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 ...

The overall goal of this method is to direct the Differentiation of Human Pluripotent Stem Cells toward the Primitive or Definitive Hematopoietic Program using a stage specific signal manipulation approach. This method can address many key questions in the field, such as understanding the signal and transcriptional regulators of human definitive hematopoiesis. The main advantage of this technique is that it uses stage specific manipulation instantly.Demonstrating this procedure is Carissa Dege, a post doc in my laboratory. To begin the protocol, obtain previously prepared Human Pluripotent Stem Cell Lines or HPSCs grown on mouse embryonic fiberblasts or MEFs to 70%to 80%confluency in a 37-degree Celsius incubator with five percent CO2. Aspirate the HPSC media and add one milliliter of 37-degree Celsius 0.25%Trypsin EDTA to each well for one minute.After one minute, aspirate the Trypsin and add one milliliter of stock media to each well. Using a cell scraper, scrape the cells off the plate. Then, add one milliliter of wash buffer to each well.With a two-milliliter serological pipette, triturate the cells three to five times to break up large clumps. And transfer the cells to a 50-milliliter conical tube. Centrifuge the HPSCs at 330 x G for five minutes.After the spin, add HPSC media and re-suspend the cells. Next, add two milliliters of re-suspended HPSCs to each well of the MAT-coded plasticware and incubate the cells overnight. After 24 hours, aspirate the media and replace it with 37-degree Celsius 0.05%Trypsin EDTA for one minute.Aspirate the Trypsin EDTA. Add one milliliter stock media and scrape the cells vigorously with a cell scraper. Then add one milliliter of wash media.With a two-milliliter serological pipette, triturate the cells three times and transfer them to a 50-milliliter conical tube. Centrifuge the cells at 220 x G for five minutes. Then aspirate the media, add 20 milliliters of wash media and use a five milliliter serological pipette to gently re-suspend the cells.After centrifugation and aspiration, carefully re-suspend the Embryoid Bodies or EBs in Day Zero media. Dispense two milliliters of differentiation media containing EBs into each well of a six-well low-adherence cell culture dish using a ten-milliliter serological pipette. Place the EBs into a 37-degree Celsius five percent CO2, five percent O2 multigas incubator.Twenty four hours later, add two milliliters Day One media. Incubate the cells overnight in a multigas incubator. After 18 hours, gently harvest the EBs with a five-milliliter serological pipette and spin them at 100 x G for five minutes.Wash and combine the entire culture with ten milliliters of Iscove's Modified Dulbecco Medium or IMDM. After spinning the culture again at 100 X G for five minutes to remove debris, aspirate the media. Gently re-suspend the EBs with Day Two Media using a P1000 pipette.Then dispense two milliliters of the EBs per well into six-well low-adherence cell culture plates. Add three micromolar CHIR99021 or IWP2 to specify definitive or primitive hematopoetic progenitors respectively. Incubate the plate in a 37-degree Celsius multigas incubator for 30 hours.After 30 hours, isolate the EBs with a two-milliliter serological pipette by transferring them to a 50-milliliter conical tube and centifuging them at 330 x G for five minutes. Gently re-suspend the EBs and wash them with 10 milliliters of IMDM. Re-suspend the EBs in Day Three Media and dispense two milliliters into each well of low-adherence six-well plates.Incubate the cells in a 37-degree Celsius multigas incubator for 72 hours. After 72 hours of incubation, add two milliliters of Day Six Media to each well. Then incubate the EBs in a 37-degree Celsius multigas incubator for an additional 48 hours.Isolate the CHIR99021-treated EBs on day eight of differentiation and proceed to the next section. Centrifuge the EBs at 330 x G for five minutes. Aspirate the supernatant.Next add two milliliters of 0.25%Trypsin EDTA to the EBs. Vortex the EBs for five seconds and incubate the Ebs in a 37-degree Celsius water bath for eight minutes. Then add five milliliters of stop solution and centrifuge the EBs.Then aspirate the supernatant. Re-suspend the EBs in five milliliters of Collagenase II.Vortex the tube for five seconds and then incubate the EBs in a 37-degree Celsius water bath. After 60 minutes, vortex the EBs for five seconds and add five milliliters of stop solution.After centrifugation, aspirate the supernatant and re-suspend the cells in one milliliter of FACS buffer. Pass the cells thru a 40-micron cell strainer to remove undissociated cell clumps. Proceed with antibody labelling and FACS isolation of CD34-positive hemogenic endothelial progenitors.Following FACS isolation, resuspend the cells in HE media. Distribute the cells in 50-microliter aliquots in a 96-well low-adherence culture plate and then incubate the cells overnight. The next morning, the hematopoietic progenitors should aggregate into clumps of six to ten cells.Gently transfer the 50-miroliter volumes of re-aggregated cells into the center of a 24-well MAT-coded plate. Gently place the plate in the multigas incubator for four to six hours until the cells are attached. After four to six hours, to allow for cells to attach, slowly add one milliliter of freshly prepared HE media to each well.Incubate the cells in the multigas incubator until the hematopoetic cells are visible. Next harvest the definitive hematopoetic progenitors by gently removing the HE media from the cells and passing through a 40-micron cell strainer to collect flow-through. For the remaining adherent cells, add 0.5 milliliters 0.25%Trypsin EDTA to the cells and incubate them in a 37-degree Celsius incubator for five minutes.After Trypsinization, add 0.5 milliliters of stop solution to each well. Pass the cells through the same 40-micron cell strainer to pool all adherent and non-adherent cells together and spin the cells. Add 2, 000 to 10, 000 FACS isolated cells to one well of the OP9 DL4 cells in OP9 media.Incubate in a 37-degree Celsius, 5%CO2 incubator. To passage the cells every four to five days, triturate the cells with a one-milliliter pipette. Pass through a 40-micron filter to remove clumps.After centrifugation, aspirate the media. Re-suspend the cells in two milliliters of fresh T-cell media and place them on fresh OP9 DL4 cells. After at least 21 days of cold culture, triturate the cells vigorously and pass through a 40-micron filter to remove cell debris.Centrifuge at 330 x G for five minutes and aspirate the supernatant and then continue with standard flow cytometry. Flow cytometry shows that IWP2-treated differentiation cultures, yield a distinct CD34 positive, CD43 negative and CD34 low CD43 positive population. While CHIR-treated differentiation cultures have less than 1%CD43 positive cells on day eight of differentiation.Primitive hematopoietic progenitors derived under IWP2 conditions isolated on day nine will give rise to predominantly primitive erythroid and myeloid progenitors in the methylcellulose assays. The CD34 positive CD43 negative population derived from the CHIR99021 treatment is a heterogeneous population containing endothelial progenitors as well as CD34 positive, CD43 negative, CD73 negative, CD184 negative hemogenic endothelium, which when isolated by FACS will initially form a monolayer of endothelial-like cells that then undergoes an endothelial to hematopoietic transition resulting in round, refractile, non-adherent hematopoietic cells. These hematopoietic cells can be assessed by flow cytometry for the expression of CD34 and CD45.Hematopoietic progenitors isolated from the EHT assays can be used in methylcellulose assays and give rise to large, burst-like, erythroid colony forming units, small erythroid colony forming units and myeloid progenitors. These representative flow cytometric analyses of the T-lymphocyte assays potential from CHIR99021-derived CD34 positive, CD43 negative, CD73 negative, CD184 negative hematopoietic progenitors and IWP2-derived CD34 positive, CD43 negative hematopoietic progenitors show the presence of a CD45 positive, CD56 negative, CD4 positive, CD8 positive population in CHIR-derived progenitors indicative of the hematopoietic potential within the input CD34 positive population. After watching this video, you should have good understanding of how to differentiate human pluripotent stem cells towards primitive or definitive amount of hematopoietic progenitors.

directed-differentiation-primitive-definitive-hematopoietic

Research

JoVE Journal

Developmental Biology

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

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

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

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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 a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

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

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

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

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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 transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

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

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...