Name: direct induction of hemogenic endothelium and blood by overexpression of transcription factors in human pluripotent stem cells
Uploaded: 2015-12-03T10:30:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells at JoVE.com

We thank Matt Raymond for editorial assistance. This work was supported by funds from the National Institute of Health (U01HL099773, R01HL116221, and P51 RR000167) and The Charlotte Geyer Foundation.

1.病毒制剂和转录因子组合<ol><li>制备用含有编码蛋 ​​白质的DNA的供ETV2，GATA1，GATA2，TAL1和LMO2（表1）PSIN-EF1α启动慢病毒表达质粒的解决方案。 </li><li>测量通过记录紫外吸收用分光光度计在230nm，260nm处，和280nm用于慢病毒生产的质粒制备物的浓度和纯度。注:DNA制备展示A260 / 280和A260 / 230的值大于1.8通常被认为是良好的品质。低级A260 / 280值可以指示蛋白污染，而较低的A260 / 230值指示与盐或某些溶剂如苯酚杂质。推荐质粒浓度为1-3微克/微升。 </li><li>在此前公布的协议11说明生产慢病毒的转导。感应HE与泛粒细胞潜力，需要共同表达ETV2和GATA1，或ETV2和GATA2的。感应HE与赤和巨核细胞潜力需要GATA2和TAL1。加入LMO2的显著改善赤型巨核细胞从hPSCs在GATA2和TAL1 10的存在的产率。 </li></ol> 2. hPSCs文化协议<ol><li>传代之前，锐边成长的多能干细胞集落紧至70-80％汇合。马克和删除传代细胞前自发分化的殖民地。 </li><li>稀释商用细胞外基质凝胶（ 见表 2）根据制造商的说明书和外套6孔板过夜，在4℃，或者用于至少一个使用前小时，在37℃。前传代细胞，吸出基质中，添加2毫升新鲜商业完整culturemedium（ 见表 2），并保持在平板37 ℃，5％的CO 2，直至细胞准备播种。 </li><li>通道hPSCs <ol><li> Aspira从6孔板与hPSCs之一汇合井，并添加1.5的预温热的Dispase II溶液忒介质（2毫克/毫升的DMEM / F12中，通过0.22微米的过滤器过滤灭菌）。孵育5-7分钟，在37℃，5％的CO 2，直至菌落的边缘开始从表面抬起。 </li><li>抽吸分散酶II溶液并小心两次加入，然后吸移2毫升新鲜DMEM / F12培养基洗涤菌落。然后，用5ml的血清学玻璃吸管，游离hPSCs用3ml新鲜商业完全培养基（ 见表 2）通过吸取细胞悬液上下数次。 </li><li>添加0.5 ml的细胞悬液，每孔一个6孔板含有1.5 ml的商用完全培养基的（ 见表 2）。搅动板来回和从右到左几次，以确保均匀的细胞分布境的板放入培养箱时。保持细胞在37℃，5％CO 2的18-24小时。 </li><li>第二天换液，以新鲜的商业完全培养基（ 见表 2），并检查hPSCs形态。成功的传代导致小，良好的附着的菌落保留HPSC形态。 hPSCs必须以2.5-3毫升freshmedium每被馈送孔，每天传代每4-5天在不超过70-80％汇合更多。 注意:如果人iPS细胞维持在小鼠胚胎成纤维细胞（MEFs），它们需要被转移至无饲养条件和传代转导前2-3次，以确保有效的分化。 </li></ol></li></ol>从hPSCs（0天，hPSCs的转导）3.感应Hematoendothelial Pprecursors的<ol><li>为了制备反应介质结合1.3商业完整无血清培养基（ 见表 2），病毒，聚凝胺，和Y27632 ROCK抑制剂（表1）:添加1.3微升10毫ROCK抑制剂以1.3毫升培养基至最终concentra灰为10μM，以及聚凝胺至终浓度6微克/毫升。 <ol><li>在0.5-1.0 MOI每个细胞每种病毒的最终浓度（感染复数）添加病毒浓缩物（S）的一个适当的量。保持反应介质在冰上或在4℃下，直到单细胞悬液是准备好了。 注:在GATA1 / ETV2，GATA2 / TAL1每种病毒的相同数量的MOI和GATA2 / TAL1 / LMO2的反应混合物是最佳的诱导分化。然而，在ETV2 / GATA2转导培养倍增教学语言GATA2，相对于ETV2，提高差异化。因此，对于这些培养物，比1:为ETV2的MOI和GATA2 1 MOI 2建议（例如，如果病毒的浓缩物，估计大约6.8×10 7个粒子/ ml，加10微升ETV2和20微升GATA2病毒浓缩物至每ETV2 / GATA2反应0.68×10 6细胞在6孔板的35毫米孔）。 </li></ol></li><li>在单个小区停赛制备hPSCsension。 <ol><li>如在第2节第4天生长在无饲养条件hPSCs下面的最后一段，观察倒置显微镜下细胞密度和形态。 hPSCs应在紧菌落边缘锋利成长没有自发分化和达到〜60-70％汇合的转导的天。 </li><li>吸出培养基，加入1.5-2毫升每孔商用细胞解离试剂（ 表1）和在37℃，5％CO 2的5-7分钟。 </li><li>收集细胞分化成商业完全培养基等体积的（ 见表 2）与10μM的ROCK抑制剂的，计数细胞并确定存活率。离心沉淀细胞，在200×g离心5分钟，然后重悬细胞在商业完全培养基用10μM的ROCK抑制剂的每毫升3.4-5×10 6个细胞的浓度。 注:细胞活力是成功的病毒转导和随后分化的关键。通常情况下，转导前细胞存活率超过90％。 </li></ol></li><li>加入200微升细胞悬浮液，含有0.68-1×10 6个细胞 ，成1.3 3.1制备的反应介质的溶液中。 </li><li>吸在从2.2制备的过夜涂覆6孔板的基质溶液，将反应混合物转移6孔板的一个孔和均匀分布的细胞。在37℃，5％CO 2的24小时。 24小时后，至少80％的细胞应附到基质。 </li></ol>从hPSCs 4.感应Hematoendothelial前体（1-7天） <ol><li>后24小时转导，除去含病毒的培养基，并与商业的不完整的细胞培养基洗涤附着的细胞（见表1），然后每商业不完全细胞培养介质中含有孔加入3毫升的造血细胞因子:SCF 100纳克/毫升，TPO 50纳克/毫升，20的bFGF纳克/毫升（以下称为3F-介质）。 </li><li>替换总培养基用新鲜3F-介质上天2,3和4天以去除死细胞和碎片。 4天后，当浮动造血细胞不断涌现，更换一半3F-媒体隔日在保持总量以4ml。 注:PSIN-EF1α启动慢病毒表达质粒掺入嘌呤霉素抗性基因，从而允许阳性筛选转导细胞。在1μg/ ml的嘌呤霉素的可在第一1-2天的分化被加入到培养基，以消除任何残余的未分化hPSCs。 </li><li>观察第4天的细胞形态在倒置显微镜下:紧细胞簇与典型的内皮形态的开始出现（ 图2A，3A）。圆形的血细胞似乎从第5天到分化的第7天，而一些地区可能保留hPSCs形态。如下所述分析HE和造血细胞分化。 注:成功hematoendothelial不同iation导致产生松散地附着到底层平面的内皮细胞显示为折射圆形细胞的血细胞。这些细胞增殖活跃地形成小聚集体，并最终分离和浮子（图2A和3A）。现场的血细胞可以从基于其反射光，并扩大培养条件的能力死者和垂死的细胞在视觉上区分开来。 </li></ol> 5.分化Hemogenic内皮细胞（HE）阶段的分析。 <ol><li>用显微镜观察细胞的内皮形态检测何的天3和4分化之间的形成。有没有漂浮的血细胞的培养在这个阶段的分化。何的存在可以通过免疫荧光染色确认，用VE-钙粘蛋白，CD73，CD226和CD43的抗体流式细胞仪分析。 <ol><li>要在第3天评估流式细胞仪分析使用细胞HE形成4日从一个实验。通过将培养物与商业细胞解离试剂收集细胞（ 见表 1）5-10分钟，在37℃，5％的CO 2，然后用高达每染色反应1×10 5个细胞（流式细胞术中所述染色步骤12）。 注:VE-钙粘蛋白+ HE细胞获得的早期造血标志物CD226表达，但缺乏CD73和CD43 6的表达。与此相反，非何细胞表达CD73（ 图2B，3B）。 </li></ol></li><li>免疫荧光染色HPSC源性何。 <ol><li>洗涤细胞用磷酸盐缓冲盐水（PBS）的附单层，然后固定细胞在4％多聚甲醛为30〜60分钟，在室温下进行。 </li><li>洗涤细胞再次用PBS清洗，然后透化使用在PBS中的0.1％的Triton X-100进行20分钟，在室温下进行。 </li><li>在PBS 2洗涤细胞三次，然后在孵育封闭缓冲液由PBS中，用10％胎牛血清（FBS），30至120分钟。 </li><li>制备染色溶液，同时含有初级抗体（1:1000稀释）的小鼠抗人CD43和兔抗人VE-钙粘蛋白在PBS有5％FBS，（ 表1）。 </li><li>从细胞吸出封闭缓冲液并添加每染色缓冲孔1ml与添加的初级抗体;孵育3小时，在室温下，或过夜，在4℃。 </li><li>加入2毫升的PBS有5％FBS洗涤细胞三次。 </li><li>制备第二染色缓冲液，含有二抗以1:500在PBS中稀释有5％FBS:驴抗兔缀合绿色荧光染料，和抗小鼠缀合红色荧光染料（见表1）。加入1毫升每二次染色缓冲井和在室温下孵育1小时。 </li><li>无血清洗涤三次，用PBS。染色细胞300nM的DAPI工作液中的dh2 O 10-15分钟。 </li><li>用PBS洗涤细胞两次，加2-3毫升的PBS成很好地与着色细胞。观察荧光绿，红，蓝镜过滤器。 </li></ol></li></ol>诱导造血前体6.分析<ol><li>维持分化培养物，直到漂浮细胞显著扩大，达10-14天，更换一半3F-介质，如在3.1中描述，每两天。 </li><li>分离并收集由用市售细胞解离试剂处理的细胞分化的细胞的单层（见表1）在37 5-10分钟 ℃，5％的CO 2。 </li><li>执行流式细胞仪使用抗VE钙粘蛋白，CD43和CD45抗体，以评估造血培养12。需要注意的是一个大级分的细胞，至多ETV2和GATA2转导的细胞的40％，共表达VE-CAD和CD43。以下抗体可用于检测以下电子特定细胞谱系xpansion或诱发血液祖细胞分化:CD235a（红细胞），CD41a（巨核细胞），CD32（所有类型的骨髓细胞），CD66b（中性粒细胞）和CD163（巨噬细胞）。 </li><li>使用根据制造商的说明市售克隆培养基（ 表1）进行的CFC-测定。 <ol><li>转移1-2×10 4个细胞于3ml商业克隆培养基（ 见表 3）以评估集落形成细胞和类型的造血集落的数目。最佳接种密度允许单个菌落分别增长相互不重叠的鉴定和分离。接种密度可根据分化效率实验之间有所不同，但应不超过每毫升克隆形成商业介质的1×10 4个细胞。 </li><li>混合细胞克隆商业中由温和的震荡，而传输3毫升悬浮液中，以两张35毫米超低附着菜CFC的试验，1.5毫升悬浮液中每道菜。均匀分布介质和孵育在37℃，5％CO 2的14天。避免干扰的菜;观察第7天及测定第10天的集落形成。 </li><li>根据自己的第14天的形态识别和计数造血集落。 </li></ol></li></ol>

<ol>
	<li>Zape, J. P., Zovein, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemogenic+endothelium:+origins,+regulation,+and+implications+for+vascular+biology.">Hemogenic endothelium: origins, regulation, and implications for vascular biology.</a> Semin Cell Dev Biol. 22, 1036-1047 (2011).</li><li>Swiers, G., Rode, C., Azzoni, E., de Bruijn, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+history+of+hemogenic+endothelium.">A short history of hemogenic endothelium.</a> Blood Cells, Mol &amp; Dis. 51, 206-212 (2013).</li><li>Kennedy, 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=T+lymphocyte+potential+marks+the+emergence+of+definitive+hematopoietic+progenitors+in+human+pluripotent+stem+cell+differentiation+cultures.">T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2, 1722-1735 (2012).</li><li>Wang, 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=Endothelial+and+hematopoietic+cell+fate+of+human+embryonic+stem+cells+originates+from+primitive+endothelium+with+hemangioblastic+properties.">Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties.</a> Immunity. 21, 31-41 (2004).</li><li>Rafii, 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=Human+ESC-derived+hemogenic+endothelial+cells+undergo+distinct+waves+of+endothelial+to+hematopoietic+transition.">Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.</a> Blood. 121 (5), 770-780 (2012).</li><li>Choi, K. 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=Identification+of+the+hemogenic+endothelial+progenitor+and+its+direct+precursor+in+human+pluripotent+stem+cell+differentiation+cultures.">Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2, 553-567 (2012).</li><li>Vodyanik, M. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukosialin+(CD43)+defines+hematopoietic+progenitors+in+human+embryonic+stem+cell+differentiation+cultures.">Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.</a> Blood. 108, 2095-2105 (2006).</li><li>Wang, 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=TGFbeta+inhibition+enhances+the+generation+of+hematopoietic+progenitors+from+human+ES+cell-derived+hemogenic+endothelial+cells+using+a+stepwise+strategy.">TGFbeta inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy.</a> Cell Res. 22, 194-207 (2012).</li><li>Uenishi, 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=Tenascin+C+promotes+hematoendothelial+development+and+T+lymphoid+commitment+from+human+pluripotent+stem+cells+in+chemically+defined+conditions.">Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions.</a> Stem Cell Rep. 3, 1073-1084 (2014).</li><li>Elcheva, I., 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=Direct+induction+of+haematoendothelial+programs+in+human+pluripotent+stem+cells+by+transcriptional+regulators.">Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators.</a> Nat Commun. 5, 4372 (2014).</li><li>Tiscornia, G., Singer, O., 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=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1, 241-245 (2006).</li><li>Vodyanik, M. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoendothelial+differentiation+of+human+embryonic+stem+cells.">Hematoendothelial differentiation of human embryonic stem cells.</a> Curr Protoc Cell Biol. Chapter 23, Unit 23.6 (2007).</li><li>Cao, 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=Comparison+of+gene-transfer+efficiency+in+human+embryonic+stem+cells.">Comparison of gene-transfer efficiency in human embryonic stem cells.</a> Mol Imgn and Biol. 12, 15-24 (2010).</li></ol>

IS是创始股东和顾问Cynata。

对于hPSCs通过转录因子的过表达造血分化的上述方法，表示了快速和有效的方法为HE和髓系和erytho-magakaryocytic祖细胞的产生由hESCs和iPS细胞，由此允许生产多达30百万血细胞从百万多能干细胞10。这种方法在多个人类胚胎干细胞和iPS细胞系10表现出一致的差异。在分化ETV2和GATA2，GATA1因素以及GATA2和TAL1因素，细胞形成的血液产生内皮细胞，他和经历的内皮细胞造血转变。何的形成天3和...

人多能干细胞（hPSCs）的自我更新和分化为三个胚层，包括血细胞，使它们的有价值的工具为造血发育的机理研究，血液病的模型，药物筛选的独特能力，毒性研究，以及细胞疗法的发展。因为通过内皮的造血转变1,2血液形成从hemogenic内皮细胞（HE）的胚胎进行，HE的文化的产生是必须研究调节内皮细胞造血转变和造血规格的分子机制。目前的方法进行HE的研究是基于感应hPSCs与造血-支持基质细胞6,7或在二维培养与细胞外的hematoendothelial分化的聚集体（EBS）通过加入造血细胞因子3-5，和共培养矩阵和cytokines 8,9。这些经典的差异化方法的基础上推出的作用于细胞表面和启动的分子途径，最终导致转录程序指导hematoendothelial发展激活级联外部信号。因此，hPSCs分化在这些系统的效率依赖于有效的感应这些信号，信号转导到细胞核，将得到的特定转录调节子活化。此外，他在常规分化培养物中研究需要使用细胞分选分离HE细胞的附加步骤。在这里，我们描述了一个简单的协议，直接诱导HE和血液的造血转录因子的表达。这个方法允许有效地诱导贺在培养皿中并直接观察内皮造血过渡而不使用笨重细胞分选过程的必要性，HE的隔离。何的形成和血液从人多能干细胞可以通过超表达只是几个转录因子（TF）被有效地诱导。能够从hPSCs诱导强大的泛骨髓造血转录因子的优化组合包括ETV2和GATA1或GATA2。与此相反，GATA2和TAL1的组合诱导erythromegakaryocytopoiesis 10。通过对这些因素的过度编程hPSCs分化hPSCs直接对VE-CAD + CD43 - CD73 - HE细胞逐渐获得 ​​由早期造血标志物CD43 7表达定义的造血型。这种慢病毒为基础的方法的人多能干细胞的方法的直接编程适用于生成HE和血细胞的机理研究，内皮造血过渡研究，和造血发育和说明书的转录调控。虽然C使用，类似的结果可以用改性的mRNA 10来获得转基因的组成型表达urrent协议描述血液生产。

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 1.&lt;/strong&gt; &lt;strong&gt;Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>pSIN4-EF1a-ETV2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61061</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-GATA2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61063</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-GATA1-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61062</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-TAL1-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61062</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-LMO2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61064</td><td>Lentiviral Vector</td></tr><tr><td>Hexadimethrine bromide (Polybrene)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>107689-10G</td><td>Cationic polymer used to increase the efficiency of infection</td></tr><tr><td>Y-27632 (Dihydrochloride) ROCK inhibitor</td><td>STEMCELL Technologies</td><td>72302</td><td>RHO/ROCK pathway inhibitor Inhibits ROCK</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>A11105-01</td><td>Cell Dissociation Reagent</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Incomplete (growth factor- free) culture medium&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mTeSR1 Custom formulation&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>MCF</td><td>&lt;a name=&quot;RANGE!D12&quot;&gt;Serum-free mTeSR1 medium without bFGF and TGFb&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>human SCF</td><td>Peprotech</td><td>300-07</td><td>Premium grade</td></tr><tr><td>human TPO</td><td>Peprotech</td><td>300-18</td><td>Research grade</td></tr><tr><td>human FGF-basic</td><td>Peprotech</td><td>100-18B</td><td>Premium grade</td></tr><tr><td>CD144 (VE-cad) FITC</td><td>BD Biosciences</td><td>560411</td><td>Endothelial marker (FACS)</td></tr><tr><td>CD226 PE</td><td>BD Biosciences</td><td>338305</td><td>Hematopoietic (FACS)</td></tr><tr><td>CD43 PE</td><td>BD Biosciences</td><td>560199</td><td>Hematopoietic (FACS)</td></tr><tr><td>CD73 APC</td><td>R&amp;amp;D Systems</td><td>FAB5795A</td><td>Endothelial marker (FACS)</td></tr><tr><td>CD45 APC</td><td>BD Biosciences</td><td>555485</td><td>Hematopoietic (FACS)</td></tr><tr><td>7AAD</td><td>Life Technologies</td><td>A1310</td><td>Live/Dead assay (FACS)</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Cell fixation&amp;nbsp;</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T9284-500ML&amp;nbsp;</td><td>Permeabilization&amp;nbsp;</td></tr><tr><td>FBS</td><td>Fisher Scientific</td><td>SH3007003</td><td>Fetal bovine serum</td></tr><tr><td>Mouse anti-human CD43</td><td>BD Biosciences</td><td>551457</td><td>Pure, primary antibody for Immunofluorescence (IF) staining</td></tr><tr><td>Rabbit anti-human VE-cadherin</td><td>BenderMedSystem</td><td>BMS158</td><td>Primary (IF)</td></tr><tr><td>Anti-rabbit Alexa Fluor 488-conjugated</td><td>JacksonResearch</td><td>715-486-152</td><td>Secondary (IF)</td></tr><tr><td>Anti-mouse Alexa Fluor 594-conjugated</td><td>JacksonResearch</td><td>715-516-150&amp;nbsp;</td><td>Secondary (IF)</td></tr><tr><td>DAPI nucleic acid stain</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>D1306&amp;nbsp;</td><td>Live/Dead assay (IF)</td></tr><tr><td>Clonogenic medium MethoCult H4435 Enriched</td><td>STEMCELL Technologies</td><td>4435</td><td>CFC-assay</td></tr><tr><td>Wright Stain solution</td><td>Sigma -Aldrich</td><td>32857</td><td>Staining cytospins</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 2.&lt;/strong&gt; &lt;strong&gt;hPSCs culture.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Human Pluripotent Stem Cells (hPSCs)&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>hESCs (WA01, WA09) human Embryonic Stem cells; iPSCs (DF-19-9-7T, DF-4-3-7T) transgene-free induced Pluripotent Stem Cells</td><td>hPSCs are able to self-renew and to differentiate into cells of three germ layers</td></tr><tr><td>Complete serum-free medium for culture of hPSCs&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mTeSR1 media&amp;nbsp;&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>M500&amp;nbsp;</td><td>Serum-free&amp;nbsp; medium with growth factors for feeder free culture of ESC/iPSCs</td></tr><tr><td>Matrigel</td><td>BD Biosciences/ Corning&amp;nbsp;</td><td>356234</td><td>Matrix for maintenance of human ESC/iPSCs</td></tr><tr><td>DMEM/F-12, powder&amp;nbsp;</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>12500-062</td><td>Basal Medium&amp;nbsp;</td></tr><tr><td>HyClone Dulbecco&#039;s PBS powder</td><td>Fisher Scientific</td><td>dSH30013.04</td><td>PBS</td></tr><tr><td>Dispase II, powder</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>17105-041</td><td>Neutral protease, Cell dissociation</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 3.&lt;/strong&gt; &lt;strong&gt;Primers for detection of virus genomic integration.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primer&#039;s Name&lt;/strong&gt;</td><td>&lt;strong&gt;Forward (Fwd)&amp;nbsp; 5&amp;rsquo; --&gt;3&amp;rsquo;&lt;/strong&gt;</td><td>&lt;strong&gt;Reverse (Rev) 5&amp;rsquo;--&gt;3&amp;rsquo;&lt;/strong&gt;</td><td>&lt;strong&gt;Discription&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>pSIN EF1a Fwd</td><td>TTC CAT TTC AGG TGT CGT GA</td><td>--</td><td>EF1a promoter sequence</td></tr><tr><td>GATA1 Rev</td><td>--</td><td>TCC CTG TAG TAG GCC AGT GC</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>GATA2 Rev</td><td>--</td><td>GGT TGG CAT AGT AGG GGT TG</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>TAL1 Rev</td><td>--</td><td>AGG CGG AGG ATC TCA TTC TT</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>LMO2 Rev</td><td>--</td><td>GGC CCA GTT TGT AGT AGA GGC</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>ETV2 Rev</td><td>--</td><td>GAA CTT CTG GGT GCA GTA AC&amp;nbsp;</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr></tbody></table>

direct induction of hemogenic endothelium and blood by overexpression of transcription factors in human pluripotent stem cells

在开发过程中，造血干细胞源自血管内皮细胞的一个专门的子集，hemogenic内皮细胞（HE）。建模何发展体外为内皮造血过渡和造血规范机理研究必不可少的。在这里，我们描述了用于有效诱导贺从人多能干细胞（hPSCs）由不同组的转录因子表达的方式的方法。 ETV2和GATA1或GATA2转录因子的组合被用于诱导他以泛粒细胞潜力，而GATA2和TAL1转录因子的结合允许生产的贺红系和巨核细胞潜力。加入LMO2到GATA2和TAL1组合显着加快分化，增加红系和巨核细胞的生产。这种方法提供何感应的有效且迅速的方式从hPSCs并且允许进行观测的内皮hematopoieti的在文化菜C的过渡。该协议包括hPSCs转导程序和HE的转导后分析和血液祖细胞。

HE和从hPSCs通过转录因子过度表达血诱导的示意图示于图1。ETV2与GATA1或GATA2组合诱导泛骨髓造血，而一个GATA2，TAL1 +/- LMO2组合诱导主要赤型巨核造血。这两个TF组合直接诱发HE细胞随后转变为血液祖细胞与造血细胞分化的一个独特的光谱。从多能状态血液hPSCs分化产生，他对平均4天。圆形血细胞首次出现由分化第5天。随后，无数的漂浮的血细胞出现，并扩大在文化。为了?...

Watch this Scientific Journal Video about Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells at JoVE.com

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

direct-induction-hemogenic-endothelium-blood-overexpression

Research

JoVE Journal

Developmental Biology

7.5K 次观看.  University of Wisconsin-Madison. This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors. 

视频: Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

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

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

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

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

生物学

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

从多能干细胞生成人体血管类器官

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...