This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

hPSCs的单细胞为主的非殖民地式单层（NCM）的文化。 1，准备工作<ol><li>制成500毫升培养基为小鼠胚胎成纤维细胞（MEFs细胞）培养：补充有10％FBS，2mM L-谷氨酰胺和0.1mM非必需氨基酸（NEAA）的DMEM培养基中。 </li><li>分离小鼠胚胎成纤维细胞的CF1系以下例行协议16和培养的MEF上0.1％明胶包被的6孔细胞培养板中的DMEM培养基中衍生（的MEF）细胞。另外，购买股票的MEF第3代从商业资源。 </li><li>准备基质胶板。 <ol><li>稀释将5ml的hESC合格基质胶股票用5毫升的DMEM/F12培养基（冷藏至〜4℃），并存储为50％等分试样在-20℃下冷冻。解冻冷冻基底膜股票在冰箱中（〜4°C）O / N和进一步稀释基底膜在寒冷的DMEM/F12培养基（以产生2.5％的工作浓度）。 </li><l我&gt;外套6孔细胞培养板用1.5ml的2.5％基质胶每孔，存储涂层板在冰箱中O / N（注：2个星期内使用的基质胶涂层板）。 </li><li>从冰箱中取出基底膜板，使板在室温暖在细胞培养罩为10至30分钟（注：不暖板在细胞培养孵化器），并吸出DMEM培养基之前，电镀hPSCs。 </li></ol></li><li>使500毫升人类胚胎干细胞培养基中：80％DMEM/F12培养基，20％KSR，2mM的L-谷氨酰胺，0.1mM非必需氨基酸，0.1mM的β-巯基乙醇和4纳克FGF-2 /毫升。 </li><li>准备10毫升2X HPSC冷冻介质：60％FBS，20％DMSO和20μM的Y-27632在mT​​eSR1介质，通过过滤灭菌，并在1周内使用介质。 </li></ol> 2，协议1（基本）：成长HPSC殖民地上料机<ol><li>使用MEF通道数为5或6（指定为P5和P6）为HPSC培养，以获得一致的雷苏尔TS。有丝分裂通过用10微克/毫升丝裂霉素C 3小时，在37℃处理细胞灭活的MEF，洗涤细胞3倍用1X Dulbecco氏磷酸盐缓冲盐水（D-PBS），再分离的MEF用0.05％胰蛋白酶在0.53毫EDTA。可选地，照射的MEF为8000拉德的剂量用X射线照射。 </li><li>使用显微镜下的台盼蓝染色排除法计数细胞数。另外，使用自动细胞计数器。 </li><li>在6孔聚苯乙烯板板照射的MEF涂覆有0.1％明胶以每孔1.88×10 5个细胞（ 即 1.96×10 4个细胞/ cm 2）的密度。孵育的细胞在37℃和5％CO 2中放置24小时。 </li><li>吸用巴斯德吸管（：需要在这个时候没有洗涤步骤注）拆下MEF培养基。 </li><li>从以前的文化磨碎HPSC菌落成小团块，检查显微镜下的团块，以确保其大小不等，FROM 50到100微米的直径，和板对MEF饲养层顶HPSC团块于一体的含2ml HPSC介质。 </li><li>通过照片改变人类胚胎干细胞培养基，每日3〜5天，记录菌落生长，标记任何形态的改变还是有区别的殖民地（〜5％），并手动轻轻吸用巴斯德吸管取出标菌落。 </li><li>漂洗在MEFs上剩余的菌落两次（每次用D-PBS 2分钟），并孵育的菌落用2ml的1毫克/毫升胶原酶IV中HPSC介质为10至30分钟），并检查HPSC菌落从脱离MEF涂层表面（注意：用刮刀以帮助该拆卸过程只有在殖民地都紧紧地贴在板上）。 </li><li>加入5 ml HPSC培养基到每个孔中，以最小化的酶反应，转移菌落到15毫升管中，允许HPSC的菌落，以在室温下沉淀3〜5分钟，并确保菌落的沉淀由C的直接可视化细胞l在管的底部（注意：不要在这个步离心管）中。 </li><li>除去含残余的MEF的培养上清和游离的单细胞，重悬菌落用5ml人类胚胎干细胞培养基中，并重复该沉淀步骤两次。 </li><li>除去培养基，磨碎HPSC菌落成小团块，存储在1X HPSC冷冻介质（或CryoStore CS10冷冻介质），用于冷冻保存（1合流每冷冻小瓶孔）或盘上的6孔板的细胞传代。 </li></ol> 3协议2：转换HPSC从殖民地到喂料机NCM <ol><li>冲洗HPSC粒料在D-PBS后，孵育粒料用1ml 1X的Accutase的10分钟，并在显微镜下研究酶促反应，以确保单细胞解离。 </li><li>用含有10μM的Y-27632，然后离心以200×g离心在RT 5分钟轻轻重悬细胞于5ml mTeSR1介质的终止酶促反应（注意，不要使用过多的离心势力，造成细胞损伤）。 </li><li>加入5 ml mTeSR1介质的沉淀，重悬沉淀为单细胞，并过滤通过40微米的细胞过滤（以除去任何残留的细胞聚集体），解离的细胞。 </li><li>种子1.3-2×10 6个hPSCs到一个井一个6孔板中涂有2.5％的hESC合格基质胶（1.4-2.1×10 5个细胞/ cm 2），添加mTeSR1介质可达2.5毫升，并包括10μM Y-27632的培养基中（以便于初始24小时后单池镀覆）。另外，利用下面的小分子，以取代10微米的Y-27632增强单细胞接种：1微米Y型39983（ROCK I抑制剂），1微米phenylbenzodioxane（ROCK II抑制剂），1微米thiazovivin（一种新型的ROCK抑制剂）和2μM的JAK抑制剂1。 </li><li>与在第二天无药物mTeSR1介质（24小时内）更换培养基，从一个额外的解离细胞好，并计数细胞数，以确定单细胞接种效率（注：约50〜90％，可以在这一阶段可以实现单细胞电镀效率）。 </li><li>使细胞用3ml mTeSR1介质的成长作为单细胞形成单层几天。每日更换培养基。 </li><li>凭经验确定用于传代适应NCM取决于细胞密度的时间表。当细胞生长达到汇合在3天或4天，或在细胞 - 细胞边界消失后的4个小时的时间点通过。分离的细胞在1ml的Accutase的，用1至3个分束比（ 例如 ，1汇合的细胞以及接种于3个孔的6孔培养板），用于细胞传代，并以5代稳定适于NCM。 </li><li>细胞冷冻<ol><li>利用一个汇合井（〜5-6×10 6个细胞）一冷冻小瓶：解离的细胞从一个铺满以及用1毫升的Accutase的10分钟。 </li><li>稀瓦特第i个5毫升如上所述mTeSR1中，离心细胞。 </li><li>重悬沉淀中加入500μlmTeSR1培养基中，然后慢慢地在一个冷冻小瓶中加入500μl2X HPSC冷冻培养基（含有20μM的Y-27632）。可替换地，重悬含有2μM的JAK抑制剂1或1X CryoStor CS10介质中任一10微米的Y-27632或2μM的JAK抑制剂1的存在下，细胞在1X HPSC冷冻介质。 </li><li>将冷冻小瓶至冰 - 冷却的低温容器（底部填充isopranol）和低温容器立即转移到-80℃的冰箱中。 </li><li>转移细胞从-80℃冷冻到液氮罐（第二天），用于长期冷冻保存。 </li></ol></li><li>细胞解冻<ol><li>利用1超低温保存的小瓶板1以及一个6孔板中。 </li><li>在37℃水浴中10分钟，解冻细胞在相同的水浴中放置2分钟预温mTeSR1介质逐滴加入细胞到5ml预热mTeSR1介质中含有10μM的Y-27632，并如上所述离心。 </li><li>轻轻重悬细胞沉淀用2ml mTeSR1介质中含有10μM的Y-27632和缓慢的细胞转移到一个良好的预涂布有基质胶（注：避免产生气泡）。 </li><li>每天都在变化中，预计HPSC增长到3天或4达到汇合。 </li></ol></li></ol> 4协议3：转换HPSC殖民地基底膜上以NCM文化<ol><li>从冰箱取出基质胶包被的板，将板在组织培养橱中10〜30分钟，从该板的每个孔中除去培养基，并添加2预热mTeSR1培养基中，以每孔中。 </li><li>转让研磨HPSC团块从供料器培养（基本协议的步骤2.10），以上述的Matrigel板。加mTeSR1介质到每个孔中2.5毫升最终体积。 </li><li>改变介质每日3〜4天S运动，直到菌落达到80〜90％汇合。 </li><li>用于细胞传代：冲洗用D-PBS将菌落两次，治疗的细胞用1ml的2毫克/毫升分散酶在37℃下进行15至20分钟，沉淀物和传代细胞的团块。 </li><li>对于NCM文化：重复步骤3.1至3.9的协议2所述。 </li></ol> 5协议4：在hPSCs的NCM文化LN-521 <ol><li>使用的前一天解冻的重组LN-521溶液，在4℃和解冻的层粘连蛋白与1X的D-PBS（含Ca 2 + / Mg 2 +的）稀释至终浓度为使层粘连蛋白涂层溶液（LCS） 10微克/毫升。 </li><li>加入1 ml濒海战斗舰的一个很好的6孔板（注：避免干出在涂布过程中）。 </li><li>密封涂层板用封口膜，以防止蒸发和板存放在冰箱（4℃）O / N（注：1周内用板）。 </li><li>轻轻地取出用巴斯德吸管濒海战斗舰，但无uching的涂层表面，并添加2毫升mTeSR1介质到一个阱。 </li><li>所有温暖的培养液（含D-PBS），在37℃水浴25分钟。 </li><li>转换HPSC殖民地NCM <ol><li>如在协议2所述游离细胞聚集到单个细胞用Accutase。 </li><li>种子1.3-2×10 6个hPSCs成一个LN-521-包被的孔（1.4-2.1×10 5个细胞/ cm 2）无ROCK抑制剂的存在。 </li><li>改变介质每日3〜4天。 </li><li>分离的细胞在1ml的Accutase在3天或4为用1至3个分束比的下一个传代细胞。 </li></ol></li></ol> 6，协议5：NCM文化的质粒DNA转染<ol><li>适应HPSC殖民地NCM文化作为协议2描述4。 </li><li>板分离在12孔板hPSCs为7.5×10细胞密度5个细胞/孔在mTeSR1介质中的10μM的Y 2763的存在2。 </li><li>电镀后的细胞替换mTeSR1培养基4-8小时之间无药mTeSR1媒体。 </li><li>对于每个转染，稀2.5表达质粒微克（ 例如 ，pmaxGFP）和5μl脂质体2000在独立的Eppendorf管中每个的Opti-MEM的125微升低血清培养基。 </li><li> 5分钟后，混合的稀释试剂，孵育20分钟，在RT（以形成转染复合物）。 </li><li>添加250μl的转染复合物，以1毫升mTeSR1培养基每孔含有hPSCs并在CO 2培养箱中培养24小时，培养的细胞在37℃。 </li><li>检查荧光显微镜和照片为随机的实际转染效率计算下的转染效率。 </li></ol> 7，协议6：NCM文化的小分子RNA的转染<ol><li>通过加入在61毫升的Accutase每孔离解半汇合hPSCs在NCM条件下2天<html>孔板。 </li><li>加入10 ml mTeSR1培养基来稀释的酶反应和离心机在200×g离心5分钟。 </li><li>重悬细胞沉淀用mTeSR1介质，种子细胞以每孔7.5×10 5个细胞在12孔板中含有10μM的Y-27632的密度在mTeSR1介质，并让细胞附着4小时。 </li><li>与Y27632无mTeSR1培养基更换培养基。 </li><li>使用市售的microRNA（ 例如 ，非靶向miRIDIAN的miRNA转染对照标记DY547）。使用提供的试剂滴定浓度为0-160纳米，并按照制造商的指示。 </li><li>通过成像活细胞的DY547荧光显​​微镜在转染后24小时下优化转染效率对于每个浓度在HPSC线。 </li><li>计算基于DY547阳性细胞在所有成像细胞的百分比的转染效率。 </li></ol>。地幔“&gt; 8协议7：NCM文化的慢病毒载体转导<ol><li>重复步骤7.1至协议6 7.4。 </li><li>计算用于转导病毒颗粒的量：使用下面的公式：TU =（MOI x CN）/ VT，而TU表示总的数字变换单元，MOI等于感染的期望的多样性在阱（MOI或TU /细胞），CN指定单元格的每个数好，和VT表明该股的病毒滴度（TU /毫升）。例如，由于库存的病毒滴度为SMART-shRNA表达载体等于1×10 9 TU /毫升（每毫升变换单元），7.5×10 5个每孔的细胞在转导的时间，和20的预期MOI：使用15个微升股票的病毒颗粒为每口井的（注：建议这种情况下的瞬态慢病毒诱导）。 </li><li>预温300微升mTeSR1介质用10毫克/毫升的聚凝胺进行的30分钟的。 </li><li>加入15微升股票病毒颗粒的成包含聚凝胺的预热mTeSR1中，轻轻混匀的解决方案。 </li><li>用300μl预热的培养基中含有的病毒颗粒更换细胞培养基。 </li><li>经过4小时培养，考察turboGFP荧光（一个制造商的shRNA表达），以确定转染效率。 </li><li>加入300微升额外mTeSR1介质来换能顺利当细胞开始表达turboGFP。 </li><li>经过12-16小时培养，重新审视涡轮GFP的表达。 </li><li>与在72小时这些转导的细胞执行所需的后续实验。 </li></ol>

<ol>
	<li>Cherry, A. B., 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=Reprogrammed+cells+for+disease+modeling+and+regenerative+medicine.">Reprogrammed cells for disease modeling and regenerative medicine.</a> Annu Rev Med. 64, 277-290 (2013).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Thomson, J. 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Burridge, P. W., 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=Production+of+de+novo+cardiomyocytes:+human+pluripotent+stem+cell+differentiation+and+direct+reprogramming.">Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.</a> Cell Stem Cell. 10, 16-28 (2012).</li><li>Mallon, B. 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=StemCellDB:+The+Human+Pluripotent+Stem+Cell+Database+at+the+National+Institutes+of+Health.">StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health.</a> Stem Cell Res. 10, 57-66 (2012).</li><li>Chen, K. G., 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=Human+pluripotent+stem+cell+culture:+considerations+for+maintenance,+expansion,+and+therapeutics.">Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.</a> Cell Stem Cell. 14, 13-26 (2014).</li><li>Bendall, S. 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=IGF+and+FGF+cooperatively+establish+the+regulatory+stem+cell+niche+of+pluripotent+human+cells+in+vitro.">IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.</a> Nature. 448, 1015-1021 (2007).</li><li>Moogk, D., 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=Human+ESC+colony+formation+is+dependent+on+interplay+between+self-renewing+hESCs+and+unique+precursors+responsible+for+niche+generation.">Human ESC colony formation is dependent on interplay between self-renewing hESCs and unique precursors responsible for niche generation.</a> Cytometry A. 77, 321-327 (2010).</li><li>Chen, K. G., 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=Non-colony+type+monolayer+culture+of+human+embryonic+stem+cells.">Non-colony type monolayer culture of human embryonic stem cells.</a> Stem Cell Res. 9, 237-248 (2012).</li><li>Amit, 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=Suspension+culture+of+undifferentiated+human+embryonic+and+induced+pluripotent+stem+cells.">Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells.</a> Stem Cell Rev. 6, 248-259 (2010).</li><li>Dang, S. 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=scalable+embryonic+stem+cell+differentiation+culture.">scalable embryonic stem cell differentiation culture.</a> Stem Cells. 22, 275-282 (2004).</li><li>Serra, 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=Process+engineering+of+human+pluripotent+stem+cells+for+clinical+application.">Process engineering of human pluripotent stem cells for clinical application.</a> Trends Biotechnol. 30, 350-359 (2012).</li><li>Steiner, D., 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=propagation+and+controlled+differentiation+of+human+embryonic+stem+cells+in+suspension.">propagation and controlled differentiation of human embryonic stem cells in suspension.</a> Nat Biotechnol. 28, 361-364 (2010).</li><li>Watanabe, 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+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat Biotechnol. 25, 681-686 (2007).</li><li>Androutsellis-Theotokis, 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=Notch+signalling+regulates+stem+cell+numbers+in+vitro+and+in+vivo.">Notch signalling regulates stem cell numbers in vitro and in vivo.</a> Nature. 442, 823-826 (2006).</li><li>Jozefczuk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. , (2012).</li><li>Kozhich, O. 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=Standardized+Generation+and+Differentiation+of+Neural+Precursor+Cells+from+Human+Pluripotent+Stem+Cells.">Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells.</a> Stem Cell Rev. , (2012).</li><li>Kunova, 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=Adaptation+to+robust+monolayer+expansion+produces+human+pluripotent+stem+cells+with+improved+viability.">Adaptation to robust monolayer expansion produces human pluripotent stem cells with improved viability.</a> Stem Cells Transl Med. 2, 246-254 (2013).</li><li>Tsutsui, 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=An+optimized+small+molecule+inhibitor+cocktail+supports+long-term+maintenance+of+human+embryonic+stem+cells.">An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells.</a> Nat Commun. 2, 167 (2011).</li><li>Amps, 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=Screening+ethnically+diverse+human+embryonic+stem+cells+identifies+a+chromosome+20+minimal+amplicon+conferring+growth+advantage.">Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.</a> Nat Biotechnol. 29, 1132-1144 (2011).</li><li>Baker, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+to+culture+of+human+embryonic+stem+cells+and+oncogenesis+in+vivo.">Adaptation to culture of human embryonic stem cells and oncogenesis in vivo.</a> Nat Biotechnol. 25, 207-215 (2007).</li><li>Lee, A. 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=Tumorigenicity+as+a+clinical+hurdle+for+pluripotent+stem+cell+therapies.">Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies.</a> Nat Med. 19, 998-1004 (2013).</li><li>Domogatskaya, 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=Laminin-511+but+not+-332,+-111,+or+-411+enables+mouse+embryonic+stem+cell+self-renewal+in+vitro.">Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro.</a> Stem Cells. 26, 2800-2809 (2008).</li><li>Domogatskaya, 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=Functional+diversity+of+laminins.">Functional diversity of laminins.</a> Annu Rev Cell Dev Biol. 28, 523-553 (2012).</li><li>Rodin, 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=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nat Biotechnol. 28, 611-615 (2010).</li><li>Liew, C. G., 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=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Braam, S. 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=Genetic+manipulation+of+human+embryonic+stem+cells+in+serum+and+feeder-free+media.">Genetic manipulation of human embryonic stem cells in serum and feeder-free media.</a> Methods Mol Biol. 584, 413-423 (2010).</li><li>Braam, S. 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=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li></ol>

The authors declare that they have no competing financial interests.

有两种主要的方法来培养hPSCs 体外 ：常规菌落型培养物（细胞上馈线或细胞外基质）和hPSCs作为骨料未经进料器6的悬浮培养物。这两个殖民地式和悬浮培养方法的局限性包括累积的异质性和可继承的表观遗传变化。 NCM培养的基础上，无论是单细胞传代和高密度的细胞接种，代表了一种新的培养方法对HPSC生长6,18。尽管各单细胞传代的方法已被记载在文献中，但他们都不是?...

hPSCs的分化走向多向成体组织的能力开辟了新的途径来治疗从谁，涉及心血管，肝，胰腺和神经系统，严重的1-4患有疾病的患者。从hPSCs派生的各种细胞类型也将提供强大的移动平台，疾病模型，基因工程，药物筛选和毒理试验1,4。确保其未来的临床药理及应用的关键问题是大量的临床级hPSCs通过体外细胞培养的一代。然而，目前的文化系统是不足或固有可变的，涉及hPSCs为菌落5,6各馈线和无饲养层培养。 殖民地式增长的hPSCs股的内细胞团哺乳动物早期胚胎（ICM）的许多结构特征。将ICM容易分化成三个胚层因为在异构信号梯度的存在，多细胞环境。因此，收购异质性在胚胎发育早期被认为是分化的必要过程，但HPSC文化的不必要的功能。在HPSC文化的异质性往往被过度凋亡信号和自发分化，由于不理想的生长条件引起的。因此，在集落类型的文化，异质细胞通常在7,8菌落的周围观察到。它也已表明，细胞在人胚胎干细胞（hESC细胞）菌落表现出不同反应到的信号分子，如BMP-4 9。此外，集落培养方法由于不可控的增长率及凋亡信号通路6,9产生低电池产量以及极低的电池回收率从冷冻保存。在最近几年，各种悬浮培养物已被开发用于培养hPSCs，particul阿尔利扩张的馈线和无基质条件6,10-13大量hPSCs的。显然，不同的文化系统都有自己的优点和缺点。在一般情况下，hPSCs的异质性表示的主要缺点在菌落型的和聚集的培养方法，这是不理想的用于递送DNA和RNA的物料进入hPSCs遗传工程6中的一个。 显然，有一个迫切需要开发出规避目前的文化方法的一些不足之处的新系统。小分子抑制剂（如ROCK抑制剂Y-27632和JAK抑制剂1）提高单细胞存活的发现铺平了道路分离，HPSC文化14,15。通过使用这些小分子，我们最近开发出一种培养方法基于非集群式分离- hPSCs 9（NCM）的增长。这种新颖的培养方法结合了单细胞传代和高密度电镀方法，使我们能够在一致的增长周期产生大量同质hPSCs无重大染色体异常9。或者，NCM培养可能与不同的小分子和定义矩阵（如层粘连蛋白），以优化的培养方法在广泛的应用中实现。在这里，我们提出几个具体的协议的基础上NCM文化和划定详细程序，基因工程。为了证明NCM协议的通用性，我们还测试了NCM的文化与不同的ROCK抑制剂和单层粘连蛋白亚型521（ 即 ，LN-521）。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Countess automated cell counter&#160;&#160;</td>
			<td style="width:220px;">Invitrogen Inc.&#160;</td>
			<td style="width:187px;">C10227</td>
			<td style="width:187px;">Automatic cell counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Faxitron Cabinet X-ray System</td>
			<td style="width:220px;">Faxitron X-ray Corporation, Wheeling, IL&#160;</td>
			<td style="width:187px;">Model RX-650</td>
			<td style="width:187px;">X-ray irradiation of MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">MULTIWELL six-well plates&#160;</td>
			<td style="width:220px;">Becton Dickinson Labware</td>
			<td style="width:187px;">353046</td>
			<td style="width:187px;">Polystyrene plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11965&#8211;092</td>
			<td style="width:187px;">For MEF medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">mitomycin C</td>
			<td style="width:220px;">Roche&#160;</td>
			<td style="width:187px;">107 409</td>
			<td style="width:187px;">Mitotic inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Trypsin</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">25300-054</td>
			<td style="width:187px;">For MEF dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM/F12&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11330&#8211;032</td>
			<td style="width:187px;">For hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Opti-MEM I Reduced Serum Medium&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">31985-062</td>
			<td style="width:187px;">For hPSC transfection</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Heat-inactivated FBS</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">16000&#8211;044</td>
			<td style="width:187px;">Component of MEF medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Knockout Serum Replacer&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">10828&#8211;028</td>
			<td style="width:187px;">KSR, Component of hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">14190-144</td>
			<td style="width:187px;">D-PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Non-essential amino acids&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">11140&#8211;050</td>
			<td style="width:187px;">NEAA, component of hPSC medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">L-Glutamine&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">25030&#8211;081</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:203px;">mTeSR1 &#38; Supplements</td>
			<td rowspan="2" style="width:220px;">StemCell Technologies</td>
			<td rowspan="2" style="width:187px;">5850</td>
			<td style="width:187px;">Animal protein-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TeSR2 &#38; Supplements</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">5860</td>
			<td style="width:187px;">Xeno-free medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:220px;">Sigma&#160;</td>
			<td style="width:187px;">7522</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;"> 
			MEF (CF-1) ATCC</td>
			<td style="width:220px;">American Type Culture Collection (ATCC)&#160;</td>
			<td style="width:187px;">SCRC-1040</td>
			<td style="width:187px;">For feeder culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">hESC-qualified Matrigel</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">354277</td>
			<td style="width:187px;">For feeder-free culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Laminin-521</td>
			<td style="width:220px;">BioLamina</td>
			<td style="width:187px;">LN521-02</td>
			<td style="width:187px;">Human recombinant protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">FGF-2 (recombinant FGF, basic)</td>
			<td style="width:220px;">R&#38;D Systems, MN</td>
			<td style="width:187px;">223-FB</td>
			<td style="width:187px;">Growth factor in hPSC medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CryoStor CA10&#160;</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">7930</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Accutase</td>
			<td style="width:220px;">Innovative Cell Technologies</td>
			<td style="width:187px;">AT-104</td>
			<td style="width:187px;">1X mixed enzymatic solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">JAK inhibitor I</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">420099</td>
			<td style="width:187px;">An inhibitor of Janus kinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">688000</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0012</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-39983</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0029</td>
			<td style="width:187px;">ROCK I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Phenylbenzodioxane&#160;</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0030</td>
			<td style="width:187px;">ROCK II inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Thiazovivin</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0017</td>
			<td style="width:187px;">A novel ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">BD Falcon Cell Strainer&#160;</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">352340</td>
			<td style="width:187px;">40-&#181;m cell strainer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nalgene 5100-0001 Cryo 1&#176;C</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">C6516F-1</td>
			<td style="width:187px;">&#8220;Mr. Frosty&#8221; Freezing Container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Lipofectamine 2000&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11668-027</td>
			<td style="width:187px;">Transfection reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DharmaFECT Duo&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">T-2010-02</td>
			<td style="width:187px;">Transfection reagent</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Non-targeting miRIDIAN miRNA Transfection Control</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">IP-004500-01-05</td>
			<td style="width:187px;">Labeled with Dy547, to monitor the delivery of microRNAs&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">SMART-shRNA</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">To be determined</td>
			<td style="width:187px;">Lentiviral vector</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">pmaxGFP</td>
			<td>amaxa Inc (Lonza)</td>
			<td>Included in every transfection kit</td>
			<td style="width:187px;">Expression plasmid for transfection control</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">4-Oct</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-5279</td>
			<td style="width:187px;">Mouse IgG2b, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-1</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21702</td>
			<td style="width:187px;">Mouse IgM, differentiation marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-4</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21704</td>
			<td style="width:187px;">Mouse IgG3, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-60</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21705&#160;</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-81</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21706</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">CK8 (C51)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8020</td>
			<td style="width:187px;">Mouse IgG1, against cytokeratin 8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">&#945;-fetoprotein</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8399</td>
			<td style="width:187px;">AFP, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF-3&#946; (P-19)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-9187</td>
			<td style="width:187px;">FOXA2, goat polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Troponin T (Av-1)</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">MS-295-P0</td>
			<td style="width:187px;">Mouse IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Desmin&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">RB-9014-P1</td>
			<td style="width:187px;">Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Anti-NANOG</td>
			<td style="width:220px;">ReproCELL Inc, Japan</td>
			<td style="width:187px;">RCAB0004P-F</td>
			<td style="width:187px;">Polyclonal antibody&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Rat anti-GFAP</td>
			<td style="width:220px;">Zymed</td>
			<td style="width:187px;">13-0300</td>
			<td style="width:187px;">Glial fibrillary acidic protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Albumin (clone HSA1/25.1.3)</td>
			<td style="width:220px;">Cedarlane Laboratories Ltd. (</td>
			<td style="width:187px;">CL2513A</td>
			<td style="width:187px;">Mouse IgG1,</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Smooth muscle actin (clone 1A4)</td>
			<td style="width:220px;">DakoCytomation Inc</td>
			<td style="width:187px;">IR611/IS611</td>
			<td style="width:187px;">Mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nestin</td>
			<td style="width:220px;">Chemicon International</td>
			<td style="width:187px;">MAB5326</td>
			<td style="width:187px;">Rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TUBB3</td>
			<td style="width:220px;">Convance Inc</td>
			<td style="width:187px;">MMS-435P</td>
			<td style="width:187px;">Tuj1, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF4&#945; (C11F12)</td>
			<td style="width:220px;">Cell Signaling Technologies</td>
			<td style="width:187px;">3113</td>
			<td style="width:187px;">Rabbit monoclonal antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Paraformaldehyde (solution)</td>
			<td style="width:220px;">Electron Microscopy Sciences</td>
			<td style="width:187px;">15710</td>
			<td style="width:187px;">PFA, fixative, diluted in D-PBS</td>
		</tr>
	</tbody>
</table>

alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

NCM文化的一般模式 图1显示出高密度的单细胞接种在ROCK抑制剂Y-27632的存在后hPSCs的动态变化典型的NCM的文化模式。这些形态变化包括电镀，细胞簇的形成，并呈指数生长的细胞，然后进行细胞缩合（ 图1A）之后，细胞间的连接。代表性实验表明WA01（H1）的胚胎干细胞，镀成单细胞在1.9×10 5个细胞/ cm 2在10μMY-27632在第1天?...

Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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12.2K 次观看.  National Institutes of Health. Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.