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

Detta arbete har stötts av den institutionen för invärtesmedicin, avdelningen för hematologi, Washington University School of Medicine. CD stöddes av T32HL007088 från nationella hjärta, lungor och blod Institute. CMS stöddes av en American Society of hematologi Scholar Award.

1. reagenser <ol><li>Hämta cell linjer; hESCs eller hiPSCs 1, mus embryonala fibroblaster (MEFs) 29, OP9-DL4 stroma 30 , 31.</li> <li>REAGENSBEREDNING <ol><li>förbereda en 0,1% w/v lösning av gelatin i PBS. Sterilisera i autoklav och förvaras vid 4 ° C efter alikvotering.
		<ol><li>Förbereda den gelatin-belagd plasticware. Kappa 6-bra rätter med 1,5 mL 0,1% gelatin. Inkubera i 15 min i...

<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>

Det här protokollet beskriver en snabb, serumfritt, stroma-fri metod för differentiering av antingen primitiva eller slutgiltiga hematopoetiska progenitorceller. Mesodermala specifikation av antingen primitiva eller slutgiltiga hematopoetiska progenitorceller kan uppnås på ett tillförlitligt sätt med våra protokoll, som unikt utnyttjar småmolekylära hämmare av kanoniska WNT signalering. Scenen-specifika WNT aktivering av den GSK3β-hämmaren CHIR9902133 ger upphov till slutgiltig hematop...

Mänskliga pluripotenta stamceller (hPSCs) definieras som omfattar både mänskliga embryonala stamceller (hESCs) och mänskliga inducerade pluripotenta stamceller (hiPSCs), och har den unika funktionen av inte bara genomgå självförnyelse under lämpliga odlingsbetingelser, men också förmåga att differentieras till alla celltyper härrör från de tre groddar skikt: endoderm, mesoderm och ektoderm1. På grund av dessa unika förmågor hålla hPSCs stort löfte för regenerativ medicin, sjukdom modellering och cellbaserade terapier2. Medan flera celltyper har varit framgångsrikt differentierade från hPSCs, är en betydande utmaning specifikationen i vitro endast vuxen-liknande hPSC-härrör hematopoetiska stamceller (Förenta) och slutgiltig hematopoetiska progenitorceller.
Ett sannolikt hinder för utvecklingen av mänskliga Förenta från hPSCs är förekomsten av flera hematopoetiska program inom det mänskliga embryo3. Det första programmet som framträder, kallas ”primitiva blodbildning”, påbörjar inom embryoniskt gulesäcken vävnaden och är bäst kännetecknas av övergående produktion av erytroblast stamfäder (EryP-CFC), makrofager och megakaryocyter. Bland annat detta program ger inte upphov till Förenta, inte heller ger det upphov till T- och B lymfoida stamfäder. Gulesäcken ger dock övergående upphov till begränsad slutgiltiga hematopoetiska progenitorceller, såsom den erythro-myeloida stamceller (EMP4,5,6,7,8) och den erytroid-brist lymfoida-primade multipotenta stamceller (LMPP9). Men är varken EMPs eller LMPPs fullt multipotenta eller kan HSC-liknande engraftment i vuxna mottagare. Däremot senare i utvecklingen anges klassiskt definierade ”slutgiltiga” hematopoetiska programmet i regionen aorta-gonad-mesonephros av embryot korrekt, ger upphov till alla vuxna hematopoetiska härstamningar, inklusive HSC. Specifikationen av dessa inom embryonala slutgiltiga hematopoetiska celler uppstår i en Notch-beroende mode, via en endothelial-till-hematopoetiska övergång från hemogenic endotel (han)3,10,11 ,12,13,14. Bortsett från beredning kapacitet, kan den multilineage potentiella och Notch-beroende av dessa celler användas att skilja dessa slutgiltiga hematopoetiska progenitorceller från EMP och den LMPP (recenserade i referenser3,13 ).
Förstå de mekanismerna som reglerar primitiva och slutgiltiga hematopoetiska specifikation från hPSCs är sannolikt kritisk till reproducerbara produktionen av slutgiltiga hematopoetiska progenitorceller över en mängd olika hPSC linjer. Tills nyligen, fanns hPSC differentiering protokoll som kunde avskilja multipotenta primitiva och slutgiltiga hematopoetiska progenitorceller inte15,16,17,18, 19,20,21,22,23,24,25. Många metoder först med fetalt bovint serum (FBS) eller stromal samtidig kultur beskrivs hPSC differentiering, hematopoetiska potential med blandningar av primitiva och slutgiltiga hematopoetiska potentiella15,16, 17,19,22,23,25. Vidare, många serumfritt hematopoetiska protokoll har beskrivit signal kraven för specifikation av mesoderm från hPSCs som hamnar hematopoetiska potentiella18,20,21, 24. eftersom dessa metoder gav ändå upphov till heterogena blandningar av båda programmen, deras användning i kliniska tillämpningar och förståelse utvecklingsmässiga mekanismer kan dock begränsas.
Vi har nyligen byggt på dessa studier, har beskrivits scenen-specifik signal kraven för ACTIVIN/NODAL och WNT signalering i primitiva och slutgiltiga hematopoetiska specifikation från hPSC-derived mesoderm18,26 . Den senare var särskilt unik, eftersom dess användning av scenen-specifika WNT-signal manipulering möjliggör specifikation av antingen uteslutande primitiva eller uteslutande slutgiltiga hematopoetiska progenitorceller26. Under mesoderm specifikation, hämning av kanoniska WNT signalering med PORCN-hämmaren IWP2 resulterar i specifikationen av CD43+ EryP-CFC och myeloida med inga detekterbara lymfoida potential. I skarp kontrast, stimulering av kanoniska WNT signalering med GSK3β-hämmaren CHIR99021, under samma skede i differentiering resulterade i en total avsaknad av detekterbara CD43+ EryP-CFC, medan samtidigt leder till den specifikation av CD34+CD43− han. Denna population besatt myeloisk, HBG-uttrycker erytroid och T-lymfoida potential. Efterföljande analyser identifierat detta han saknas uttryck för CD7327,28 och CD18428, och dess hematopoetiska potential var NOTCH-beroende28. Ytterligare, encelliga klonal analyser visat att dessa slutgiltiga hematopoetiska härstamningar kan härledas från multipotenta enstaka celler28. Sammantaget tyder dessa studier på att scenen-specifika WNT signalering manipulation kan ange antingen ren primitiva hematopoetiska progenitorceller, eller multipotenta NOTCH-beroende slutgiltiga hematopoetiska progenitorceller.
Här beskriver vi vår differentiering strategi som ger uteslutande primitiva eller slutgiltiga hematopoetiska progenitorceller, via manipulation av kanoniska WNT signalering under mesodermala mallning och deras nedströms hematopoetiska härstamning analyser. Detta protokoll är av stort värde att utredare som är intresserade av produktionen av antingen primitiva eller slutgiltiga hematopoetiska progenitorceller från hPSCs för regenerativ medicin program.

<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

En av de stora målen för regenerativ medicin är generation och underhåll av hematopoetiska stamceller (Förenta) härrör från mänskliga pluripotenta stamceller (hPSCs). Tills nyligen har ansträngningar differentieras hPSCs till Förenta huvudsakligen genererat hematopoetiska progenitorceller som saknar HSC potential, och istället likna gulesäcken blodbildning. Dessa resulterande hematopoetiska progenitorceller kan ha begränsad nytta för in vitro- sjukdom modellering av olika vuxna hematopoetiska störningar, särskilt de av de lymfoida härstamningar. Vi har dock nyligen beskrivit metoder för att generera erythro-myelosuppression-lymfoida Multilinjärt slutgiltiga hematopoetiska progenitorceller från hPSCs med en stage-specifika riktad differentiering-protokollet, som vi beskriver här. Genom enzymatisk dissociation av hPSCs på basalmembranet matrix-belagda plasticware bildas embryoid organ (EBs). EBs särskiljs till mesoderm av rekombinant BMP4, som därefter anges till slutgiltig hematopoetiska programmet av den GSK3β-hämmaren, CHIR99021. Alternativt, primitiva blodbildning anges av den PORCN-hämmaren, IWP2. Blodbildning drivs vidare genom tillägg av rekombinant VEGF och stödjande hematopoetiska cytokiner. De resulterande hematopoetiska progenitorceller genereras med den här metoden har potential att användas för sjukdom och utvecklingsmässiga modellering, in vitro.

En schematisk föreställande induktion av primitiva och slutgiltiga hematopoetiska progenitorceller från hPSCs illustreras i figur 1. Mesoderm mönstring av kanoniska WNT signalering uppstår under dagarna 2-3 av differentiering, följt av hematopoetiska stamceller specifikation.
Representativa flöde flödescytometrisk analys och kolonibildande metylcellulosa analyser av hPSC-härrör hematopoeti...

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

Här presenterar vi mänskliga pluripotenta stamceller (hPSC) kultur-protokoll, används för att differentiera hPSCs in CD34+ hematopoetiska progenitorceller. Denna metod använder scenen-specifika manipulation av kanoniska WNT signalering ange celler uteslutande att antingen slutgiltig eller primitiva hematopoetiska program.

Regisserad differentiering av primitiv och slutgiltig hematopoetiska progenitorceller från mänskliga pluripotenta stamceller

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

Research

JoVE Journal

Developmental Biology

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Tredimensionell Kvantifiering av Dendritutskotten från Pyramidal nervceller Härstammar från Human inducerade pluripotenta stamceller

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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

Storskalig produktion av Cardiomyocytes från Human pluripotenta stamceller med hjälp av en mycket reproducerbar liten molekyl baserad differentiering protokoll

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

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

Effektiv Differentiering av pluripotenta stamceller till NKX6-1<sup&gt; +</sup&gt; Pankreasstamfäder

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In vitro-differentiering av mänskliga pluripotenta stamceller till trophoblastic celler

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

Snabb, regisserad differentiering av Retinal Pigment epitelial celler från mänskliga embryonala eller inducerade pluripotenta stamceller

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

Generation av första hjärtat fält-liknande hjärt stamfäder och ventrikulära-liknande hjärtmuskelcellerna från mänskliga pluripotenta stamceller

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

Effektiv och skalbar riktad differentiering av kliniskt kompatibel korneal limbala epitelial stamceller från mänskliga pluripotenta stamceller

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

Effektiv differentiering av humana pluripotenta stamceller till leverceller

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

I Vitro Generation av Somite derivat från mänskliga inducerade stamceller pluripotenta

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

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

Övergående behandling av humana pluripotenta stamceller med DMSO för att främja differentiering

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