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>

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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 1. Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.
<table>
	<tbody>
		<tr>
			<td>pSIN4-EF1a-ETV2-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61061</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-GATA2-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61063</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-GATA1-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61062</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-TAL1-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61062</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>pSIN4-EF1a-LMO2-IRES-Puro&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #61064</td>
			<td>Lentiviral Vector</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide (Polybrene)&#160;</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 rowspan="2">StemPro Accutase Cell Dissociation Reagent</td>
			<td rowspan="2"><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td rowspan="2">A11105-01</td>
			<td rowspan="2">Cell Dissociation Reagent</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Incomplete (growth factor- free) culture medium&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; mTeSR1 Custom formulation&#160;</td>
			<td>WiCell Research Institute (Madison, WI)</td>
			<td>MCF</td>
			<td><a name="RANGE!D12">Serum-free mTeSR1 medium without bFGF and TGFb&#160;</a></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&#38;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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>Cell fixation&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML&#160;</td>
			<td>Permeabilization&#160;</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&#160;</td>
			<td>Secondary (IF)</td>
		</tr>
		<tr>
			<td>DAPI nucleic acid stain</td>
			<td><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td>D1306&#160;</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>
	</tbody>
</table>

Table 2. hPSCs culture.
<table>
	<tbody>
		<tr>
			<td>Human Pluripotent Stem Cells (hPSCs)&#160;</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&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; mTeSR1 media&#160;&#160;</td>
			<td>WiCell Research Institute (Madison, WI)</td>
			<td>M500&#160;</td>
			<td>Serum-free&#160; medium with growth factors for feeder free culture of ESC/iPSCs</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences/ Corning&#160;</td>
			<td>356234</td>
			<td>Matrix for maintenance of human ESC/iPSCs</td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder&#160;</td>
			<td><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td>12500-062</td>
			<td>Basal Medium&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s PBS powder</td>
			<td>Fisher Scientific</td>
			<td>dSH30013.04</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td rowspan="2">Dispase II, powder</td>
			<td rowspan="2"><a href="http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html">Life Technologies</a></td>
			<td rowspan="2">17105-041</td>
			<td rowspan="2">Neutral protease, Cell dissociation</td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>
Table 3. Primers for detection of virus genomic integration.
<table>
	<tbody>
		<tr>
			<td>Primer&#39;s Name</td>
			<td>Forward (Fwd)&#160; 5&#8217; --&#62;3&#8217;</td>
			<td>Reverse (Rev) 5&#8217;--&#62;3&#8217;</td>
			<td>Discription&#160;</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>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>GATA2 Rev</td>
			<td>--</td>
			<td>GGT TGG CAT AGT AGG GGT TG</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>TAL1 Rev</td>
			<td>--</td>
			<td>AGG CGG AGG ATC TCA TTC TT</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>LMO2 Rev</td>
			<td>--</td>
			<td>GGC CCA GTT TGT AGT AGA GGC</td>
			<td>&#160;Coding Region&#160;</td>
		</tr>
		<tr>
			<td>ETV2 Rev</td>
			<td>--</td>
			<td>GAA CTT CTG GGT GCA GTA AC&#160;</td>
			<td>&#160;Coding Region&#160;</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

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

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

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 ...

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.