茉莉Kresse的熟练技术援助深表感谢。

1.人类ESC分化对定形内胚层<ol><li>培养在培养箱中的人胚胎干细胞（ESC），在37℃和5％ 的 CO 2。 </li><li>涂覆新的6孔细胞培养板用1ml基底膜基质孵育培养餐具在RT至少30分钟。有关具体细节，请谈谈各自制造商的说明。 </li><li>确认该培养的人胚胎干细胞已达到使用低倍率（ 例如，4X）在显微镜下的80％-90％汇合。吸通过吸走培养基，用无菌玻璃巴斯德吸管从空腔的介质。用磷酸盐缓冲盐水（PBS）溶液洗涤细胞一次。对于这一点，加入2毫升的PBS至每孔轻轻摇动板和吸出溶液，以去除死细胞和细胞碎片。 </li><li>加入1毫升的无酶传代解决方案，试剂的细胞团的解离温柔。在37℃的细胞D 5％的CO 2，直到细胞显示中断的迹象明显分成小簇。 注:培养时间取决于所使用的试剂。在材料部分中提到的无酶传代溶液，温育时间大约是7分钟。 </li><li>加入1ml的DMEM / F-12培养基并通过上下抽吸用1毫升枪头破坏剩余的细胞聚集成单个细胞。使用此冲洗从表面的细胞和细胞转移到一个离心管中。检索所有细胞，用1ml的DMEM / F-12培养基中洗每个孔，并在介质加入到离心管中。 </li><li>离心细胞5分钟，在300×g的。吸出上清液并重新悬浮于5毫升的ES细胞培养基含有10μM的Rho激酶（ROCK）抑制剂的细胞。 </li><li>算使用血球在显微镜下的细胞和种子150000 - 400,000个单元格每6孔或在另一块板的布局，这取决于所用的胚胎干细胞系。使用CULTURE培养基含有10μM的ROCK抑制剂，以避免细胞凋亡和培养在培养箱中的细胞在37℃和5％的CO 2。 </li><li>大约24小时后播种抽吸介质用无菌玻璃巴斯德吸管，并加2ml原条诱导培养基中。 注意:此培养基含有高级RPMI-1640培养基1％谷氨酰胺，0.2％FCS，5μMCHIR-99021和50ng / ml激活素A的最终浓度。通常，使用两毫升培养基用于培养在6孔平板上。 </li><li>接种后48小时，更换为内胚层诱导培养基的媒介。培养细胞的另一个48小时，每天更换培养基这个网上平台。 注意:此培养基含有高级RPMI-1640培养基1％谷氨酰胺，0.2％FCS和50ng / ml激活素A。正在朝着定型内胚层致力于细胞表达表面标志CXCR4。 CXCR4的染色可以用于量化的DE-致力于细胞的数目。 </li></ol><p类="jove_title"&gt; 2。 CXCR4 +定形内胚层细胞的染色流式细胞仪分析<ol><li>收获前的最后24小时，但至少1小时的细胞中添加10μMROCK抑制剂的培养基。 </li><li>涂层的细胞培养器皿，将用于重新播种与基底膜基质， 即用于免疫荧光染色定量PCR分析或室载玻片12孔板。孵育所述板或幻灯片在室温至少30分钟。 </li><li>吸与用于染色和/或分选的分化的细胞的孔中的无菌玻璃巴斯德吸管的介质。 </li><li>加入1ml无酶传代溶液试剂为细胞团的温和解离。孵育细胞在37℃和5％的CO 2，直至细胞显示中断的明显迹象成小簇。 注:培养时间取决于所使用的试剂。在马泰提到的无酶传代溶液ALS部，温育时间大约是7分钟。 </li><li>加入1ml的DMEM / F-12培养基并破坏通过上下吹打使用1毫升尖剩余细胞聚集成单个细胞。使用此冲洗从表面的细胞和细胞转移到一个离心管中。检索所有细胞，用1ml的DMEM / F-12培养基中的w / o FCS洗每个孔，并在介质加入到离心管中。 </li><li>算使用血球在显微镜下的细胞。从6孔板的三个孔细胞应后分化三天导致10-15×10 6个细胞。离心细胞5分钟，在300×g的。 注意:得到的细胞的确切数目将取决于用于分化多能干细胞系。 </li><li>吸出上清液并重新悬浮在含有10μM的ROCK抑制剂PEB缓冲器细胞。使用100微升该缓冲液中达10 7个细胞。添加10μM的ROCK抑制剂上staini的天NG。使用该缓冲液用于所有下游应用（被称为PEB缓冲液+ RI）。 注:PEB缓冲液含有0.5％BSA和2mM EDTA的PBS中。如果有更多的细胞被染色，相应地调整缓冲体积。 </li><li>每10 7个细胞加入10微升CXCR4-APC抗体对100微升，这大致表示1:10的稀释度。 注意:一个APC连接的抗体的使用不是强制的。相反，它可以根据所用的微珠被取代。如果有更多的细胞被染色相应地调节抗体量。 </li><li>通过用手指轻轻翻转管混合并在冰箱中在4℃孵育15分钟。重悬以1-2毫升PEB缓冲器细胞。离心细胞5分钟，在300×g的。 </li><li>吸用无菌玻璃巴斯德吸管将上清液和重悬细胞沉淀在每10 7个细胞80微升的PEB，并添加20微升抗APC微珠。 注意: </s仲&gt;如果更多的细胞是被染色，相应地调整微珠体积。 </li><li>通过用手指轻轻翻转管混合并在冰箱中在4℃孵育15分钟。重悬以1-2毫升PEB缓冲液+ RI细胞。离心细胞5分钟，在300×g的。吸出缓冲器。悬浮于500μlPEB缓冲区+ RI。 </li></ol> 3. CXCR4 +细胞磁选<ol><li>放置在磁场中的中等大小的磁列按照制造指令。预冲洗用500μlPEB缓冲区+ RI列。申请的全部细胞悬浮液上柱。收集通过，因为不是所有细胞中的流动将与柱结合。确保不打扰列CXCR4 +细胞的最佳检索。 </li><li>洗柱用500μlPEB缓冲+ RI三次。通过收集第一流动并与收集的细胞来自步骤3.1相结合。从磁场去除磁列，并将其放置在一个合适的收集管。加入1ml PEB缓冲+ RI在柱子上。为了洗脱细胞柱塞用力按下到列中。 </li><li>可选的:分别收集通过样品的所有流动和使用各20微升分析使用流式细胞仪的CXCR4 +细胞的数目。其数量应与每个洗涤步骤下降。 注:在至少2×10 4个活，门控细胞应该算为可靠的结果。 </li><li>重复步骤3.1-3.3从3.1步收集流过样品，并通过使用样品新列第一，由步3.2。不要重复使用前一列。通过使用柱塞空气被压入柱，其阻断它。 </li><li>算使用血球在显微镜下的细胞。根据不同的分化达6×10 6个细胞的效率可以最初排序和另一个1×10 6个细胞使用10 7个细胞的过程时使用的第二列。 </LI&gt; <li>离心细胞，在300×g离心5分钟。吸出上清液，用无菌玻璃巴斯德吸管和来自步骤1.9与附加10μM的ROCK抑制剂悬浮在1ml内胚层的诱导培养基中的细胞。 </li><li>算使用血球在显微镜下的细胞。种子的细胞以合适的密度， 即，〜4×10 5个细胞每一个12孔板的孔（表观3.6 cm 2的表面）或者〜每8孔腔室载玻片孔1.5×10 5个细胞。 </li></ol> 4.可选:纯净定形内胚层人口分析<ol><li>对于免疫荧光染色固定来自步骤3.7纯化的细胞大致与4％多聚甲醛和染色为定形内胚层（DE）和/或多能性标记蛋白接种后24小时。 注:常用的DE标志物包括FOXA2和SOX17，常用的多能性标志物包括OCT3 / 4，NANOG和SOX2 6,8。 </li><li>佛- [R RT-qPCR的分析，收获纯化细胞直接或24小时后播种，提取总-RNA和反向从提取的总-RNA样品的cDNA转录。使用10纳克的cDNA作为每RT-qPCR的反应（一式三份）模板来分析DE和多能性标记基因6,8的表达。 注:常用的标记基因在步骤4.1提及。循环条件是95℃5分钟，并在95℃15秒的40个循环，在60℃1分钟，接着通过熔化曲线分析。 </li></ol>

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The authors declare that they have no competing financial interests.

目前使用的分化协议很少导致100％的分化的细胞。对于仍然有要解决的原因，某些细胞抗蚀分化过程。取决于所用的分化方案的效率和ESC的倾向排队甚至分化成定形内胚层后，通常观察到残余的多能细胞的一定数量。这些残余细胞会削弱下游分化或进一步的分析，如转录组学，蛋白质组学和miRNA表达分析。残留的多能干细胞或其它不想要的谱系也可以表现出可与分化目标干扰旁分泌的影响。因此，除去这些细胞可能导致改善的再现性。 CXCR4 +分化后表达内胚层细胞的纯化可用于富集这些人口并消除抵制分化过程的细胞。表面DE标志物蛋白的CXCR4可用于定形内胚层细胞的特异性纯化。眼下的MACS纯化方法可以小于2小时内完成，并可以在每一个细胞培养室简单的台式格式，无需昂贵的实验室设备和设备来执行。 FACS纯化是一种常用的技术，但采用了苛刻的条件。 FACS中纯化细胞通常保持悬浮于延长的时间周期和细胞是受其他具有挑战性的因素， 例如，在流式细胞仪装置的喷嘴的高压。在比较MACS过程是快速和温柔。这提高了细胞的纯化过程后重新播种时重新连接的能力。为了进一步简化这个复位ROCK抑制剂是之后和提纯，以防止细胞凋亡之前加入到培养基。是影响再附着的另一个重要因素是在哪些细胞被重新接种密度。这强烈依赖新生所用的细胞线DS。用于本研究中播种细胞数是在我们手中工作良好代表性的细胞数。然而，也有可能被需要为不同的细胞系调整这些数字。这两项措施，增加了一个ROCK抑制剂和细胞数的对补播的评价，是对进一步细胞培养物的分选后的成功至关重要。 随着使用DE分化协议可以不排序8获得通常&gt; 70％CXCR4 +细胞。用于DE生成协议的性能必然影响随后的MACS纯化方案的效率。在一般情况下，有效的分化（&gt; 80％的CXCR4 +细胞）MACS分选（高达95％）后得到较高纯度。因此，该纯化方法的使用减少了未分化的细胞基本上但100％纯度不能达到的数量。在未来，谱系特异性表面标记物可以被定义吨帽子将允许更终末分化细胞的纯化。在MACS分选程序是实现这一目的的主要选择。

多能干细胞，如胚胎干细胞（ESC）具有分化成几乎人体的任何细胞类型的能力。因此， 在体外分化协议可以被用于产生许多成人细胞类型，如心肌1，肝细胞2，β细胞3，肺上皮4或神经元细胞5。这使得胚胎干细胞对各种退行性疾病3的潜在治疗的有价值的工具。 胚胎干细胞的朝向肺，肝脏和胰腺的成人组织中体外分化需要一个伪原肠胚成让人想起定形内胚层（DE）6的细胞。因为向上述体细胞类型的下游分化是显著效率较低，最佳的内胚层分化被视为限速7。被朝内胚层系致力于细胞进行查在他们的基因表达谱racteristic变化。多能性主控调节基因被下调，而其他转录因子如FOXA2，SOX17，HNF1B，该GATA家族的成员和表面受体CXCR4被高度上调6，8，9。CXCR4已知由SMAD2被反式表达/ 3，交点/ TGF-β信号传导和SOX17的下游，由于在其启动子区域10特异性结合位点。因此，它是在一些报告6,8 11-13中使用的非常合适的标记。这些表达的变化反映了伪原肠事件，其中胚胎干细胞首先获取原始的条纹状的细胞群的特点，并随后提交到内胚层胚层6。 然而，分化的协议很少是100％有效，因为一些细胞可能会抵制分化过程或向其他意外谱系分化14。这些细胞可能负面influeNCE进一步分化。此外，剩余的未分化细胞海港供以后移植实验很大的风险，并可能导致畸胎瘤15-17。 除去这些不想要的细胞早期的表面标记物CXCR4的可用于该朝向DE 18致力于细胞的纯化。在这里，我们描述了从DE分化培养物的CXCR4 +细胞的阳性选择的方法。对于这一点，表面标志物CXCR4的是由再依次结合至磁微珠的抗体结合。不像FACS分选过程中的恶劣条件下，磁性标记DE样细胞然后可以很容易地在一个台式格式使用温和的纯化方法进行纯化。这个协议提供了用于抵制对DE分化过程中除去细胞群的一个简单的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Hues8 human embryonic stem cell line</td>
			<td >Harvard Department of stem cell &#38; regenerative biology</td>
			<td >NA</td>
			<td >Suitable cell line for endoderm generation</td>
		</tr>
		<tr >
			<td >Hes3 human embryonic stem cell line</td>
			<td >ES Cell International</td>
			<td >NA</td>
			<td >Suitable and robust cell line for endoderm generation</td>
		</tr>
		<tr >
			<td >mTeSR1</td>
			<td >Stemcell Technologies</td>
			<td >5850</td>
			<td >ESC culture medium</td>
		</tr>
		<tr >
			<td >FCS</td>
			<td >Biowest</td>
			<td >S1860</td>
			<td ></td>
		</tr>
		<tr >
			<td >Advanced RPMI 1640</td>
			<td >Life Technologies</td>
			<td >12633012</td>
			<td ></td>
		</tr>
		<tr >
			<td >CD184 (CXCR4)-APC, human</td>
			<td >Miltenyi Biotec</td>
			<td >130-098-357</td>
			<td ></td>
		</tr>
		<tr >
			<td >anti-APC MicroBeads</td>
			<td >Miltenyi Biotec</td>
			<td >130-090-855&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >OctoMACS Separator</td>
			<td >Miltenyi Biotec</td>
			<td >130-042-109</td>
			<td >magnetic field</td>
		</tr>
		<tr >
			<td >Y-27632</td>
			<td >Selleck Chemicals</td>
			<td >S1049</td>
			<td >ROCK inhibitor</td>
		</tr>
		<tr >
			<td >CHIR-99021</td>
			<td >Tocris Bioscience</td>
			<td >4423</td>
			<td ></td>
		</tr>
		<tr >
			<td >Activin A</td>
			<td >Peprotech</td>
			<td >120-14</td>
			<td ></td>
		</tr>
		<tr >
			<td >Gentle Cell Dissociation Reagent</td>
			<td >Stemcell Technologies</td>
			<td >7174</td>
			<td >Enzyme-free passaging solution, alternative: Trypsin/EDTA</td>
		</tr>
		<tr >
			<td >Matrigel*</td>
			<td >Corning</td>
			<td >354277</td>
			<td >basement membrane matrix 
			 * solve and store in aliquots at -80 &#176;C as outlined in the suppliers manual. Upon use, thaw on ice, dilute in 25 ml ice-cold knockout DMEM/F-12. 
Add 1 ml to each well of a 6-well plate and incubate for 45 min at room temperature. 
Remove the matrigel and use immediately.
			</td>
		</tr>
		<tr >
			<td >MS Columns</td>
			<td >Miltenyi Biotec</td>
			<td >30-042-201</td>
			<td ></td>
		</tr>
		<tr >
			<td >MACS Separator</td>
			<td >Miltenyi Biotec</td>
			<td >130-042-302</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human FOXA2 FW gggagcggtgaagatgga</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human FOXA2 REV tcatgttgctcacggaggagta</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human GSC FW gaggagaaagtggaggtctggtt</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human GSC REV ctctgatgaggaccgcttctg</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >SOX17 TaqMan assay</td>
			<td >Applied Biosystems</td>
			<td >Hs00751752_s1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human SOX7 FW gatgctgggaaagtcgtggaagg</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human SOX7 REV tgcgcggccggtacttgtag</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human POU5F1 FW cttgctgcagaagtgggtggagg</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human POU5F1 REV ctgcagtgtgggtttcgggca</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human Nanog FW ccgagggcagacatcatcc</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human Nanog REV ccatccactgccacatcttct</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human TBP FW caa cag cct gcc acc tta cgc tc</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human TBP REV agg ctg tgg ggt cag tcc agt g</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human TUBA1A FW ggc agt gtt tgt aga ctt gga acc c</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human TUBA1A REV tgt gat aag ttg ctc agg gtg gaa g</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human G6PD FW agg ccg tca cca aga aca ttc a</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Human G6PD REV cga tga tgc ggt tcc agc cta t
</td>
			<td >Life Technologies</td>
			<td >NA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-SOX2</td>
			<td >Santa Cruz Biotechnology</td>
			<td >sc-17320</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-FOXA2</td>
			<td >MerckMillipore</td>
			<td >07-633</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-SOX17</td>
			<td >R&#38;D Systems</td>
			<td >AF1924</td>
			<td ></td>
		</tr>
	
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >NA = not applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a quick and efficient method for the purification of endoderm cells generated from human embryonic stem cells

多能干细胞，如胚胎干细胞（ESC）的分化能力允许对细胞替代疗法的潜在的治疗应用。终末分化的细胞类型可以用于各种退行性疾病的治疗。 在体外 ，这些细胞向肺，肝脏和胰腺的组织的分化需要作为第一步定型内胚层细胞的产生。这个步骤是限速为朝向末端成熟细胞类型，如产生胰岛素的β细胞，肝细胞或其它内胚层来源的细胞类型进一步分化。即朝向内胚层谱系定型细胞高度表达的转录因子如FOXA2，SOX17，HNF1B，该GATA家族的成员，且表面受体CXCR4多种。然而，分化的协议很少是100％的效率。这里，我们描述了一种用于一CXCR4阳性细胞群的分化后的纯化入DE通过使用磁微珠。这种净化此外删除不需要的谱系的细胞。平缓的纯化方法是快速，可靠的，并且可以用于改善下游应用和分化。

在分化时 胚胎干细胞进行基因和蛋白质表达的急剧变化。 图1描绘了可用于验证成功定形内胚层分化典型的标记基因。对于基因表达分析的主要目标是GSC，FOXA2和 SOX17在一个相对的基因表达分析尤其是FOXA2和SOX17相比未分化的胚胎干细胞时，增加了&gt; 2000倍。GSC原条的形成过程中已经表示24小时内很早但它仍然引起第四天&gt; 100倍。这些基因的表达被在使用时的CXCR4的表面标记6排序随之增加。多能性主调节剂如OCT3 / 4（POU5F1）和NANOG是显著下调虽然这些基因的表达仍然是四天后可检测的。这籼稻有些细胞不充分与CHIR-99021和激活A. SOX7基因表达的治疗方案作出回应工商业污水附加费通常是给歧视DE胚外内胚层的形成。在第一种情况下SOX17是共表达与SOX7和在后一种情况下SOX17是共表达与FOXA2。 图1中的结果表明，与DE沿着一些胚外细胞可能分化过程中形成的，尽管我们尚未能染色SOX7 +细胞。 图2示出了不同细胞群体是胚胎干细胞分化成的DE后存在。在步骤1中胚胎干细胞接种为单细胞，然后通过用CHIR-99021和激活素A分化为四天。将DE的IF染色标记物SOX17和FOXA2（ 图2A）表明，这两种标志物是均匀地共expresse核D中。这被认为是DE承诺的标志。然而，某些细胞抗蚀分化过程，并表示无论两个DE标记蛋白（ 图2A）的。事实上，两个不同的细胞群体四天分化后观察。虽然许多细胞表达DE标记FOXA2仍有剩余表达该多能性标记物SOX2（ 图2B）的细胞。这两种蛋白质在两个独立的群体所表达并可以观察到（ 图2B）没有共表达。 在第三天或分化四个表面蛋白的CXCR4用于排序的DE-致力于CXCR4 +群体，从而除去剩余的多能干细胞和其它不想要的谱系。取决于所用的细胞系的分化效率&gt; 80％的CXCR4 +细胞可使用在步骤1中8所概述的分化方案来获得</suP&gt;。这里使用的HES3线得到66％±3％的CXCR4阳性细胞向DE分化后排序后的排序和92％±4％（n = 3时）。 图3染色和CXCR4的MACS纯化后描绘流式细胞术数据+细胞之前示出了使用免疫荧光（IF）染色前和MACS纯化后共同DE和多能性标记蛋白的表达。分化得到（ 图3A，中图），使用在步骤2中描述的CXCR4的免疫荧光染色大约60％的CXCR4 +细胞三个一天。 CXCR4 +细胞在第四季度与APC结合抗CXCR4抗体染色移动到Q1。在MACS纯化步骤3中描述后，CXCR4 +群体富集到85％。纯化过程，然而，这并不排除从培养所有不需要的谱系（ 图3A，右图）。 在MACS纯化后，的CXCR4阳性细胞可以接种用于进一步分析或任选可以进行第二轮分选，以得到更高的纯度，但减少的生存能力。分化多能性标记SOX2（绿色染色）的广泛的表达在此之前由IF染色检测。与此相反，DE标志物FOXA2（红色染色）不能被检测（ 图3B）。在图3C中的CXCR4 +细胞染色与FOXA2（绿色）的抗体和被共染色的多能性标记物SOX2（红色）。纯化CXCR4 +细胞接种后检测到只有少数SOX2 +细胞。大多数种子细胞是积极为DE标志FOXA2。 <img alt="图1" src="/files/ftp_upload/53655/53655fig1highres.jpg" width="700" /> 图四天定形内胚层分化期间1中不同的多能性和定形内胚层标志物的基因的基因表达的代表性的变化。 （一个） </被用于内胚层分化后的基因表达分析的协议STRONG&gt;插图。随机是指以培养条件没有任何生长因子，RP使用引用协议7，用激活素A孤身独处的治疗和CA-A表示的分化（CHIR-99021和激活素A）的协议，该协议用 ​​于MACS排序（见8详情）。在CA-A协议使用的媒体都在这里被称为原条诱导培养基和内胚层诱导培养基。（B）描绘由GSC，SOX17和FOXA2的RT-qPCR的，这是没有根据定型内胚层的承诺表示测量的基因表达进一步MACS分选。POU5F1（OCT3 / 4）和NANOG是多能性调节剂和在分化通常下调，而SOX7在胚外内胚层中表达。基因表达标准化对三名稳定表达的看家基因（&lt;EM&gt; TBP，TUBA1A，G6PD）导致CNRQ值。在未分化的细胞中的上述基因的表达设定为1和变化被绘制为基因表达的倍数变化。数值是平均值±SEM，N = 4-5。 ANOVA加邦弗朗尼的事后检验。 ** P≤0.01随机和＃P≤0.05相比，## P≤0.01与RP（此图中的所有数据最近被刊登在8）进行比较。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53655/53655fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53655/53655fig2.jpg" /> 图内胚层分化后2.不同的细胞群。 （一）DE染色标志物SOX17和分化的D4各自的抗体FOXA2揭示其均匀的共定位。然而，一些抵制的细胞分化过程，不表达这些标记（仅适用于蓝核染色）。绿色和红色染色的合并示于黄色。（B）的 DE分化后有两种不同的细胞群。 DE样细胞表达FOXA2而该抵制的分化过程仍细胞表达的多能性标记SOX2。 （AB）细胞核用DAPI（蓝色）复染色。在面板（A）比例尺是50微米，并在面板（B）为100微米。成像在670纳米（红，cy5），520纳米（绿色，FITC）和433纳米（蓝色，DAPI）进行，分别为。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53655/53655fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53655/53655fig3highres.jpg" width="700" /> 图3. MACS DE分化的细胞群体和代表性ST排序排序后癌宁。 （A）+ CXCR4染色细胞（Q1）的MACS排序后充实。 CXCR4-细胞（Q4）的数量减少。取决于细胞系所使用的分化方案收率&gt; 80％的CXCR4阳性细胞8和MACS可以用来进一步丰富它们。（B）的未分化细胞表达多能标记物SOX2（绿色），而不是对DE标记FOXA2（红）。（C）的MACS分选只有极少数未分化SOX2 +细胞后留（红色），而排序的人口几乎一致地表达DE标志FOXA2（绿色）。 （BC）核用DAPI（蓝色）。在面板（C）比例尺为100μm，并适用于所有的面板。成像在670纳米（红，cy5），520纳米（绿色，FITC）和433纳米（蓝色，DAPI）进行，分别为。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53655/53655fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells at JoVE.com

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

在这里，我们描述了该朝向为下游应用和进一步分化的改善的定形内胚层致力于分化的人类胚胎干细胞的纯化方法。

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

a-quick-efficient-method-for-purification-endoderm-cells-generated

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.3K 次观看.  Hannover Medical School. 在这里，我们描述了该朝向为下游应用和进一步分化的改善的定形内胚层致力于分化的人类胚胎干细胞的纯化方法。 

视频: A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

科研

发育生物学

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

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

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

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

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

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.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...