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

Quest'opera è stata sostenuta dal dipartimento di medicina interna, divisione di ematologia, Washington University School of Medicine. CD è stato supportato da T32HL007088 dal cuore nazionale, polmone e sangue Institute. CMS è stato supportato da una società americana di ematologia Scholar Award.

1. reagenti <ol><li>Obtain cella linee; hESCs o hiPSCs 1, mouse fibroblasti embrionali (MEFs) 29, OP9-DL4 stroma 30 , 31.</li> <li>preparazione dei reagenti <ol><li>preparare una soluzione di 0,1% p/v di gelatina in PBS. Sterilizzare in autoclave e dopo aliquotare e conservare a 4 ° C.
		<ol><li>Preparare il plasticware rivestite con gelatina. Cappotto piatti 6 pozzetti con 1,5 mL di soluzio...

<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>

Questo protocollo descrive un metodo rapido, privo di siero, stroma-free per la differenziazione dei progenitori ematopoietici primitivi o definitivi. Mesodermal specifica dei progenitori ematopoietici primitivi o definitivi può essere raggiunto in modo affidabile utilizzando il nostro protocollo, che sfrutta in modo univoco piccole molecole inibitrici di segnalazione WNT canonico. L'attivazione di WNT fase-specifici dall'inibitore GSK3β CHIR9902133 dà luogo a definitiva mesoderma ematopoietico...

Cellule staminali pluripotenti umane (hPSCs) sono definite nel senso che comprende sia le cellule staminali embrionali umane (hESC) e cellule staminali umane pluripotenti indotte (hiPSCs) e hanno la capacità unica di non solo in fase di auto-rinnovamento in condizioni di crescita appropriata, ma anche, la capacità di differenziarsi in tutti i tipi di cellule derivate da tre strati di germe: ectoderma, mesoderma ed endoderma1. A causa di queste abilità uniche, hPSCs tenere grande promessa per la medicina rigenerativa, la modellazione di malattia e le terapie basate sulle cellule2. Mentre più tipi di cellule sono stati differenziati con successo dai hPSCs, una sfida significativa è la specifica in vitro di esclusivamente adulto-come hPSC-derivato cellule staminali ematopoietiche (HSCs) e definitivi progenitori ematopoietici.
Un probabile ostacolo allo sviluppo di HSC umane da hPSCs è la presenza di più programmi ematopoietiche entro l' embrione umano3. Il primo programma che emerge, definito "primitivo ematopoiesi," nasce all'interno del tessuto del sacco vitellino extraembryonic e meglio è caratterizzato dalla sua produzione transitoria di progenitori dell'eritroblasto (EryP-CFC), macrofagi e megacariociti. In particolare, questo programma non dà luogo a HSCs, né dà origine ai progenitori linfoidi T e B. Tuttavia, il sacco vitellino transitoriamente dar luogo a progenitori ematopoietici definitivi con restrizioni, come il progenitore eritro-mieloide (EMP4,5,6,7,8) e degli eritrociti-carenti linfoide-innescato multipotenti progenitrici (LMPP9). Tuttavia, EMPs né LMPPs sono completamente multipotenti, o in grado di attecchimento di HSC-come nei destinatari adulti. Al contrario, più avanti in sviluppo, il programma ematopoietico "definitivo" classicamente definito è specificato nella regione dell'aorta-gonade-Mesonefro dell'embrione propriamente detto, dando vita a tutte le filiere emopoietiche adulti, tra cui l'HSC. La specifica di queste cellule ematopoietiche definitive intra-embrionali si verifica in una tacca-dipendente, tramite una transizione endoteliali--emopoietici dal hemogenic dell'endotelio (lui)3,10,11 ,12,13,14. A parte la capacità di ricostituzione, il potenziale di multilineage e tacca-dipendenza di queste cellule può essere utilizzato per distinguere questi progenitori ematopoietici definitivi da EMP e il LMPP (rivisto in riferimenti3,13 ).
Comprendere i meccanismi che disciplinano primitivo e definitiva specifica ematopoietica da hPSCs è probabilmente fondamentale per la produzione riproducibile dei progenitori ematopoietici definitivi attraverso una varietà di linee di hPSC. Fino a poco tempo, protocolli di differenziazione hPSC che potrebbero separare multipotenti progenitori ematopoietici primitivi e definitivi non esisteva15,16,17,18, 19,20,21,22,23,24,25. Molti approcci utilizzando siero bovino fetale (FBS) e/o stromale co-coltura prima delineato il potenziale ematopoietico di differenziazione hPSC, con miscele di primitivo e definitiva ematopoietiche potenziali15,16, 17,19,22,23,25. Ulteriormente, molti protocolli senza siero ematopoietiche sono descritti i requisiti di segnale per la specifica del mesoderma da hPSCs che continuano ad avere potenziale ematopoietico18,20,21, 24. Tuttavia, come questi metodi ancora ha dato luogo a miscele eterogenee di entrambi i programmi, il loro uso in applicazioni cliniche e comprensione dei meccanismi dello sviluppo può essere limitato.
Recentemente abbiamo costruito su questi studi, dopo aver delineato i requisiti del segnale di fase-specifici per ACTIVIN/NODAL e segnalazione di WNT nella specifica ematopoietico primitivo e definitiva dal mesoderma hPSC-derivato18,26 . Quest'ultimo era particolarmente unico, come il suo utilizzo di manipolazione del segnale WNT fase-specifici permette per la specifica di essere esclusivamente primitivo o di progenitori ematopoietici definitivo26esclusivamente. Durante la specifica di mesoderma, l'inibizione di segnalazione WNT canonico con l'inibitore PORCN IWP2 risultati nella specifica di CD43+ EryP-CFC e dei progenitori mieloidi, con nessun potenziale linfoide rilevabile. In netto contrasto, stimolazione della canonica WNT segnalazione con l'inibitore di GSK3β, CHIR99021, durante la stessa fase di differenziazione ha provocato la completa assenza di CD43 rilevabile+ EryP-CFC, mentre contemporaneamente conduceva per la specifica di CD34+CD43− HE. Questa popolazione ha posseduto mieloide, HBG-che esprimono degli eritrociti e potenziale T-linfoide. Successive analisi hanno identificato questo aveva come manca l'espressione di CD7327,28 CD18428e suo potenziale ematopoietico tacca-dipendente28. Più ulteriormente, unicellulare clonale analisi hanno dimostrato che queste filiere emopoietiche definitivi potrebbero essere derivati da cellule multipotenti singola28. Presi insieme, questi studi indicano che fase-specifici WNT segnalazione manipolazione possibile specificare puri primitivi progenitori ematopoietici, oppure multipotenti progenitori ematopoietici definitivi di NOTCH-dipendente.
Qui, descriviamo la nostra strategia di differenziazione che produce esclusivamente primitivi o definitivi progenitori ematopoietici, attraverso la manipolazione delle canoniche WNT segnalazione durante mesodermal patterning e loro a valle lineage ematopoietico saggi. Questo protocollo è di grande valore per i ricercatori che sono interessati nella produzione di progenitori ematopoietici primitivi o definitivi da hPSCs per applicazioni di medicina rigenerativa.

<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

Uno degli obiettivi principali per la medicina rigenerativa è la generazione e la manutenzione di cellule staminali ematopoietiche (HSCs) derivati da cellule staminali pluripotenti umane (hPSCs). Fino a poco tempo, gli sforzi per differenziare hPSCs in HSCs hanno generato principalmente progenitori ematopoietici che mancano di potenziale HSC e invece assomigliano vitellino ematopoiesi. Questi progenitori ematopoietici risultanti potrebbero essere limitato utilità per la modellazione in vitro malattia di vari disordini ematopoietici adulti, in particolare quelli dei lignaggi linfoidi. Tuttavia, recentemente abbiamo descritto i metodi per generare progenitori ematopoietici definitivi multilineare eritro-mielo-linfoidi da hPSCs utilizzando un protocollo di differenziazione diretto fase-specifici, che descriviamo qui. Attraverso la dissociazione enzimatica di hPSCs su plasticware rivestite con matrice della membrana dello scantinato, si formano corpi embryoid (EBs). EBs si differenzino per mesoderma BMP4 ricombinante, che viene successivamente specificato al programma ematopoietico definitivo dall'inibitore GSK3β, CHIR99021. In alternativa, ematopoiesi primitivo viene specificato dall'inibitore PORCN, IWP2. Ematopoiesi è ulteriormente guidato attraverso l'aggiunta del ricombinante VEGF e citochine ematopoietiche solidale. I progenitori ematopoietici risultanti generati utilizzando il metodo hanno il potenziale per essere utilizzato per malattia e modelli inerenti allo sviluppo, in vitro.

Un disegno schematico raffigurante l'induzione di progenitori ematopoietici primitivi e definitivi da hPSCs è illustrato nella Figura 1. Mesoderma patterning di Canonica WNT segnalazione si verifica durante i giorni 2-3 di differenziazione, seguita dalla specifica di progenitori emopoietici.
L'analisi cytometric di flusso rappresentativi e saggi di metilcellulosa di culture di differenziamento emat...

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

Qui, presentiamo pluripotenti umane protocolli di coltura della cellula formativa (hPSC), utilizzata per differenziare hPSCs in CD34+ progenitori ematopoietici. Questo metodo utilizza la modifica di fase-specifici di Canonica WNT segnalazione per specificare le celle esclusivamente al programma ematopoietica sia definitiva o primitivo.

Regia di differenziazione dei progenitori ematopoietici primitivi e definitivo da cellule staminali pluripotenti umane

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

Quantificazione tridimensionale di spine dendritiche da piramidale neuroni derivate da cellule staminali umane pluripotenti indotte

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

Ricerca

Biologia dello sviluppo

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

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

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

Differenziazione efficiente delle pluripotenti cellule staminali per NKX6-1<sup&gt; +</sup&gt; progenitori pancreatici

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

La differenziazione in vitro di pluripotenti umane Cellule Staminali in cellule trofoblastiche

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

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

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

Generazione del primo cuore campo-come progenitori cardiaci e ventricolare-come cardiomiociti da cellule staminali pluripotenti umane

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

Efficiente e scalabile di differenziazione diretto di clinicamente compatibile corneale Limbal cellule staminali epiteliali da cellule staminali pluripotenti umane

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

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

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

Generazione in Vitro di Somite derivati da umani indotta da cellule staminali pluripotenti indotte

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

Trattamento transitorio delle cellule staminali pluripotenti umane con DMSO per promuovere la differenziazione

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