Name: development, expansion, and in vivo monitoring of human nk cells from human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs)
Uploaded: 2013-04-23T13:00:00
Description: Watch this Scientific Journal Video about Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs at JoVE.com

Forfatterne vil gerne takke Melinda Hexum til indvielse af spin EB-protokollen inden for vores lab. Vi vil gerne takke de øvrige medlemmer af laboratoriet, herunder Laura E. Bendzick, Michael Lepley og ZhenYa Ni for deres tekniske bistand med dette arbejde. Forfatterne vil også gerne takke Brad Taylor på Caliper Life Sciences for hans ekspert teknisk rådgivning.

1.. Tilpasning hESCs eller iPSCs i TrypLE til Spin EB Cultures <ol><li> Den indledende dissociation med TrypLE fungerer bedst, hvis ES / iPS kolonier fra kollagenasebehandlede passerede kulturer er relativt små. Udgangspunktet ES / iPS populationer bør celler passerede længere end 4-5 dage tidligere. 4-5 dage før initiering af TrypLE passage, passerer ES-celler ved en densitet, som vil tillade cellerne at være ~ 70% sammenflydende i fire dage tid. Bruge almindelige ES medier til dyrkning af TrypLE-passerede ES-ce...

<ol>
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P., Azzola, L., 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=Forced+aggregation+of+defined+numbers+of+human+embryonic+stem+cells+into+embryoid+bodies+fosters+robust,+reproducible+hematopoietic+differentiation.">Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation.</a> Blood. 106, 1601-1603 (2005).</li><li>Vodyanik, M. A., Bork, J. A., Thomson, J. A., Slukvin, I. 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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=Sensitive+in+vivo+imaging+of+T+cells+using+a+membrane-bound+Gaussia+princeps+luciferase.">Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase.</a> Nature Medicine. 15, 338-344 (2009).</li><li>Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Proteins+and+Their+Applications+in+Imaging+Living+Cells+and+Tissues.">Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues.</a> Physiological Reviews. 90, 1103-1163 (2010).</li><li>Knorr, D. A., Bock, A., Brentjens, R. J., Kaufman, D. 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hESCs er en ideel platform til at studere diverse celletyper og holde bemærkelsesværdigt potentiale for klinisk oversættelse. Vi bruger en defineret, spin-EB tilgang til at differentiere embryonale / iPSCs til bloddannende stamceller. Spin EB tilgang har givet konsekvent afledning af hæmatopoietiske stamceller og differentiering til NK-celler endnu, variation, findes stadig i differentiering effektiviteten på tværs cellelinjer og skal muligvis modificeres til generering af andre hæmatopoietiske cellelinier. Mens ...

Humane embryonale stamceller (hESCs) og induceret pluripotente stamceller (iPSCs), er udifferentierede, pluripotente celler, som kan ubegrænset selvfornyelse og multi-slægt differentiering. hESCs succes er blevet differentieret i modne og funktionelle undergrupper af hver kim lag, herunder celler af den hæmatopoietiske system 1-3. Naturlige dræberceller (NK-celler) er lymfocytter i det medfødte immunsystem, der kan afledes af hESCs ved dannelse af embryoid organer (EBS) 4,5 eller co-kultur med stromale cellelinier 1,2,6-8. NK-celler besidder anti-viral og anti-tumor egenskaber og har potentiale til at være effektiv mod en bred vifte af maligniteter, da de ikke kræver forudgående antigenstimulering til at udføre deres effektorfunktioner. Således hESC afledt NK-celler er en attraktiv kilde til celler til immunterapi. Derudover afledning af NK-celler fra hESCs giver et genetisk medgørlige til undersøgelse normal udvikling &lt;em&gt; in vitro. Fordi de giver et genetisk medgørligt system kan embryonale afledt NK celler eksperimentelt modificeret til at udtrykke fluorescerende og bioluminescerende reportere giver en optimal model til at studere NK celle effektorfunktioner in vitro og in vivo. hESC-afledte NK-celler besidder aktivitet mod en række mål, herunder HIV 9, leukæmi (K562) og andre kræfttyper 7,8. Men evnen effektivt udlede nok NK-celler er i stand til at behandle patienter fortsat en vigtig barriere for klinisk oversættelse og er i mindre grad, en begrænsning for omfattende prækliniske in vivo studier af NK-celle udvikling og anti-cancer-funktioner. Her bruger vi et spin EB tilgang til at udlede hæmatopoietiske stamfædre fra hESCs 4,5,10. Efter elleve dage spin EB overføres til NK-celle kultur med eller uden automater til 28 dage. Efter 4 uger i NK-celle dyrkningsmedium, er NK-celler transferred til co-kultur med K562-celler modificeret til at udtrykke membranbundet interleukin 21 (IL-21), der tjener som kunstige antigenpræsenterende celler (aAPCs). Tilpasning en protokol til udvidelse af perifert blod NK-celler ved hjælp af disse kunstige APC&#39;er 11,12, vi er i stand til at udvide NK-celler 2-logs og samtidig bevare en moden fænotype og cytotoksiske kapaciteter. Denne proces med udvikling og ekspansion giver tilstrækkelige embryonale afledt NK-celler til omfattende in vivo karakterisering. Til in vivo studier, er vi i stand til ikke-invasivt overvåge langsigtet engraftment og kinetik af indsprøjtet ildflueluciferase udtrykker (Fluc +), embryonale-afledte NK-celler ved hjælp af bioluminescens billeddannelse. Desuden er vi i stand til at følge NK celleinteraktioner med tumorceller ved hjælp af en dobbelt, selvlysende eller fluorescerende billeddannelse ordningen. En tidligere undersøgelse fra vores gruppe brugte bioluminescence billeddannelse i en anti-tumor model til efterfølgelse tumor progression og cleArance af udsving + K562-celler in vivo 7.. Nu ved ingeniør vore hESCs at udtrykke ildflueluciferase 13,14 vi kan følge biofordelingen og handel med NK-celler til K562 tumorceller, som udtrykker den nyligt karakteriseret fluorescerende protein, turboFP650 15.. Vi har valgt dette dobbelte reporter system for at samtidigt følge de to cellepopulationer in vivo (figur 1). De fleste dual imaging modeller har været dual-luciferase-systemer, men disse systemer kan teknisk udfordrende på grund af de levering krav coelenterazin, underlaget kræves for ekspression af de fleste Renilla og Gaussia luciferase journalister 16-18. Fluorescerende reportere har tilladt nem overvågning af mange cellelinjer og konstruktioner in vitro, men har haft begrænset succes til in vivo billeddannelse på grund af overlapning mellem væv og pels autofluorescens og emission spektre af mange commonly brugt fluorescerende reportere, herunder GFP, DsRed og TdTomato 15,19. Denne bekymring har tilskyndet til udviklingen af langt rødt fluorescerende proteiner, som tillader en bedre væv penetrans og højere specifik signal i forhold til baggrunden 15,19. TurboFP650 det fluorescerende protein er vist i dette system, er langt rødt flyttet og overvinder mange af de spørgsmål, der er forbundet med billeddiagnostiske fluorescerende proteiner i levende dyr. Denne metode for udvikling og udvidelse NK-celler afledt fra hESCs har givet os mulighed for yderligere at karakterisere embryonale afledt NK-celler in vitro og in vivo, som er nødvendig for bedre at forstå NK-cellernes funktion og klinisk vigtigt at forbedre de nuværende NK celle adoptiv behandlingsformer. Det er også modtagelig for udledning og udvidelse af iPSC-afledte NK-celler. Den dobbelte fluorescerende og bioluminiscerende imaging-ordningen er bredt anvendelig til andre systemer end den anti-tumor model, vi har vist her.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>BPEL media for Spin EB generation</td>
		</tr>
		<tr>
			<td>Iscove's Modified Dulbecco's Medium (IMDM)</td>
			<td>Fisher Scientific</td>
			<td>SH3022801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture w/ Glutamax I</td>
			<td>Invitrogen</td>
			<td>31765035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A3311</td>
			<td>Commercially available BSA can be cytotoxic to ES cells. Deionizing the solution can reduce the potential for cytotoxicity.</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L1012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L2376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Synthechol 500X solution</td>
			<td>Sigma-Aldrich</td>
			<td>S5442</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>a-monothioglycerol (a-MTG)</td>
			<td>Sigma-Aldrich</td>
			<td>S5442</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protein-free hybridoma mix II</td>
			<td>Invitrogen</td>
			<td>12040077</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic Acid 2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax I</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium 100X solution (ITS)</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>NK Differentiation Media </td>
		</tr>
		<tr>
			<td>Dubecco's Modified Eagle Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Media</td>
			<td>Invitrogen</td>
			<td>CX30315</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15% Human AB serum</td>
			<td>Valley Biomed</td>
			<td>HP1022HI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ng/ml Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 uM ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20 ng/ml ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 uM 2-mercaptoethanol (BME)</td>
			<td>Gibco</td>
			<td>21985</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 mM L-glutamine</td>
			<td>Gibco</td>
			<td>CX30310</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1% Pen-Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Cytokines</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>(all cytokines used fresh from frozen aliquots)</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>PeproTech, Inc.</td>
			<td>300-07</td>
			<td>40 ng/ml in BPEL media; 20 ng/ml in NK medium). As noted by the Elefanty protocol, and as we discovered with a bad lot of BMP4, there can be lot differences with cytokines in regards to hematopoietic differentiation.Especially if buying cytokines in bulk, obtain a sample of the lot to test prior to purchase. Compare head-to-head with old lot.</td>
		</tr>
		<tr>
			<td>rhBMP-4</td>
			<td>R &amp;D Systems</td>
			<td>314-BP</td>
			<td>20 ng/ml in BPEL media</td>
		</tr>
		<tr>
			<td>rhVEGF</td>
			<td>R&amp;D Systems</td>
			<td>293-VE</td>
			<td>20 ng/ml in BPEL media</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>PeproTech, Inc.</td>
			<td>200-02</td>
			<td>1 x 105 U/ml given to mice after NK cell injection; 50 U/ml used in NK cell expansion protocol</td>
		</tr>
		<tr>
			<td>IL-15</td>
			<td>PeproTech, Inc.</td>
			<td>200-15</td>
			<td>10 ng/ml given to mice after NK cell injection (first 7 days only)</td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>PeproTech, Inc.</td>
			<td>200-03</td>
			<td>5 ng/ml in NK media</td>
		</tr>
		<tr>
			<td>IL-7</td>
			<td>PeproTech, Inc.</td>
			<td>200-07</td>
			<td>20 ng/ml in NK media</td>
		</tr>
		<tr>
			<td>Flt-3-Ligand</td>
			<td>PeproTech, Inc.</td>
			<td>300-19</td>
			<td>10 ng/ml in NK media</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>In vivo Imaging</td>
		</tr>
		<tr>
			<td>D-Luciferin Sodium Salt</td>
			<td>Gold BioTechnology</td>
			<td>Lucna-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TurboFP650 plasmid</td>
			<td>Evrogen, Moscow, Russia</td>
			<td>FP731</td>
			<td>Subcloned into a Sleeping Beauty transposon based plasmid driven by the mCAGs promoter. Cells were then sorted on their expression of turboFP650 by FACS. Cells and plasmids can be obtained from our lab.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>IVIS Spectrum Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Membrane bound IL-21 expressing artificial antigen presenting cells</td>
			<td>MD Anderson, Houston, TX</td>
			<td>&nbsp;</td>
			<td>contact: Dean A. Lee. <a href="http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank">http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood</a></td>
		</tr>
		<tr>
			<td>Firefly luciferase expressing hESCs</td>
			<td>University of Minnesota, Minneapolis, MN</td>
			<td>&nbsp;</td>
			<td>contact: Dan S. Kaufman . H9 cells modified with a Sleeping Beauty transposon based method (references 13 and 14). Expression of firefly luciferase is driven by the mCAGGS promoter. Following the firefly luciferase gene is an IRES element at the 5' end of a GFP:zeocin fusion construct.</td>
		</tr>
		<tr>
			<td>TurboFP650 expressing K562 cells</td>
			<td>University of Minnesota, Minneapolis, MN</td>
			<td>&nbsp;</td>
			<td>contact: Dan S. Kaufman. Description under plasmid comments section</td>
		</tr>
	</tbody>
</table>
Materials Table.

development, expansion, and in vivo monitoring of human nk cells from human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs)

Vi præsenterer en metode til at udlede naturlige dræberceller (NK-celler) fra udifferentierede hESCs og iPSCs hjælp af en feeder-fri tilgang. Denne metode giver anledning til høje niveauer af NK-celler efter 4 ugers kultur og kan undergå yderligere 2-log ekspansion med kunstige antigenpræsenterende celler. hESC-og iPSC-afledte NK-celler udviklet i dette system har en moden fænotype og funktion. Produktionen af ​​et stort antal genetisk modificerbare NK-celler er gældende for både grundforskning mekanistisk samt anti-tumor studier. Ekspression af ildflueluciferase i hESC-afledte NK-celler giver en non-invasiv metode til at følge NK-celle transplantation, distribution og funktion. Vi beskriver også en dual-imaging ordning, der giver mulighed for separat overvågning af to forskellige cellepopulationer til mere udpræget karakterisere deres interaktion in vivo. Denne metode til afledning, ekspansion, og dual in vivo imaging giver en pålidelig metode til at frembringe NK-celler, og deres evalueringtion, der er nødvendig for at forbedre de nuværende NK celle adoptivforældre behandlingsformer.

Den generation af hæmatopoietiske stamceller ved hjælp af spin-EB tilgang giver mulighed for optimal NK-celle udvikling fra hESCs og iPSCs. Som vist i figur 2, dag 11 spin-EB indeholder høje procentdele af progenitorceller, der udtrykker CD34, CD45, CD43 og CD31. Høje niveauer af CD34 og CD45 giver mulighed for direkte overførsel til NK forhold uden behov for sortering eller støtte stromaceller. Hvis der er suboptimal spin-EB differentiering, anbefales det, at stromale celler, såsom EL08-1D2 anve...

Watch this Scientific Journal Video about Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs at JoVE.com

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

Denne protokol beskriver udviklingen, ekspansion, og in vivo billeddannelse af NK-celler afledt fra hESCs og iPSCs.

Udvikling, Expansion, og<em&gt; In vivo</em&gt; Overvågning af menneskerettigheder NK-celler fra humane embryonale stamceller (hESCs), og og induceret pluripotente stamceller (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Use of Human Perivascular Stem Cells for Bone Regeneration

Brug af menneskelige perivaskulær stamceller til Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

Alginat mikrokapselsammensætning som en 3D-platform for Formering og differentiering af humane embryonale stamceller (embryonale) til forskellige linier

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Transplantation af induceret pluripotente stamcelle-afledt Mesoangioblast-lignende Myogenic Stamfadere i Mouse Modeller af muskel regenerering

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Bestilling enkelte celler og Single Embryoner i 3D Indespærring: En ny enhed for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Effektiv Generation og redigering af Feeder-gratis IPSCs fra pancreas celler ved hjælp af CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Isolering af voksne humane Dermal fibroblaster fra abdominal hud og generering af inducerede pluripotente stamceller ved hjælp af en ikke-integrerings metode

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Skalerbar generation af modne cerebellar organoider fra humane pluripotente stamceller og karakterisering ved immunstaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Guidet differentiering af modne nyre podocytter fra menneskeskabte pluripotente stamceller under kemisk definerede forhold

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Single-Cell Optisk Action Potentiel måling i human induceret pluripotente stamcelle-afledte kardiomyocytter

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

En kultur metode til at opretholde hvilende humane hæmatopoietiske stamceller

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...