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

우리는 프레드 허 친 슨 흐름 Cytometry 코어에 FACS에 앤드류 버거, 스테이 시 Dozono, 그리고 브라이언 라덴을 감사 하 고 싶습니다. 이 작품 보조 공동 그랜트 #HL099997, 그리고 NIDDK 부여 #RC2DK114777 #HL100395, 건강 NHLBI UO1의 국가 학회 교부 금에 의해 지원 되었다. 브랜든 Hadland 알렉스의 레모네이드 스탠드 재단 희망과 현대 바퀴 기초에 의해 지원 됩니다.

여기에 설명 된 모든 메서드는 기관 동물 관리 및 사용 위원회 (IACUC)의 프레드 허 친 슨 암 연구 센터에 의해 승인 되었습니다.
1. AGM-EC Monolayers 공동 문화에 대 한 준비
<ol><li>24 시간 이전에 관련해 해 부 정렬, AGM-EC 문화 미디어와 소독 필터를 확인 합니다.</li>
	<li>물 (100 µ L/잘)에 0.1% 젤라틴 96 잘 접시의 각 음을 추가 하 고 15 분 Aspirate는 젤라틴에 대 한 실 온에서 품 어 두고 ...

<ol>
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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. 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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>

배아 개발 HSC 창세기 연구 HSC hemogenic 선구자 아직 이식 성인 받는 사람의 장기 multilineage 조 혈 재구성을 제공 하기 위해 능력 부족에서 가능성을 감지 하는 수단을 필요로 합니다. 이 프로토콜에서 선물이 배아 hemogenic 선구자의 클론 분석 결과 혈관 틈새 ECs stromal 공동 문화 지원 HSC에 선구자의 성숙, AGM에서 이식 분석 실험에서 후속 기능 분석. 허가 정렬 하는 인덱스의 phenotypic 매개 변수 사전-HSC ?...

클론 연구 노화1동안 HSC 하위 및 HSC 행동의 변화에 새로운 통찰력을 제공 하는 성인 HSCs의 장기 engraftment 속성에서이 밝혀졌다. 그러나, 배아 HSCs의 비슷한 연구와 그들의 선구자 되었습니다 더 많은 도전. HSCs 과도 과정에서 내 피 hemogenic 라고 알려진 선구자의 인구에서 발생 하는 초기 배아 개발 중으로 조 혈 전환2내 피. 강력 하 고, 장기 이식 조건된 성인 받는 사람에 따라 multilineage engraftment를 제공 하는 그들의 능력에 의해 정의 된 첫 번째 HSC 배아 이날 10.5 (E10.5) murine 태아에 매우 낮은 주파수3 이후까지 검색 되지 않습니다. . 그들의 발전 동안 3300 HSC (pre-HSC) hemogenic 피에서 발생 하는 이식 분석 실험4,5 에서 효율적인 engraftment 허용 성인 HSC의 속성을 획득 하기 전에 성숙 겪어야 한다 , 6. 희귀 HSC 기원, 다양 한 erythroid와 조 혈 창시자의 연구를 왜곡 골수성, 림프 잠재력 사전-HSC7,8에서 HSC의 출현 전에 이미 감지는. 따라서, 다른 조 혈 창시자에서 사전-HSC 구별 방법 clonally 세포를 분리 하 여 이식 분석 실험에서 engraftment 속성을 검색 하기 위해 HSC에 그들의 성숙에 대 한 충분 한 신호를 제공 필요 합니다.
다양 한 접근 HSC에 비보 전 또는 vivo에서 성숙 하 여 사전-HSC의 검출을 허용 하는 기술 되었다. 방법을 ex vivo는 첫 번째 HSC 개발9에서 감지 되는 AGM 지역과 같은 배아 조직의 문화에 달려 있었다. 이 방법, 분리, 통합 하는 프로토콜에 정렬, AGM 조직의 재 집계는 허용 파라 대동맥에 E11.5 E9.5에서 개발 하는 동안 HSC 선구자를 포함 하는 정렬 된 인구의 특성 splanchnopleura (P-Sp) / 관련해 지역4,,510; 그러나, 이러한 방법 클론 분석에 필요한 단일 세포 수준에서 일어나 높은 처리량 분석에 순종 하지 않습니다. 마찬가지로, 신생아 쥐에 이식 하 여 성숙 vivo에서 어디는 microenvironment HSC 선구자의 초기 단계 지원을 위해 더 적합 한 것으로 추정 되는 또한 활성화 노른자위 sac 및 AGM에서 정렬 된 인구의 연구 / P-Sp (P-Sp는 AGM에 선구자 지역) 사전-HSC, 하지만이 방법의 특성을 가진 또한 단일에 대 한 강력한 플랫폼을 제공 하는 실패 세포 분석11,12.
Rafii 외 에서 연구 보여주었다 그 내 피 세포 Akt 활성화 (EC) 기질 성인 HSC 자체 갱신 생체 외에서13,,1415의 지원에 대 한 틈새 기질을 제공할 수 있습니다. 우리는 최근 Akt 활성화 EC 관련해 지역 (AGM-EC)에서 파생 된 hemogenic 선구자, E9 성인 engrafting HSC 개발에서 뿐만 아니라 후속 빠르면 절연의 성숙에 대 한 적합 한 생체 외에서 틈새를 제공 한다 결정 생성 된 HSC16의 자체 갱신. 감안할 때이 시스템 사용 간단한 2 차원 공동 문화, 그것은 개별적으로 고립 된 hemogenic 선구자의 HSC 잠재력의 클론 분석을 위해 쉽게 적응.
우리는 최근 클론 hemogenic 선구자의 HSC 잠재력 AGM-EC 공동 문화 및 이식에서 이후의 기능 분석 murine 배아에서 개별 hemogenic 선구자의 인덱스 정렬 결합 하 여 분석 결과에 대 한 접근을 보고 17분석 실험. 인덱스 정렬 세포 형광 활성화의 모드 phenotypic의 모든 매개 변수 (즉, 앞으로 분산형 (FSC-A), 측면 살포 (SSC-A), 형광 매개 변수) 각 개별적으로 정렬된 셀 같은 정렬 (FACS) (인덱스)를 기록 하는이 기능 소급 정렬 다음 후속 기능적 분석 상관 될 수 있다. FACS 소프트웨어 위치/잘 두었다는 96 잘 접시의 각 셀에 대 한 두 phenotypic 정보를 기록 합니다. 이 기술은 이전 우아하게 사용 되었습니다 성인 HSC에서이 확인, 추가 HSC, 장기 engrafting 하위 집합에 대 한 풍부 하 고 연관 HSC의 phenotypic 매개 변수 transcriptional phenotypic 매개 변수 결정 단일 속성 셀 레벨18,19. 여기, 우리는 배아 개발의 초기 단계 동안 독특한 phenotypic 매개 변수 식별 및 사전-HSC의 혈통 기여를 가능 하 게이 방법의 상세한 방법론을 제공 합니다.

<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

배아 개발 하는 동안 조 혈 줄기 세포 (HSC) 창세기를 공부 하는 능력은 초기 배아에 HSC 선구자의 희귀 한 고 기능의 장기 multilineage engraftment 잠재력을 식별 하는 분석 실험의 부족에 의해 제한 되어 개별 putative HSC 선구자입니다. 여기, 우리가 격리와 단일 세포 수준에서 기능적으로 검증된 HSC 선구자의 방법론을 설명 합니다. 첫째, 우리 활용 개별적으로 정렬 된 각 셀의 정확한 phenotypic 매개 변수 카탈로그 인덱스 정렬 phenotypic 감 적의 조합 실험 분석에 대 한 추가 마커 HSC 선구자에 대 한 풍부 하 게 사용 하 여. 둘째, 각 인덱스 정렬 셀이 공동 지 원하는 multilineage, 장기 engraftment에서 가능성과 기능성 HSC에 HSC 선구자 비 engrafting의 성숙 대동맥-생식-mesonephros (AGM) 지역에서 혈관 틈새 기질과 교양 이식 분석 실험입니다. 이 방법을 수 있도록 그들의 기능적인 engraftment 잠재력 클론 hemogenic 선구자의 phenotypic 속성 또는 HSC 전조의 상세한 분석을 위한 수단을 제공 하는 transcriptional 프로필 등의 다른 속성의 상관 관계 단일 세포 수준에서 개발입니다.

그림 1A 는 실험적인 디자인의 회로도 보여준다. P-Sp/관련해 조직 해 부, 풀링, 그리고 콜라에 해리, 일단 그들은 인덱스 정렬에 대 한 VE Cadherin와 EPCR 항 체와 얼룩이 있습니다. 사전-HSC VE Cadherin+EPCR높은 (그림 1B)에 정렬 셀 농축 됩니다. 다른 형광 색소 활용 된 항 체는 소급 인덱스 정렬 하는 동안 각 셀에 대 한 ?...

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

단일 셀 인덱스 정렬 내 피 세포 틈새 공동 문화 결합을 사용 하 여 배아 조 혈 줄기 세포 선구자의 클론 분석

여기 우리 murine 배아 개발 하는 동안 조 혈 줄기 세포 선구자의 클론 분석을 위한 방법론을 제시. 우리는 내 피 세포 공동 문화 및 phenotypic 속성 및 단일 조 혈 일어나 engraftment 잠재력 하 이식 배아 대동맥-생식-mesonephros 지역에서 단일 셀의 인덱스 정렬 결합.

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.

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쥐 배아 Hemogenic 내피 세포의 분리

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

경쟁 이식은 조혈 줄기 세포의 체력을 평가하는

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

인간의 배아 또는 유도 만능 줄기 세포에서 망막 안료 상피 세포의 급속 한, 감독 차별화

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

전체 산 Epifluorescence를 사용 하 여 마우스 Embryogenesis 동안 심장 챔버의 분석

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...