Name: clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture
Uploaded: 2018-05-08T13:00:00
Description: Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

Vi vil gjerne takke Andrew Berger, Stacey Dozono og Brian Raden i Fred Hutchinson Flow cytometri kjernen for hjelp med FACS. Dette arbeidet ble støttet av den nasjonale institutter for helse NHLBI UO1 grant #HL100395, tilknyttede samarbeidende Grant #HL099997, og NIDDK gir #RC2DK114777. Brandon Hadland støttes av Alex's Lemonade Stand Foundation og Hyundai Hope på hjul fundament.

Alle metodene som er beskrevet her er godkjent av institusjonelle Animal Care og bruk Committee (IACUC) av Fred Hutchinson Cancer Research Center.
1. utarbeidelse av AGM-EC Monolayers for co kultur
<ol><li>24 timer før generalforsamlingen disseksjon og Sorter gjøre AGM-EC kultur medier og filteret sterilisere.</li>
	<li>Legg 0,1% gelatin i vannet (100 µL/vel) hver brønn av en 96-brønns plate og ruge ved romtemperatur i 15 min. leveringstanken gelatin og la platen i vev kultur hette med ...

<ol>
	<li>Copley, M. R., Beer, P. A., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+heterogeneity+takes+center+stage.">Hematopoietic stem cell heterogeneity takes center stage.</a> Cell Stem Cell. 10 (6), 690-697 (2012).</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>Kumaravelu, 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=Quantitative+developmental+anatomy+of+definitive+haematopoietic+stem+cells/long-term+repopulating+units+(HSC/RUs):+role+of+the+aorta-gonad-mesonephros+(AGM)+region+and+the+yolk+sac+in+colonisation+of+the+mouse+embryonic+liver.">Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.</a> Development. 129 (21), 4891-4899 (2002).</li><li>Taoudi, 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=Extensive+hematopoietic+stem+cell+generation+in+the+AGM+region+via+maturation+of+VE-cadherin+CD45++pre-definitive+HSCs.">Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs.</a> Cell Stem Cell. 3 (1), 99-108 (2008).</li><li>Rybtsov, 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=Hierarchical+organization+and+early+hematopoietic+specification+of+the+developing+HSC+lineage+in+the+AGM+region.">Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.</a> J Exp Med. 208 (6), 1305-1315 (2011).</li><li>Rybtsov, 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=Tracing+the+origin+of+the+HSC+hierarchy+reveals+an+SCF-dependent,+IL-3-independent+CD43(-)+embryonic+precursor.">Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.</a> Stem Cell Reports. 3 (3), 489-501 (2014).</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>Lin, Y., Yoder, M. C., Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+progenitor+emergence+in+the+murine+embryo+and+yolk+sac+precedes+stem+cell+detection.">Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection.</a> Stem Cells Dev. 23 (11), 1168-1177 (2014).</li><li>Medvinsky, A., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+hematopoiesis+is+autonomously+initiated+by+the+AGM+region.">Definitive hematopoiesis is autonomously initiated by the AGM region.</a> Cell. 86 (6), 897-906 (1996).</li><li>Sheridan, J. M., Taoudi, S., Medvinsky, A., Blackburn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+generation+of+reaggregated+organotypic+cultures+that+permits+juxtaposition+of+defined+cell+populations.">A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.</a> Genesis. 47 (5), 346-351 (2009).</li><li>Yoder, M. C., Hiatt, K., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+repopulating+hematopoietic+stem+cells+are+present+in+the+murine+yolk+sac+at+day+9.0+postcoitus.">In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.</a> Proc Natl Acad Sci U S A. 94 (13), 6776-6780 (1997).</li><li>Arora, 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=Effect+of+developmental+stage+of+HSC+and+recipient+on+transplant+outcomes.">Effect of developmental stage of HSC and recipient on transplant outcomes.</a> Dev Cell. 29 (5), 621-628 (2014).</li><li>Butler, J. M., Rafii, S. <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+a+vascular+niche+for+studying+stem+cell+homeostasis.">Generation of a vascular niche for studying stem cell homeostasis.</a> Methods Mol Biol. 904, 221-233 (2012).</li><li>Butler, J. 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=Endothelial+cells+are+essential+for+the+self-renewal+and+repopulation+of+Notch-dependent+hematopoietic+stem+cells.">Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells.</a> Cell Stem Cell. 6 (3), 251-264 (2010).</li><li>Kobayashi, 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=Angiocrine+factors+from+Akt-activated+endothelial+cells+balance+self-renewal+and+differentiation+of+haematopoietic+stem+cells.">Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells.</a> Nat Cell Biol. 12 (11), 1046-1056 (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=Endothelium+and+NOTCH+specify+and+amplify+aorta-gonad-mesonephros-derived+hematopoietic+stem+cells.">Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.</a> J Clin Invest. 125 (5), 2032-2045 (2015).</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+Common+Origin+for+B-1a+and+B-2+Lymphocytes+in+Clonal+Pre-+Hematopoietic+Stem+Cells.">A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.</a> Stem Cell Reports. 8 (6), 1563-1572 (2017).</li><li>Wilson, N. 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=Combined+Single-Cell+Functional+and+Gene+Expression+Analysis+Resolves+Heterogeneity+within+Stem+Cell+Populations.">Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.</a> Cell Stem Cell. 16 (6), 712-724 (2015).</li><li>Schulte, 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=Index+sorting+resolves+heterogeneous+murine+hematopoietic+stem+cell+populations.">Index sorting resolves heterogeneous murine hematopoietic stem cell populations.</a> Exp Hematol. 43 (9), 803-811 (2015).</li><li>Morgan, K., Kharas, M., Dzierzak, E., Gilliland, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+early+hematopoietic+stem+cells+from+murine+yolk+sac+and+AGM.">Isolation of early hematopoietic stem cells from murine yolk sac and AGM.</a> J Vis Exp. (16), (2008).</li><li>deBruijn, M. F., 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=Hematopoietic+stem+cells+localize+to+the+endothelial+cell+layer+in+the+midgestation+mouse+aorta.">Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta.</a> Immunity. 16 (5), 673-683 (2002).</li><li>Lorsbach, R. 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=Role+of+RUNX1+in+adult+hematopoiesis:+analysis+of+RUNX1-IRES-GFP+knock-in+mice+reveals+differential+lineage+expression.">Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression.</a> Blood. 103 (7), 2522-2529 (2004).</li><li>Holmes, R., Z&#250;&#241;iga-Pfl&#252;cker, 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), pdb.prot5156 (2009).</li><li>Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+wave+of+B+lymphopoiesis+develops+independently+of+stem+cells+in+the+murine+embryo.">The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo.</a> Ann N Y Acad Sci. 1362, 16-22 (2015).</li><li>Kobayashi, 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=Functional+B-1+progenitor+cells+are+present+in+the+hematopoietic+stem+cell-deficient+embryo+and+depend+on+Cbf&#946;+for+their+development.">Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbf&#946; for their development.</a> Proc Natl Acad Sci U S A. 111 (33), 12151-12156 (2014).</li><li>Yoshimoto, 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=Autonomous+murine+T-cell+progenitor+production+in+the+extra-embryonic+yolk+sac+before+HSC+emergence.">Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.</a> Blood. 119 (24), 5706-5714 (2012).</li><li>Inlay, M. 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=Identification+of+multipotent+progenitors+that+emerge+prior+to+hematopoietic+stem+cells+in+embryonic+development.">Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development.</a> Stem Cell Reports. 2 (4), 457-472 (2014).</li><li>Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell-independent+hematopoiesis:+emergence+of+erythroid,+megakaryocyte,+and+myeloid+potential+in+the+mammalian+embryo.">Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo.</a> FEBS Lett. 590 (22), 3965-3974 (2016).</li></ol>

Studiet av HSC genesis under embryonale utviklingen nødvendiggjør måte å oppdage HSC i hemogenic forløpere ennå manglet kompetanse til å gi langsiktig multilineage blodkreft rekonstituering i transplantert voksen mottakere. I denne protokollen presenterer vi en klonal analysen av embryonale hemogenic prekursorer av stromal co kultur på vaskulær nisje ECs fra generalforsamlingen, som støtter modning av forløpere til HSC, med påfølgende funksjonell analyse i transplantasjon analyser. Innlemmelse av indeks sort...

Klonal undersøkelser avdekket heterogenitet i egenskapene for langsiktige engraftment for voksen HSCs, gir ny innsikt i HSC subtyper og endringer i HSC virkemåte under aldring1. Men har lignende studier av embryonale HSCs og deres forløpere vært mer utfordrende. Under embryonale utviklingen, HSCs oppstå fra en populasjon av prekursorer kjent som hemogenic endotelet i en forbigående prosess referert til som den endothelial blodkreft overgang2. Den første HSC definert av deres evne til å gi robust, langsiktig multilineage engraftment etter transplantasjon i betinget voksen mottakere, oppdages ikke før etter embryonale dag 10.5 (E10.5) i murine embryo, på veldig lav frekvens3 . I løpet av deres utvikling, må forløpere til HSC (pre-HSC) som oppstår fra hemogenic endotelet gjennomgå modning tidligere å erverve egenskaper for voksen HSC som gir mulighet for effektiv engraftment i transplantasjon analyser4,5 , 6. skjule studiet av sjeldne HSC opprinnelse, en rekke blodkreft progenitors med erythroid, myelogen og lymfoide potensialet er allerede oppdaget før fremveksten av HSC pre-HSC7,8. Dermed krever skille pre-HSC fra andre blodkreft progenitors metoder å isolere clonally cellene og gi dem signaler tilstrekkelig for deres modning til HSC å oppdage engraftment egenskapene i transplantasjon analyser.
Flere tilnærminger har blitt beskrevet som tillater for påvisning av pre-HSC av ex vivo eller i vivo modningsprosess for HSC. Ex vivo metoder har avhengig kultur embryonale vev, som generalforsamlingen regionen, hvor den første HSC oppdages i utvikling9. Bygge på disse metodene, protokoller som innlemme dissosiasjon, har sortering og ny samling av generalforsamlingen vev tillatt karakterisering av sorterte populasjoner som inneholder HSC forløpere under utviklingen fra E9.5 til E11.5 i para-aorta splanchnopleura (P-Sp) / generalforsamlingen regioner4,5,10; men er disse ikke mottakelig for høy gjennomstrømming analyse av prekursorer på enkeltcelle nivå kreves for klonal analyse. Tilsvarende i vivo modning av transplantasjon i nyfødte mus, hvor microenvironment antas å være mer egnet til støtte for tidligere stadier av HSC forløpere, har aktivert studier av sorterte befolkningsgrupper fra eggeplomme sac og generalforsamlingen / P-Sp (P-Sp er regionen forløperen til generalforsamlingen) med karakteristikker av pre-HSC, men disse metodene også mislykkes å gi en solid plattform for enkelt celle analyse11,12.
Studier fra Rafii et al. vist at Akt-aktivert endothelial celle (EC) stroma kan gi en nisje substrat for støtte av voksen HSC selvtillit fornyelse i vitro13,14,15. Vi har nylig funnet ut at Akt-aktivert EC avledet fra regionen generalforsamling (GF-EC) gir en egnet i vitro nisje for modning av hemogenic forløpere, isolert så tidlig som E9 i utvikling, til voksen-engrafting HSC, samt den etterfølgende selvtillit fornyelse genererte HSC16. Gitt at dette systemet bruker en enkel 2-dimensjonal co kultur, er det lett tilpasses klonal analyse av HSC potensialet i individuelt isolert hemogenic prekursorer.
Vi har nylig rapportert en tilnærming til analysen HSC potensialet i klonal hemogenic forløpere ved å kombinere indeks sortering av personlige hemogenic prekursorer fra murine embryo med AGM-EC co kultur og påfølgende funksjonell analyse i transplantasjon Søk17. Indeks sortering er en modus av fluorescens-aktivert celle sortering (FACS) som poster (indekser) alle fenotypiske parametere (dvs.frem xy (FSC-A), siden xy (SSC-A), fluorescens parametere) av hver individuelt sorterte celle slik at disse funksjoner kan samsvares ettertid med påfølgende funksjonell analyse etter sortering. FACS programvaren registrerer både fenotypiske informasjon for hver celle og posisjon/vel i 96-brønnen platen der den ble plassert. Denne teknikken har tidligere blitt elegant brukt til å identifisere heterogene i voksen HSC, bestemme fenotypiske parametere som ytterligere berike for langsiktig engrafting delsettet med HSC og relatere fenotypiske parametere av HSC med transcriptional egenskaper ved singelen celle nivå18,19. Her gir vi detaljerte metodikk i denne tilnærmingen som gjør ID unik fenotypiske parametere og avstamning bidrag av pre-HSC i tidlige stadier av embryonale utvikling.

<table>
	<tbody>
		<tr>
			<td>AGM-EC culture media </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for culture of endothelial cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td>Use to dissociate AGM-EC monolayers</td>
		</tr>
		<tr>
			<td>Gelatin 0.1% in water</td>
			<td>StemCell Technologies</td>
			<td>7903</td>
			<td>Use to adhere AGM-EC monolayers to plastic</td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td>Use to co-culture AGM-EC monolayers and index sorted clones</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Use for dissection and FACS staining</td>
		</tr>
		<tr>
			<td>500 ml filter bottles</td>
			<td>Fisher Scientific</td>
			<td>9741202</td>
			<td>Use to sterile filter Endothelial media</td>
		</tr>
		<tr>
			<td>AGM-EC culture media (500 mls)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td>400 mls</td>
		</tr>
		<tr>
			<td>Hyclone Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH30088.03</td>
			<td>100 mls</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>5 mls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td>50 mg dissolved in 10 mls media</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>StemCell Technologies</td>
			<td>7100</td>
			<td>5 mls</td>
		</tr>
		<tr>
			<td>Endothelial Mitogen (ECGS)</td>
			<td>Alfa Aesar</td>
			<td>J64516 (BT-203)</td>
			<td>Add 10 mls media to dissolve and add</td>
		</tr>
		<tr>
			<td>Materials for AGM index sort</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (0.25%)</td>
			<td>StemCell Technologies</td>
			<td>7902</td>
			<td>Use for dissociation of embryonic tissues</td>
		</tr>
		<tr>
			<td>3ml syringe</td>
			<td>BD Biosciences</td>
			<td>309657</td>
			<td>Use to sterile filter antibody solutions for FACS</td>
		</tr>
		<tr>
			<td>0.22 &#181;M syringe-driven filter</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td>Use to sterile filter antibody solutions for FACS</td>
		</tr>
		<tr>
			<td>5 ml polystyrene tube with cell-strainer cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>Use to remove cell clumps prior to FACS</td>
		</tr>
		<tr>
			<td>DAPI (prepared as 1 mg/ml stock in H2O)</td>
			<td>Millipore</td>
			<td>268298</td>
			<td>Use to exclude dead cells from sort</td>
		</tr>
		<tr>
			<td>anti-mouse CD16/CD32 (FcR block)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>Use to block non-specific staining to Fc receptors</td>
		</tr>
		<tr>
			<td>Antibodies for AGM index sort</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD144 PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-1441-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG1 kappa Isotype Control, PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4301-81</td>
			<td>Isotype control for CD144 PE-Cyanine7</td>
		</tr>
		<tr>
			<td>Anti-mouse CD201 (EPCR) PerCP-eFluor710</td>
			<td>eBioscience</td>
			<td>46-2012-80</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PerCP-eFlour710</td>
			<td>eBioscience</td>
			<td>46-4031-80</td>
			<td>Isotype control for CD201 PerCP-eFluor710</td>
		</tr>
		<tr>
			<td>Anti-mouse CD41 PE</td>
			<td>BD Biosciences</td>
			<td>558040</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG1 kappa Isotype Control, PE</td>
			<td>BD Biosciences</td>
			<td>553925</td>
			<td>Isotype control for CD41 PE</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 FITC</td>
			<td>eBioscience</td>
			<td>11-0451-85</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, FITC</td>
			<td>eBioscience</td>
			<td>11-4031-81</td>
			<td>Isotype control for CD45 FITC</td>
		</tr>
		<tr>
			<td>AGM-serum free media (10mls)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-Vivo 20</td>
			<td>Lonza</td>
			<td>04-448Q</td>
			<td>10 mls</td>
		</tr>
		<tr>
			<td>recombinant murine stem cell factor (SCF)</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>recombinant human FLT3 Ligand (FLT3L)</td>
			<td>Peprotech</td>
			<td>300-19</td>
			<td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td>
		</tr>
		<tr>
			<td>recombinant human thrombopoietin (TPO)</td>
			<td>Peprotech</td>
			<td>300-18</td>
			<td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td>
		</tr>
		<tr>
			<td>recombinant murine interleukin-3 (IL3)</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td>
		</tr>
		<tr>
			<td>Materials for Staining co-cultured cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate, V-bottom</td>
			<td>Corning</td>
			<td>3894</td>
			<td>Use for FACS analysis</td>
		</tr>
		<tr>
			<td colspan="3">Antibodies for analysis of Index sorted clones co-cultured with endothelial cells</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PerCP-Cyanine5.5</td>
			<td>eBioscience</td>
			<td>45-0451-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PE-Cyanine5.5</td>
			<td>eBioscience</td>
			<td>35-4031-80</td>
			<td>Isotype control for CD45 PerCP-Cyanine5.5</td>
		</tr>
		<tr>
			<td>Anti-mouse CD201 (EPCR) PE</td>
			<td>eBioscience</td>
			<td>12-2012-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PE</td>
			<td>eBioscience</td>
			<td>12-4031-81</td>
			<td>Isotype control for CD201 PE</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6A/E (Sca-1) APC</td>
			<td>eBioscience</td>
			<td>17-5981-83</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, APC</td>
			<td>eBioscience</td>
			<td>17-4321-81</td>
			<td>Isotype control for Ly-6A/E APC</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 FITC</td>
			<td>eBioscience</td>
			<td>11-4801-81</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, FITC</td>
			<td>eBioscience</td>
			<td>11-4321-41</td>
			<td>Isotype control for F4/80 FITC</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6G/C (Gr1) FITC</td>
			<td>BD Biosciences</td>
			<td>553127</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, FITC</td>
			<td>BD Biosciences</td>
			<td>556923</td>
			<td>Isotype control for Ly-6G/C and CD3 FITC</td>
		</tr>
		<tr>
			<td>Materials for tail vein injection for transplant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 ml insulin syringes with 29G 1/2&#34; needles</td>
			<td>BD Biosciences</td>
			<td>309306</td>
			<td>Use for tail vein injection for transplantation</td>
		</tr>
		<tr>
			<td>Antibodies for Peripheral Blood analysis following translant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6G/C (Gr1) PerCP</td>
			<td>Biolegend</td>
			<td>108426</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2b kappa Isotype Control, PerCP</td>
			<td>Biolegend</td>
			<td>400629</td>
			<td>Isotype control for Ly-6G/C PerCP</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 PE</td>
			<td>eBioscience</td>
			<td>12-4801-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, PE</td>
			<td>eBioscience</td>
			<td>12-4321-80</td>
			<td>Isotype control for F4/80 PE</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 FITC</td>
			<td>BD Biosciences</td>
			<td>555274</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 APC</td>
			<td>BD Biosciences</td>
			<td>550992</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, APC</td>
			<td>BD Biosciences</td>
			<td>553932</td>
			<td>Isotype control for CD19 APC</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45.1 (A20) PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0453-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4321-81</td>
			<td>Isotype control for CD45.1 PE-Cyanine7</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45.2 (104) APC-eFluor780</td>
			<td>eBioscience</td>
			<td>47-0454-82</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Rat IgG2a kappa Isotype Control, APC-eFluor780</td>
			<td>eBioscience</td>
			<td>47-4321-80</td>
			<td>Isotype control for CD45.2 APC-eFluor780</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria II with DIVA software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II with plate reader</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td>Fisher Scientific</td>
			<td>S17040</td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>Multi-channel pipette</td>
			<td>Fisher Scientific</td>
			<td>14-559-417</td>
			<td>For dispensing cells</td>
		</tr>
		<tr>
			<td>FACS analysis software</td>
			<td>FlowJo/BD Biosciences</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
	</tbody>
</table>

clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture

Muligheten til å studere blodkreft stem cell (HSC) genesis under embryonale utviklingen har vært begrenset av sjeldenhet av HSC forløpere i tidlig embryo og mangel på analyser som funksjonelt identifiserer langsiktige multilineage engraftment potensialet i individuelle antatte HSC prekursorer. Her beskriver vi metodikk som gjør at isolering og karakterisering av funksjonelt validert HSC forløpere på enkeltcelle nivå. Først bruke vi indeksen sortering for å katalogisere presis fenotypiske parameteren for hver individuelt sorterte celle, med en kombinasjon av fenotypiske markører for å berike for HSC forløpere med ekstra markører for eksperimentanalyse. Andre er hver indeks-sortert celle co kulturperler med vaskulær nisje stroma fra aortabuen-gonad-mesonephros (GF) regionen, som støtter modning av ikke-engrafting HSC forløpere til funksjonelle HSC med multilineage, langsiktige engraftment i transplantasjon analyser. Denne metoden gjør det mulig for korrelasjon av fenotypiske for klonal hemogenic forløpere med sine funksjonelle engraftment potensial eller andre egenskaper som transcriptional profil, sørge for detaljert analyse av HSC forløper utvikling på enkeltcelle nivå.

Figur 1A viser en skjematisk av eksperimentell design. Når P-Sp/AGM vev dissekert, samlet og avstand i collagenase, er de farget med antistoffer mot VE-Cadherin og EPCR for indeks sortering. Pre-HSC er beriket i celler sortert på VE-Cadherin+EPCRhøy (figur 1B). Andre fluorochrome-konjugerte antistoffer kan inkluderes ettertid analysere flere fenotypiske parametere, som er registrert for hver celle i indek...

Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Klonal analyse av embryonale stamcelleforskningen blodkreft forløpere bruke enkeltcelle indeks sortering kombinert med endotelial celle nisje co kultur

Her presenterer vi metodikk for klonal analyse av blodkreft stamcelletransplantasjon forløpere under murine embryonale utviklingen. Vi kombinerer indeks sortering av enkeltceller fra embryonale aorta-gonad-mesonephros-regionen med endothelial celle co kultur og transplantasjon som karakteriserer fenotypiske egenskaper og engraftment potensialet i enkelt blodkreft forløpere.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

The overall goal of this method is to identify the phenotypic properties and engraftment potential of clonal Hematopoietic Stem Cell or HSC precursors during murine embryonic development. This method can help answer key questions in the field of developmental hematopoiesis about the unique lineage contributions of clonal HSC precursors during embryonic development. The main advantage of this technique is that hemogenic precursors are analyzed at the single cell level.This allows identification of the unique phenotypic properties that distinguish rare HSC precursors. Begin by adding Aorta-Gonad-Mesonephros, AGM, tissues dissected from embryos between 9.5 and 11.5 days post coitum to a 15 milliliter conical tube containing 10 milliliters of PBS supplemented with 10%FBS on ice. When the tissues have settled to the bottom of the tube, aspirate the supernatant and immediately add one milliliter of 0.25%collagenase to the samples.After 25 minutes in a 37 degree Celsius water bath, stop the reaction with one milliliter of PBS plus FBS and use a one milliliter pipette tip to triturate the tissues 20 to 30 times until a single cell suspension is obtained. Then dilute the cells with eight more milliliters of PBS plus FBS and collect the cells by centrifugation. Resuspend the pellet in 500 microliters of blocking buffer for five minutes on ice.Then add 500 microliters of the antibody cocktail of interest. After a 15 to 20-minute incubation on ice, wash the cells in eight milliliters of PBS plus FBS and resuspend the pellet in one milliliter of fresh PBS plus FBS. Filter the cells through a 35 micrometer cell strainer into a five milliliter tube, washing the strainer with an additional three milliliters of PBS plus FBS to collect all of the cells and centrifuge the cells again.Then resuspend the pellet in 500 microliters of PBS plus FBS and place the cells on ice. To set the gates for sorting in single cell and index sorting mode, load a small aliquot of cells onto the flow cytometer and set the acquisition to the lowest flow rate. Open a forward scatter area versus side scatter area plot and set a gate to include cells of varying size.Open a forward scatter area versus forward scatter width plot and a side scatter area versus side scatter width plot and set a gate in each plot to exclude the doublets. Set a forward scatter area versus DAPI plot to gate the live cells. Then create an anti-vascular endothelial Cadherin versus anti-endothelial protein C receptor plot and gate the vascular endothelial Cadherin positive and high endothelial protein C receptor expressing cells.To index sort the aorta-gonad-mesonephros cells, place a 96-well plate precultured with AGM endothelial cells in AGM serum-free medium plus cytokines into the plate block of the flow cytometer and load the experimental sample. Begin the sample acquisition and confirm that the index sorting single cell mode is selected to sort a single cell into each well of the 96-well plate. When one cell has been sorted into each well of the plate, place the plate into a tissue culture incubator set to 37 degrees Celsius and 5%CO2 for up to seven days.At the end of the co-culture period, hematopoietic colonies of various sizes and morphologies can be visualized using an inverted microscope at a 100X magnification. To analyze the clones by flow cytometry, use a multichannel pipette to dissociate the hematopoietic cells from the endothelial layers without detaching the endothelial cells. Then transfer 100 microliters of cells into individual wells of a new 96-well v-bottom plate and return the original 96-well plate containing the remaining 100 microliters of cells to the tissue culture incubator.Pellet the cells in the v-bottom plate by centrifugation and remove the supernatant by inverting and flicking the plate in a single rapid motion. Resuspend the pellets in each well with 50 microliters of blocking buffer at four degrees Celsius. After five minutes, add the antibody cocktail of interest to each well for a 20-minute incubation at four degrees Celsius.At the end of the incubation, wash each well with 200 microliters of PBS plus FBS, flicking the plate at the end of the centrifugation to discard the supernatant. Resuspend the pellets in 50 microliters of PBS plus FBS and analyze the cells according to standard flow analysis protocols to screen for colonies that contain hematopoietic stem cell potential. Selecting the wells that contain cells with hematopoietic stem cell phenotype by flow analysis, use a multichannel pipette to resuspend the remaining 100 microliters of cells from the original co-culture and add five times 10 to the fourth rescue bone marrow cells in 100 microliters of PBS plus 2%FBS to each well.After adoptive transfer of each clone into individual lethally irradiated congenic recipient animals, collect peripheral blood samples from the engrafted animals at regular intervals post transplant. To identify the clones with long-term engraftment, analyze the samples by flow cytometry according to standard flow analysis protocols. The phenotypic properties of each clone can then be correlated with its engraftment properties.Pre-hematopoietic stem cells can be enriched by vascular endothelial Cadherin positivity and a high level of endothelial protein C receptor expression. Staining with other fluorochrome conjugated antibodies allows the retrospective analysis of additional phenotypic parameters that can be recorded for each cell during index sorting. Following index sorting into aorta-gonad-mesonephros endothelial cell co-cultures, the individual clones integrate into the aorta-gonad-mesonephros endothelial cell layer before the clones begin to round up and form hematopoietic clusters.After five to seven days of co-culture, colonies of various sizes and morphologies can be detected. Here, four different colonies obtained following co-culture are shown with three colonies demonstrating different proportions of phenotypic hematopoietic stem cells along with other more differentiated cell types and the fourth colony lacking cells with hematopoietic stem cell marker expression. Adoptive transfer of the clone cell progeny allows assessment of the long-term multi-lineage engraftment capability of each clone following donor contribution to the myeloid B and T cell compartments of the peripheral blood of the recipient over time.Although most colonies containing cells with a hematopoietic stem cell phenotype achieved long-term engraftment, transplantation is necessary to confirm a functional multi-lineage engraftment as some clones lose engraftment over time. The functional hematopoietic stem cell potential of each clone can then be correlated back to that clone's original index sorting parameters to identify its precise phenotypic characteristics. Following this procedure, other methods like secondary culture of hematopoietic progeny on stroma supporting B and T cell development in vitro can be performed to answer additional questions about the lymphocyte potentials of individual hemogenic precursors.

Research

JoVE Journal

Developmental Biology

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Isolation of Murine Embryonic Hemogenic Endothelial Cells

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Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

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