Name: defined xeno-free and feeder-free culture conditions for the generation of human ipsc-derived retinal cell models
Uploaded: 2018-09-06T13:00:00
Description: Watch this Scientific Journal Video about Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models at JoVE.com

Forfatterne vil gerne takke medlemmerne af Goureau's team for deres input under Opsætning af de metoder, der beskrives her, og G. Gagliardi og M. Garita for deres kritiske læsning. Dette arbejde blev støttet af tilskud fra ANR (GPiPS: ANR-2010-RFCS005; SightREPAIR: ANR-16-CE17-008-02), nethinden Frankrig Association og den teknologioverførsel selskab SATT Lutech. Det blev også udført inden for rammen af LABEX LIFESENSES (ANR-10-LABX-65) understøttes af ANR inden for programmet Investissements d'Avenir (ANR-11-IDEX-0004-02).

Den protokol, der er beskrevet i denne hvidbog følger retningslinjerne i Institut de la Vision videnskabsetisk Komité. Institut de la Vision har været tilladt manipulering af menneskelige modellen ifølge den nuværende franske forordning. Præparatbehandling følger patienten databeskyttelse i overensstemmelse med grundprincipperne i Helsinki, og nationale bestemmelser efter den etiske godkendelse af "Comité de Protection des Personnes (CPP) Ile-de-France V".
1. forberedelse af dyrkningsmed...

<ol>
	<li>Zhao, C., Wang, Q., Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+retinal+diseases:+recapitulating+development+to+replace+degenerated+cells.">Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells.</a> Development. 144, 1368-1381 (2017).</li><li>Dalkara, D., Goureau, O., Marazova, K., Sahel, 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=Let+There+Be+Light:+Gene+and+Cell+Therapy+for+Blindness.">Let There Be Light: Gene and Cell Therapy for Blindness.</a> Human Gene Therapy. 27, 134-147 (2016).</li><li>Wright, L. S., Phillips, M. J., Pinilla, I., Hei, D., Gamm, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+as+custom+therapeutics+for+retinal+repair:+Progress+and+rationale.">Induced pluripotent stem cells as custom therapeutics for retinal repair: Progress and rationale.</a> Experimental Eye Research. 123, 161-172 (2014).</li><li>Leach, L. L., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+Stem+Cells+Retinal:+Methods+for+Deriving+Retinal+Pigment+Epithelium+and+Implications+for+Patients+with+Ocular+Disease.">Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients with Ocular Disease.</a> Stem Cells. 33, 2363-2373 (2015).</li><li>Gill, K. P., Hewitt, A. W., Davidson, K. C., P&#233;bay, A., Wong, R. C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+Retinal+Ganglion+Cell+Differentiation+From+Pluripotent+Stem+Cells.">Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells.</a> Translational Vision Science and Technology. 3, 2 (2014).</li><li>Giacalone, J. C., Wiley, L. 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=Concise+review:+Patient-specific+stem+cells+to+interrogate+inherited+eye+disease.">Concise review: Patient-specific stem cells to interrogate inherited eye disease.</a> STEM CELLS Translational Medicine. 5, 132-140 (2016).</li><li>Nakano, T., Ando, 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=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10, 771-785 (2012).</li><li>Reichman, S., Terray, 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=From+confluent+human+iPS+cells+to+self-forming+neural+retina+and+retinal+pigmented+epithelium.">From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 8518-8523 (2014).</li><li>Meyer, J. S., Howden, S. 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=Optic+vesicle-like+structures+derived+from+human+pluripotent+stem+cells+facilitate+a+customized+approach+to+retinal+disease+treatment.">Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.</a> Stem Cells. 29, 1206-1218 (2011).</li><li>Zhong, X., Gutierrez, 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=Generation+of+three-dimensional+retinal+tissue+with+functional+photoreceptors+from+human+iPSCs.">Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.</a> Nature Communications. 5, 4047 (2014).</li><li>Mellough, C. B., Collin, 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=IGF-1+Signalling+plays+an+important+role+in+the+formation+of+three+dimensional+laminated+neural+retina+and+other+ocular+structures+from+human+embryonic+stem+cells.">IGF-1 Signalling plays an important role in the formation of three dimensional laminated neural retina and other ocular structures from human embryonic stem cells.</a> Stem Cells. 33, 2416-2430 (2015).</li><li>Gonzalez-Cordero, A., Kruczek, 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=Recapitulation+of+human+retinal+development+from+human+pluripotent+stem+cells+generates+transplantable+populations+of+cone+photoreceptors.">Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors.</a> Stem Cell Reports. 9, 820-837 (2017).</li><li>Eiraku, M., Takata, N., 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Meyer, J. S., Shearer, R. L., 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=Modeling+early+retinal+development+with+human+embryonic+and+induced+pluripotent+stem+cells.">Modeling early retinal development with human embryonic and induced pluripotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. , 16698-16703 (2009).</li><li>Reichman, S., Slembrouck, 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=Generation+of+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+ips+cells+in+xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human ips cells in xeno-free and feeder-free conditions.</a> Stem Cells. 35, 1176-1188 (2017).</li><li>Chen, G., Gulbranson, D. 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=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8, 424-429 (2011).</li><li>Croze, R. H., Buchholz, D. 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=ROCK+inhibition+extends+passage+of+pluripotent+stem+cell-derived+retinal+pigmented+epithelium.">ROCK inhibition extends passage of pluripotent stem cell-derived retinal pigmented epithelium.</a> Stem Cells Translational Medicine. 3, 1066-1078 (2014).</li><li>Eberle, D., Schubert, S., Postel, K., Corbeil, D., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+integration+of+transplanted+CD73-positive+photoreceptor+precursors+into+adult+mouse+retina.">Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina.</a> Investigative Ophthalmology &amp; Visual Science. 52, 6462-6471 (2011).</li></ol>

Denne protokol beskriver hvordan man producerer ÅV celler og retinal organoids, som indeholder retinal RGCs og fotoreceptorer, fra humane pluripotente stamceller i xeno-fri og feeder-fri betingelser. Kompatibel med god fremstilling af praksis (GMP) proces, metoden dyrkede præsenteres her tillader en stor produktion af iPSC-afledte retinale celler som ÅV celler, RGCs og fotoreceptorer for udviklingen af stamceller-baserede behandlinger og medicin Discovery tilgange for den fremtidige behandling af retinale degenerative...

Nethinden er en integreret del af det centrale nervesystem (CNS) og har en begrænset kapacitet til at regenerere spontant efter en traumatisk skade eller sygdomme. Derfor, degenerative sygdomme forårsager endelige retinal celletab, såsom aldersrelateret Macula degeneration (AMD), retinitis pigmentosa (RP), glaukom og diabetisk retinopati, typisk føre til uoprettelige blindhed. Redning degenererede nethinden er en stor udfordring som stamcelle-baserede behandlinger har til formål at erstatte de beskadigede eller mistede celler er en af de mest lovende metoder1,2,3. Pluripotente stamceller som humane embryonale stamceller (økonomiske) celler eller menneskelige-induceret pluripotente stamceller (iPSCs) har kapacitet til at blive udvidet på ubestemt tid i kulturen, og de har potentiale til at producere alle celletyper. Fremskridt i vores forståelse af retinal udvikling og forbedring af in vitro- protokoller for menneskelige iPSC differentiering har resulteret i generation af retinale organoids7,8,9, 10,11,12. Alle de store retinale celler, herunder retinal ganglion celler (RGCs), fotoreceptorer, og retinal pigmenteret (ÅV) epitelceller, har med succes blevet differentieret fra menneskelige og sociale råd og iPSCs4,5, 6. baseret på SFEB (serum-fri kultur af embryoid krop-lignende aggregater) metode udviklet af Eiraku et al. 13, selvstændig dannelsen af retinale organoids kan fås fra ESC - eller iPSC-afledte embryoid krop-lignende aggregater i definerede ekstracellulære matrix komponenter7,10,14. Men disse protokollerne er indviklede, der kræver et stort antal trin ikke altid kompatible med den store produktion af celler til terapeutiske tilgange eller stof screening. Således, at valget af metoden til at producere menneskelige retinale celler er kritisk og metoden skal være robust, skalerbar og effektiv.
Her, beskriver baseret på vores tidligere publikation15, vi hvert trin i en enkel og effektiv generation af retinale celler via retinale organoid selvstændig dannelse fra vedhængende menneskelige iPSCs dyrkes i et feeder-fri og xeno tilstand. Startende fra rutinemæssige kulturer af vedhængende menneskelige iPSCs, kræver denne protokol kun en simpel successive medium forandring for at tillade generation af både iPS-afledte ÅV (hiRPE) celler og neuroretinal strukturer i 4 uger. Efter en manuel isolering, hiRPE kan udvides og de retinale strukturer kan blive kulturperler som flydende organoids hvor de retinale stamceller er i stand til at differentiere i alle retinal celletyper i sekventiel rækkefølge i overensstemmelse med i vivo menneskelige retinogenesis. Endelig, for forskning avancement eller kliniske oversættelse, vi beskriver en kryopræservering metode tillader langtidsopbevaring af hele retinale organoids og hiRPE celler uden at det påvirker deres fænotypiske egenskaber og funktionalitet.

<table>
	<tbody>
		<tr>
			<td>Vitronectin (VTN-N) Recombinant Human Protein, Truncated</td>
			<td>ThermoFisher Scientific</td>
			<td>A14700</td>
			<td>Coating</td>
		</tr>
		<tr>
			<td>CTS Vitronectin (VTN-N) Recombinant Human Protein, Truncated</td>
			<td>ThermoFisher Scientific</td>
			<td>A27940</td>
			<td>Coating</td>
		</tr>
		<tr>
			<td>Essential 8 Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A1517001</td>
			<td>medium</td>
		</tr>
		<tr>
			<td>Essential 6 Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A1516401</td>
			<td>medium</td>
		</tr>
		<tr>
			<td>CTS (Cell Therapy Systems) N-2 Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>A1370701</td>
			<td>supplement CTS</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502048</td>
			<td>supplement</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>ThermoFisher Scientific</td>
			<td>17504044</td>
			<td>supplement</td>
		</tr>
		<tr>
			<td>CTS B-27 Supplement, XenoFree</td>
			<td>ThermoFisher Scientific</td>
			<td>A1486701</td>
			<td>supplement CTS</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320074</td>
			<td>medium</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140035</td>
			<td>supplement</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>antibiotic</td>
		</tr>
		<tr>
			<td>CellStart CTS</td>
			<td>ThermoFisher Scientific</td>
			<td>A1014201</td>
			<td>Matrix CTS</td>
		</tr>
		<tr>
			<td>Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix</td>
			<td>ThermoFisher Scientific</td>
			<td>A1569601</td>
			<td>Matrix</td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>Stemcell Technologies</td>
			<td>7174</td>
			<td>dissociation solution</td>
		</tr>
		<tr>
			<td>Cryostem Freezing Media</td>
			<td>clinisciences</td>
			<td>05-710-1D</td>
			<td>Cryopreservation medium</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2)</td>
			<td>Preprotech</td>
			<td>100-18B</td>
			<td>FGF2</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2) animal free</td>
			<td>Preprotech</td>
			<td>AF-100-18B</td>
			<td>FGF2 Xeno free</td>
		</tr>
		<tr>
			<td>AGANI needle 23G</td>
			<td>Terumo</td>
			<td>AN*2332R1</td>
			<td>Needle</td>
		</tr>
		<tr>
			<td>Flask 25 cm&#178; Tissue Culture Treated</td>
			<td>Falcon</td>
			<td>353109</td>
			<td>T-25 cm&#178;</td>
		</tr>
		<tr>
			<td>24 well plate Tissue Culture Treated</td>
			<td>Costar</td>
			<td>3526</td>
			<td>24-well plate</td>
		</tr>
		<tr>
			<td>6 well plate Tissue Culture Treated</td>
			<td>Costar</td>
			<td>3516</td>
			<td>6-well plate</td>
		</tr>
	</tbody>
</table>

defined xeno-free and feeder-free culture conditions for the generation of human ipsc-derived retinal cell models

Produktionen af specialiserede celler fra pluripotente stamceller giver et kraftfuldt værktøj til at udvikle nye tilgange til regenerativ medicin. Brug af human-induceret pluripotente stamceller (iPSCs) er særligt attraktive for neurodegenerativ sygdom undersøgelser, herunder retinal dystrophies, hvor iPSC-afledte retinal cell modeller markerer et stort skridt fremad til at forstå og bekæmpe blindhed. I dette papir beskriver vi en enkel og skalerbar protokol for at generere, modne og cryopreserve retinale organoids. Baseret på mellemlang skiftende, den største fordel ved denne metode er at undgå flere og tidskrævende trin kræves almindeligvis i en guidet differentiering af iPSCs. Efterligne de tidlige faser af retinal udvikling af successive ændringer af definerede medier på vedhængende menneskelige iPSC kulturer, denne protokol giver mulighed for den samtidige generation af selvstændige danner neuroretinal strukturer og retinal pigmenteret epitel (ÅV) celler i en reproducerbare og effektiv måde i 4 uger. Disse strukturer, som indeholder retinal stamceller (fjernprocedurekald (RPC)) kan være let isoleret til yderligere modning i et flydende kultur tilstand gør det muligt for differentiering af fjernprocedurekald (RPC) til de syv retinal celletyper i den voksne menneskelige nethinde. Derudover beskriver vi hurtig metoder for kryopræservering af retinale organoids og ÅV celler til langtidsopbevaring. Kombineret sammen, vil de metoder, der beskrives her være nyttigt at producere og bank iPSC-afledte retinale celler eller væv til både grundforskning og klinisk forskning.

Det første skridt for menneskelige iPSC differentiering dyrket i feeder-fri betingelser16 er at lukke selvfornyelse maskiner bruge Bi medium for at tilskynde en spontan differentiering (figur 1A). Derefter, på D2, Bi medium er suppleret med en N2 supplement til at guide differentiering iPSCs celler mod de neurale og retinal lineages. Denne proces fører til fremkomsten af neuroretinal knopper på omkring D28 (fig...

Watch this Scientific Journal Video about Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models at JoVE.com

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Definerede Xeno-fri og Feeder-fri kultur betingelser for den Generation af menneskelige iPSC-afledte Retinal Cell modeller

Produktionen af specialiserede retinale celler fra pluripotente stamceller er et vendepunkt i udviklingen af stamcelle-baseret terapi for retinal sygdomme. Den nuværende papir beskriver en simpel metode til en effektiv generation af retinale organoids og retinal pigmenteret epitel til grundlæggende, translationel og klinisk forskning.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generering af induceret pluripotente stamceller fra humane perifere T-celler Brug af Sendai Virus i Feeder-fri Betingelser

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Feeder-fri Udledning af melanocytter fra menneskelige pluripotente stamceller

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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

Storstilet produktion af cardiomyocytter fra menneskelige pluripotente stamceller Ved hjælp af en meget reproducerbar lille molekyle-Based Differentiering Protocol

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

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Indsamling af serum- og Feeder-fri mus embryonale stamceller-conditioned Medium for en celle-fri tilgang

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Produktion og administration af terapeutiske mesenchymstamceller / Stromal Cell (MSC) Spheroids Primed i 3-D kulturer Under Xeno-frie betingelser

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Defineret og skalerbar Generation af hepatocyt-lignende celler fra menneskelige pluripotente stamceller

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Generation af iPSC-afledte menneskelige hjerne organoider til Model Tidlig forstyrrelser i nervesystemets

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Omprogrammering primære fostervand og membran celler til Pluripotency i Xeno-fri betingelser

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Hemogen endothelium differentiering fra humane pluripotente stamceller i en feeder-og Xeno-fri defineret tilstand

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