Aldrich</td>
			<td>A9525</td>
			<td></td>
		</tr>
		<tr>
			<td>Losartan Potassium</td>
			<td>Tocris</td>
			<td>3798</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 PerCP Cy5.5 Clone RPA-T4</td>
			<td>BD Biosciences</td>
			<td>560650</td>
			<td>Dilution 1:100; T cell assay</td>
		</tr>
		<tr>
			<td>CD8 PE Clone RPA-T8</td>
			<td>BD Biosciences</td>
			<td>561950</td>
			<td>Dilution 1:10; T cell assay</td>
		</tr>
		<tr>
			<td>CD34 APC Clone 8G12</td>
			<td>BD Biosciences</td>
			<td>340441</td>
			<td>Dilution 1:100; EHT assay</td>
		</tr>
		<tr>
			<td>CD34 PE-Cy7 Clone 8G12</td>
			<td>BD Biosciences</td>
			<td>348801</td>
			<td>Dilution 1:100; Hemogenic endothelium</td>
		</tr>
		<tr>
			<td>CD43 FITC Clone 1G10</td>
			<td>BD Biosciences</td>
			<td>555475</td>
			<td>Dilution 1:10; Hemogenic endothelium</td>
		</tr>
		<tr>
			<td>CD45 APC-Cy7 Clone 2D1</td>
			<td>BD Biosciences</td>
			<td>557833</td>
			<td>Dilution 1:50; T cell assay</td>
		</tr>
		<tr>
			<td>CD45 eFluor450 Clone 2D1</td>
			<td>BD Biosciences</td>
			<td>642284</td>
			<td>Dilution 1:50; EHT assay</td>
		</tr>
		<tr>
			<td>CD56 APC Clone B159</td>
			<td>BD Biosciences</td>
			<td>555518</td>
			<td>Dilution 1:20; T cell assay</td>
		</tr>
		<tr>
			<td>CD73 PE Clone AD2</td>
			<td>BD Biosciences</td>
			<td>550257</td>
			<td>Dilution 1:50; Hemogenic endothelium</td>
		</tr>
		<tr>
			<td>CD184 APC Clone 12G5</td>
			<td>BD Biosciences</td>
			<td>555976</td>
			<td>Dilution 1:50; Hemogenic endothelium</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>BD Biosciences</td>
			<td>564907</td>
			<td>Dilution 1:10,000; T cell assay</td>
		</tr>
		<tr>
			<td>OP9 DL4 cells</td>
			<td></td>
			<td></td>
			<td>Holmes, R. and J.C. Zuniga-Pflucker. Cold Spring Harb Protoc, 2009. 2009(2): p. pdb prot5156</td>
		</tr>
		<tr>
			<td>MethoCult H4034</td>
			<td>Stemcell Technologies</td>
			<td>4034</td>
			<td>&#34;MeC&#34;</td>
		</tr>
		<tr>
			<td>Milli-Q water purification system</td>
			<td>EMD Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2 incubator</td>
			<td></td>
			<td></td>
			<td>Set at 37 C</td>
		</tr>
		<tr>
			<td>Multigas incubator</td>
			<td></td>
			<td></td>
			<td>Set at 37 C, 5% CO2, 5% O2</td>
		</tr>
		<tr>
			<td>6 well tissue culture plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well tissue culture plate</td>
			<td>Corning</td>
			<td>353226</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well low-adherence tissue culture plate</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well low-adherence tissue culture plate</td>
			<td>Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt-end needle, 16 gauge</td>
			<td>Corning</td>
			<td>305198</td>
			<td></td>
		</tr>
		<tr>
			<td>3 cc syringes</td>
			<td>Corning</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene test tube</td>
			<td>Corning</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene test tube</td>
			<td>Corning</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene conical</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene conical</td>
			<td>Corning</td>
			<td>430921</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL serological pipette</td>
			<td>Corning</td>
			<td>357507</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipette</td>
			<td>Corning</td>
			<td>4487</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipette</td>
			<td>Corning</td>
			<td>4489</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Corning</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL cryovials</td>
			<td>Corning</td>
			<td>430488</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL test tube with 40 &#181;M cell strainer</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;M cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS AriaII or equivalent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSRii or equivalent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>TreeStar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td>Set at 37 C</td>
		</tr>
		<tr>
			<td>0.22 &#181;M filtration system</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 C refrigerator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-20 C Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-80 C Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

A schematic depicting the induction of primitive and definitive hematopoietic progenitors from hPSCs is illustrated in Figure 1. Mesoderm patterning by canonical WNT signaling occurs during days 2 - 3 of differentiation, followed by hematopoietic progenitor specification.
Representative flow cytometric analysis and colony forming methylcellulose assays of hPSC-derived hematopoietic differentiation c...

Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

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Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

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In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

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Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

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Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

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Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

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