This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1 배아 바디 (EB) 형성 <ol><li> 마우스 배아 섬유 아 세포 (MEFs)에 유지 된 ESCS을 젤라틴 - 적응. 통로 ESCS MEFs을 제거하기 위해 0.1 % 젤라틴으로 코팅 여섯 잘 조직 배양 접시에 세 번. <ol><li> (표 1) 미분화 세포를 유지하기 위해 함께 LIF ESC 유지 매체를 세포 성장. 확인 세포가 결코 80 %의 합류를 초과하지합니다. 분화 세포 (MEFs에서 제거한 후 10 이하 통로) 낮은 통로를 사용합니다. </li></ol></li><li> ESCS는 EB의 분화 배양되기 전에 날, 통로 세포는 그래서 그들은 약 50-70% 합류 다음 날이 될 것입니다. 세포의 다 능성을 유지하는 ESC 유지 배지에서 세포 배양을 계속한다. </li><li> 6 웰 플레이트 잘 당 : 분화, 흡인 ESC 유지 보수 매체와의 일에 1 ㎖의 인산염 완충 생리 식염수 (137 mM의 염화나트륨, 2.7 밀리미터의 KCl, 11.9 mM의 인산염 완충액 pH 7.4의 PBS)으로 씻는다. <ol><li> 0.25 %의 tryps의 200 μl를 추가/ 각 EDTA 잘에서 3 분 동안 37 ° C에서 배양한다. </li><li> 차별화 매체의 800 μL와 트립신 / EDTA를 비활성화합니다. 피펫 위아래 P200 팁 2 ㎖의 혈청 학적 피펫에서 휴식 - 최대 세포를 단일 세포 현탁액으로. 미디어 피펫 팁 인터페이스 사이에 이동되지 않도록 P200 팁 끝에 딱 맞는 있는지 확인합니다. </li><li> 피펫은 위아래로 약 5 ~ 10 배. 피펫 팅하는 동안 거품을 만들 않도록주의하십시오. </li></ol></li><li> 15 ML 원뿔 튜브에 세포를 전송합니다. PBS 10 ML에 볼륨을 가져와. 실온에서 5 분 동안 315 XG에 원심 분리기. </li><li> 상층 액을 제거합니다. LIF를 포함 남은 용지를 제거하는 5 ㎖의 PBS로 두 번 세척 할 것. 각 세척 단계와 1.4에서와 같이 원심 분리기. <ol><li> 분화 매체 (표 2) 2 ㎖의 최종 세포 펠렛을 재현 탁. </li></ol></li><li> 혈구 또는 셀 카운터 세포를 세어 10cm 페트리 접시에 6,000-10,000 세포 / ml를 판(비 조직 배양 치료). <ol><li> 각 ESC 라인에 대한 경험적으로 정확한 세포 / ml를 결정합니다. 일반적으로 세균 작업 또는 낮은 부착 판에 사용되는 멸균 페트리 접시를 사용합니다. 주 : 최상의 차별화 결과, 사채가 판에 연결하지 않고도 정지에 남아있는 구체를 형성해야한다. 최소한의 준수와 사람을 찾기 위해 판의 여러 브랜드를 테스트합니다. </li><li> 5 % CO 2와 37 ° C 조직 문화 인큐베이터에서 차별화 ESCS을 품어. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> 시약 </td><td> 주식 </td><td> 최종 농도 </td><td> 볼륨 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> 1 배 </td><td></td><td> 410 ml의 </td><td> S<html> IGMA / 알드리치 </td><td> D5796 </td></tr><tr><td> FBS (ES 선별) </td><td> 백퍼센트 </td><td> 15 % </td><td> 75 ML </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> 비 필수 아미노산 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 11140 </td></tr><tr><td> L-글루타민 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 35050 </td></tr><tr><td> Penncillin / 스트렙토 마이신 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 15070 </td></tr><tr><td> β-머 캅토 에탄올 </td><td> 14.3 M </td><td> 114 μM </td><td> 4 μL </td><td> 시그마 / 알드리치 </td><td> M3148 </td></tr><tr><td> 백혈병 억제 인자 (LIF) </td><td> 10 7 단위 / ㎖ </td><td> 1,000 개 / ㎖ </td><td> 50 μL </td><td> 밀리 포아 </td><td> ESG1107 </td></tr></tbody></table>&quot;ontent&gt; 표 1 : ESC 유지 미디어. <table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> 시약 </td><td> 주식 </td><td> 최종 농도 </td><td> 볼륨 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> 1 배 </td><td></td><td> 410 ml의 </td><td> 시그마 / 알드리치 </td><td> D5796 </td></tr><tr><td> FBS (사용자가 최적의 차별화에 대한 검사) </td><td> 백퍼센트 </td><td> 15 % </td><td> 75 ML </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> 비 필수 아미노산 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 11140 </td></tr><tr><td> L-글루타민 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> 생활 TECHNOLOGIES </td><td> 35050 </td></tr><tr><td> Penncillin / 스트렙토 마이신 </td><td> 100 배 </td><td> 1 배 </td><td> 5 ML </td><td> Life 기술 </td><td> 15070 </td></tr><tr><td> β-머 캅토 에탄올 </td><td> 14.3 M </td><td> 114 μM </td><td> 4 μL </td><td> 시그마 / 알드리치 </td><td> M3148 </td></tr></tbody></table> 표 2 : 차별화 미디어. 바이러스 감염에 대한 2 준비 EB 세포 <ol><li> 개발의 원하는 단계에서 (일 2 일 3 사이), 15 ML 원뿔 관에 10cm 페트리 접시에서 교환 사채를 전송합니다. 5 ㎖의 PBS로 접시를 씻고 원뿔 튜브에 세척을 추가합니다. </li><li> 5 분 동안 실온에서 315 XG에 사채 펠렛. 5 ㎖의 PBS와 EB 세포를 씻으십시오. </li><li> 해동 세포 분리 솔루션 (포함하는 단백질 분해 및 collagenolytic 효소) 1 ㎖를 추가하고 OCC 30 분 동안 37 ° C의 물을 욕조에 튜브 (또는 창업)을 배치(플릭 또는 빛 소용돌이로 교반부터) asional 교반. </li><li> 차별화 미디어의 한 ML을 추가합니다. 상하 피펫 P200 피펫 팁 2ml를 피펫으로. 30 ~ 60 분 동안 다시 37 ° C의 물을 욕조에 튜브를 놓습니다. </li><li> 5 분 동안 315 XG에서 세포를 스핀 다운 및 PBS로 세포를 씻어. </li></ol> 3 바이러스 감염 <ol><li> 분화 매체의 1.5 ml의 세포를 재현 탁. 6 잘 아닌 조직 문화 처리 플레이트에 세포 현탁액을 전송합니다. </li><li> 미리의 표준 기술에 의해 레트로 바이러스를 준비합니다. 바이러스 역가에 따라 세포에 바이러스 뜨는 5-100 μl를 추가합니다. 이 사채의 후속 개혁을 억제로 감염을 향상시키기 위해 폴리 브렌를 추가하지 마십시오. </li><li> 실온에서 90 분 동안 2,120 XG에서 원심 분리하여 바이러스와 세포를 Spinoculate. </li></ol> 4 매달려 방울에 바이러스 성 감염 EB 세포 차별화 <ol><li> 난의 각 웰에 차별화 미디어의 두 ML을 추가nfected EB 세포 현탁액. </li><li> 피펫 multipipettors에 대한 멸균 시약 저수지에 감염된 EB 세포 현탁액의 3.5 ML. 팔 20 μL의 15 ~ 20 행 15cm 페트리 접시의 반전 뚜껑에 방울을 피펫하는 multipipettor를 사용합니다. </li><li> 페트리 접시의 아래쪽에 멸균 PBS 또는 물 10ml를 추가합니다. 그런 다음 페트리 접시에 방울과 장소 뚜껑을 반전. 방울 챔버 가습 유지하기 위해 거꾸로 PBS 이하 물 뚜껑 매달려되어 있는지 확인합니다. </li><li> 5.0 %의 CO 2와 37 ° C 조직 문화 인큐베이터에서 접시를 놓습니다. </li><li> 이일 후, 차별화 미디어의 4.5 ml의 뚜껑을 세척 반전에 의해 삭제 교수형에 형성 사채를 수집합니다. <ol><li> 새 10cm가 아닌 조직 배양 페트리 접시로 전송 미디어 및 사채. 차별화 미디어와 10cm 플레이트로 전송의 3 ㎖로 한 번 뚜껑을 세척 할 것. </li></ol></li><li> 세포 계측법 (8)의 총 후 흐름에 의한 분석을위한 수확 세포차별화 미디어 (-LIF)에 대한 세포의 초기 전송부터 일 문화. </li></ol> 흐름 사이토 분석을위한 5 준비 셀 <ol><li> 10 ml의 피펫과 15 ML 원뿔 튜브에 세포를 전송합니다. 5 ㎖의 PBS로 접시를 씻고 같은 원뿔 튜브로 전송할 수 있습니다. </li><li> 315 X g에서 펠렛 세포. 10 ml의 PBS와 펠렛을 씻으십시오. </li><li> 해동 세포 분리 용액 1 ㎖를 추가하고 가끔 교반과 함께 30 분 동안 37 ° C의 물을 욕조에 튜브를 배치합니다. 일부 세포 표면 항원의 검출을 위해, 트립신이 대신 사용될 수있다. </li><li> 1 ml의 차별화 미디어를 추가하고 최대 피펫 및 2 ml의 피펫과 함께 아래로. 도와 피펫의 끝 P200 팁을 사용하여 해체 단일 세포 현탁액으로 EB 소화. <ol><li> 세포막 단백질을 세포 표면에 재활용하기 위해서는 60 분 동안 다시 37 ° C의 물을 욕조에 튜브를 놓습니다. </li></ol></li><li> 5 분 동안 315 XG에서 세포 펠렛. </li><li> 세포 위스콘신 씻으일 0.1 % 소 혈청 알부민 분수 PBS (PBS / BSA)에서 제조 한 V (BSA)와 특정과 세포를 배양는 형광 항체를 태그. </li></ol> 조혈 전구 세포에 대한 6 유동 세포 계측법 분석 <ol><li> 5 ㎖의 12 × 75 라운드 MM 하단 튜브에 열 여섯 EB 파생 된 세포를 놓습니다. 보상 설정 사이토 흐름을 조절하기위한, 제조사의 지시에 따라 보상 비드를 사용한다. </li><li> 315 X g에서 펠렛 세포. 튜브 잔류 PBS / BSA 용액에 뜨는에 resuspend 펠렛을 붓고. </li><li> ESC의 HPC를 검출, 형광 표지 된 항체와 세포를 배양 : 항 - 마우스 CD41-PE (R-Phycoerytherin) 및 항 - 마우스 CD117 (cKit) -APC / CY7 (알로 피코시 아닌 7). <ol><li> 항체 1을 희석 : PBS / BSA 100을. 각 샘플에 희석 항체의 100 μl를 추가합니다. 각 공급 업체, 복제, 로트 또는 항체에 대한 항체의 정확한 희석을 결정합니다. 개별적으로 세포를 라벨에 대한 최적의 희석을 결정합니다. </리&gt; </ol></li><li> 30 분 동안 어둠 속에서 얼음에 샘플을 품어. </li><li> 5 분 동안 315 XG에 2 ㎖의 PBS / BSA 및 펠릿 세포를 추가합니다. </li><li> 기음 상층 액 300 ㎕의 PBS 또는 다른 등장 버퍼에 resuspend 펠렛. </li><li> 셀 스트레이너 캡 (0.35 μM)를 통해 필터 재현 탁 세포 대 세포 집합체를 제거합니다. </li><li> 플로우 사이토에 세포를 분석 할 수 있습니다. </li></ol>

<ol>
	<li>Williams, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Desbaillets, I., Ziegler, U., Groscurth, P., Gassmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoid+bodies:+an+in+vitro+model+of+mouse+embryogenesis.">Embryoid bodies: an in vitro model of mouse embryogenesis.</a> Experimental physiology. 85, 645-651 (2000).</li><li>Hopfl, G., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+embryonic+stem+cells+into+embryoid+bodies.">Differentiating embryonic stem cells into embryoid bodies.</a> Methods Mol Biol. 254, 79-98 (2004).</li><li>Tian, X., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+towards+hematopoietic+cells:+progress+and+pitfalls.">Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.</a> Curr Opin Hematol. 15, 312-318 (2008).</li><li>Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+by+gene+targeting+in+mouse+embryo-derived+stem+cells.">Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.</a> Cell. 51, 503-512 (1987).</li><li>Mansour, S. L., Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+proto-oncogene+int-2+in+mouse+embryo-derived+stem+cells:+a+general+strategy+for+targeting+mutations+to+non-selectable+genes.">Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.</a> Nature. 336, 348-352 (1988).</li><li>Robertson, E., Bradley, A., Kuehn, M., Evans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+genes+introduced+into+cultured+pluripotential+cells+by+retroviral+vector.">Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector.</a> Nature. 323, 445-448 (1986).</li><li>Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+in+embryonic+stem+cells+and+hematopoietic+stem+cells.">Retroviral expression in embryonic stem cells and hematopoietic stem cells.</a> Mol Cell Biol. 20, 7419-7426 (2000).</li><li>Pfeifer, A., Ikawa, M., Dayn, Y., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+by+lentiviral+vectors:+lack+of+gene+silencing+in+mammalian+embryonic+stem+cells+and+preimplantation+embryos.">Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.</a> Proc Natl Acad Sci U S A. 99, 2140-2145 (2002).</li><li>Smith-Arica, J. 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=Infection+efficiency+of+human+and+mouse+embryonic+stem+cells+using+adenoviral+and+adeno-associated+viral+vectors.">Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors.</a> Cloning and stem cells. 5, 51-62 (2003).</li><li>Lakshmipathy, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Cook, B. D., Liu, S., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad1+signaling+restricts+hematopoietic+potential+after+promoting+hemangioblast+commitment.">Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment.</a> Blood. 117, 6489-6497 (2011).</li><li>Zafonte, B. T., 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=Smad1+expands+the+hemangioblast+population+within+a+limited+developmental+window.">Smad1 expands the hemangioblast population within a limited developmental window.</a> Blood. 109, 516-523 (2007).</li><li>Kyba, M., Perlingeiro, R. C., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HoxB4+confers+definitive+lymphoid-myeloid+engraftment+potential+on+embryonic+stem+cell+and+yolk+sac+hematopoietic+progenitors.">HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.</a> Cell. 109, 29-37 (2002).</li><li>Galvin, K. E., Travis, E. D., Yee, D., Magnuson, T., Vivian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+signaling+regulates+the+bone+morphogenic+protein+pluripotency+pathway+in+mouse+embryonic+stem+cells.">Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.</a> J Biol Chem. 285, 19747-19756 (2010).</li><li>Ismailoglu, I., Yeamans, G., Daley, G. Q., Perlingeiro, R. C., Kyba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+patterning+activity+of+SCL.">Mesodermal patterning activity of SCL.</a> Exp Hematol. 36, 1593-1603 (2008).</li><li>Kyba, 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=Enhanced+hematopoietic+differentiation+of+embryonic+stem+cells+conditionally+expressing+Stat5.">Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.</a> Proc Natl Acad Sci U S A. 100, 11904-11910 (2003).</li><li>Cao, 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=MiR-23a+regulates+TGF-beta-induced+epithelial-mesenchymal+transition+by+targeting+E-cadherin+in+lung+cancer+cells.">MiR-23a regulates TGF-beta-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells.</a> Int J Oncol. 41, 869-875 (2012).</li><li>Chan, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+antagonism+between+PDGF+and+the+TGFbeta+family+of+signalling+pathways+by+control+of+miR-24+expression.">Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression.</a> EMBO J. 29, 559-573 (2010).</li><li>Huang, 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=Upregulation+of+miR-23a+approximately+27a+approximately+24+decreases+transforming+growth+factor-beta-induced+tumor-suppressive+activities+in+human+hepatocellular+carcinoma+cells.">Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells.</a> Int J Cancer. 123, (2008).</li><li>Min, 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=TGF-beta-associated+miR-27a+inhibits+dendritic+cell-mediated+differentiation+of+Th1+and+Th17+cells+by+TAB3,+p38+MAPK,+MAP2K4+and+MAP2K7.">TGF-beta-associated miR-27a inhibits dendritic cell-mediated differentiation of Th1 and Th17 cells by TAB3, p38 MAPK, MAP2K4 and MAP2K7.</a> Genes Immun. 13, 621-631 (2012).</li><li>Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD41+marks+the+initiation+of+definitive+hematopoiesis+in+the+mouse+embryo.">Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.</a> Blood. 101, 508-516 (2003).</li><li>Mitjavila-Garcia, M. T., 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=Expression+of+CD41+on+hematopoietic+progenitors+derived+from+embryonic+hematopoietic+cells.">Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.</a> Development. 129, 2003-2013 (2002).</li><li>Warr, M. R., Pietras, E. M., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+hematopoietic+stem+cell+functions+during+normal+hematopoiesis+and+hematological+malignancies.+Wiley+interdisciplinary+reviews.">Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies. Wiley interdisciplinary reviews.</a> Systems biology and medicine. 3, 681-701 (2011).</li><li>Dahl, R., Ramirez-Bergeron, D. L., Rao, S., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spi-B+can+functionally+replace+PU.1+in+myeloid+but+not+lymphoid+development.">Spi-B can functionally replace PU.1 in myeloid but not lymphoid development.</a> Embo J. 21, 2220-2230 (2002).</li><li>Dahl, 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=Regulation+of+macrophage+and+neutrophil+cell+fates+by+the+PU.1:C/EBPalpha+ratio+and+granulocyte+colony-stimulating+factor.">Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor.</a> Nat Immunol. 4, 1029-1036 (2003).</li></ol>

저자는 공개 경쟁 금융 이익을 더이 없습니다.

상술 한 바와 같이, inducibly 관심 유전자를 발현 ESC 클론 독시사이클린 시스템을 사용하여 생성 될 수 있지만,이 라인의 생성 시간 소모적이고 노동 집약적이다. 이 프로토콜에서 설명하는 교환 사채 파생 ESC로부터 제조 된 단일 세포 현탁액에 대한 관심의 유전자를 표현하는 방법이다. 이 감염된 세포는 이후의 차별화를 검사 할 수 드롭을 걸어서 사채로 개편된다. 예를 (그림 3)에서는</strong...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

<table border="0" cellpadding="0" cellspacing="0" width="470">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:193px;">Reagent</td>
			<td style="width:144px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">DMEM</td>
			<td style="width:144px;">Sigma/ Aldrich</td>
			<td style="width:133px;">D5796</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">b-mercaptoethanol</td>
			<td style="width:144px;">Sigma/ Aldrich</td>
			<td style="width:133px;">M3148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">FBS (ES Screened)</td>
			<td style="width:144px;">Hyclone</td>
			<td style="width:133px;">SH30070.03E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">FBS (Defined)</td>
			<td style="width:144px;">Hyclone</td>
			<td style="width:133px;">SH30070.03</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:193px;">Non-Essential Amino Acids</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:133px;">11140</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">L-Glutamine</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:133px;">35050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Penn/Strep</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:133px;">15070</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Trypsin/EDTA</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:133px;">25200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">LIF ESGRO</td>
			<td style="width:144px;">Millipore</td>
			<td style="width:133px;">ESG1107</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">ACCUMAX</td>
			<td style="width:144px;">Millipore</td>
			<td style="width:133px;">SCR006</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Trypsin/EDTA</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:133px;">25200</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:193px;">Falcon Tissue Culture Plates 6-well</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:133px;">08-772-1B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:193px;">Falcon Non-treated Plates 6-well</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:133px;">08-772-49</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Falcon Petri dish 10cm</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:133px;">08-757-100D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Falcon Petri dish 15cm</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:133px;">08-757-148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">Falcon Tube 5mls</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:133px;">14-959-11A</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:193px;">Falcon Tube 5mls with Cell Strainer Cap</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:133px;">08-771-23</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">White sterile resevoirs</td>
			<td style="width:144px;">U.S.A. Scientific</td>
			<td style="width:133px;">111-0700</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:193px;">Rat anti-mouse CD41 PE-conjugated</td>
			<td style="width:144px;">Biolegend</td>
			<td style="width:133px;">133906</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:193px;">Rat anti-mouse CD117 APC/CY7-conjugated</td>
			<td style="width:144px;">Biolegend</td>
			<td style="width:133px;">105826</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:193px;">RW4 ESCs</td>
			<td style="width:144px;">ATCC</td>
			<td>CRL-12418</td>
		</tr>
	</tbody>
</table>

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

RW4을 젤라틴이 연구에서 사용 된 ESCS (129X1 / SVJ 마우스 변형에서 파생). 교환 사채는 차별화 삼일에 고립되었다. 최상의 결과를 위해 사채는 구형 및 비 부착 (그림 1A, B)해야한다. 바이러스가 관심의 유전자와 함께 형광 마커 GFP를 공동으로 표현하는 spinoculation 후, 교환 사채는 매달려 드롭 방식으로 개혁되었다. 사채 성공적 2.0, 2.5, 및 3.0 일 사채로부터 제조 세포 현탁액으로부터 개질...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

쥐과 배아의 레트로 바이러스 감염은 조혈 분화 분석을위한 세포 유래 배아 바디 줄기 세포

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Research

JoVE Journal

Biology

8.4K 조회수.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 

비디오: Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

분리 및 조혈 줄기 세포의 이식 (HSCs)

연구

생물학

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Murine 난황과 AGM에서 조기 조혈 줄기 세포의 분리

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Hindlimb 국소 빈혈의 치료를위한 배아 줄기 세포 유래 내피 세포

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral - 중재 최저 동안 예 VIVO</em인간 조혈 줄기 세포의> 적혈구 조혈

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Murine 조혈 줄기 세포와 리니지 - 헌신 Progenitors의 Phenotypic 분석 및 절연

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

모터 뉴런에 마우스 배아 줄기 세포의 효율적인 분화

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

차 소장 Organoid 문화에서 레트로 바이러스 감염의 비디오 프로토콜

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...