我们感谢莫纳什大学的 StemCore 设施提供 hESC 线路，以及莫纳什大学的 FlowCore 设施，为 FACS 服务提供服务。 这项工作得到了澳大利亚干细胞中心、新南威尔士州和维多利亚州政府干细胞研究资助计划的研究资助。ALL 是澳大利亚研究理事会 （ARC） 干细胞科学特别研究计划（澳大利亚干细胞）的合作伙伴研究员。

以下协议使用莫纳什大学（墨尔本）的 StemCore 设施提供的 hESC-MEL111 标准批量培养物执行。这个细胞系通常培养在bFGF补充的hESC/KOSR介质7 中一层不活化小鼠胚胎成纤维细胞（MEF），并每5-7天8天用酶分离（拼贴酶）维持。hESC 培养物在 75 厘米2 （T75） 烧瓶中增长到约 80% 的汇合度，用作此协议的输入量。下文描述的所有细胞操作程序应在 HEPA 过滤的 II 类生物安全柜中在无菌条件下执行。
在执行 TG30/GCTM-2 FACS 检测的两天前，准备 T75 培养板（第 1 节中描述），用于对后 FACS 恢复的细胞进行再培养（第 4 节中描述）。
1. MEF 的播种和 MEF 条件介质的准备
1.1 天 -2： 将 MEF 镀入 T75 烧瓶
<ol><li>派珀特 10 毫升 0.1% w/v 明胶到每个 T75 烧瓶和倾斜到涂层表面均匀。</li>
	<li>在室温下在生物安全柜中孵化 30 分钟。</li>
	<li>吸气明胶溶液。</li>
	<li>在细胞培养孵化器中加入 20 毫升 MEF 介质，在细胞培养孵化器中预平衡至 37 °C/5% CO2 30 分钟。</li>
	<li>板在以下密度下，在准备好的烧瓶上断断续续地灭活 MEF。对于 1/3 MEF 密度种子 1.4 x 104 细胞/厘米2 （T75 = 1 x 106 MEF）。对于全密度 MEF 种子 5.3 x 104 细胞/厘米2 （T75 = 4 x 106 MEF）。</li>
</ol>注意：我们经常使用被γ辐照而失活的 MEF。在米哈尔斯卡12号可以找到一个准备γ辐照的MEF的议定书。
<ol start="6"><li>在 37 °C/5% CO2 过夜孵化辐照 MEF 培养。1~3个烧瓶可以孵育1-4天，无需MEF中等变化。这些烧瓶用于重新镀上后 FACS 细胞（如协议 4 中）。完整的 MEF 烧瓶在一夜之间孵育，以便按照下面步骤 1.2 生成 CM。</li>
</ol>1.2 天 -1： 准备 MEF 条件介质 （CM）
<ol><li>从全 MEF T75 烧瓶中吸气 MEF 介质。</li>
	<li>添加预均衡 25 毫升 hESC/KOSR 介质，辅以 5 ng/ml 人类 FGF-2 每 T75 烧瓶。24 小时在 37 °C/5% CO2 中孵化。</li>
	<li>将 MEF 调节介质 （CM） 收集到无菌组织培养管中，用 10 ng/ml 人类 FGF-2 新鲜补充 CM，使用 0.22 μm 聚醚硫酮过滤器进行过滤。</li>
	<li>要生成额外的 CM，在一周内，每天重复同一 MEF 文化上的步骤 1.2.2 和 1.2.3。</li>
</ol>在执行 TG30/GCTM-2 检测的当天，用 CM 替换 1/3 MEF T75 烧瓶中的 MEF 介质，并在播种从以下程序中恢复的任何 hESC 或 hESC 衍生细胞之前预平衡至少 30 分钟。CM 要么使用新鲜，要么存储在 4 °C 下长达 2 周。与非条件介质（未显示结果）相比，CM 在我们手中使用，它增加了 FACS 后细胞的生存能力。
2. 执行 TG30/GCTM-2 FACS 分析：将 hESC 批量培养物分离到单个单元格中
注意： 前丘尔 20% FBS/DMEM-F12 介质在 4 °C。
<ol><li>从两个带有培养 hESC 线的 T75 烧瓶中吸气 hESC/KOSR 介质。</li>
	<li>用 10 毫升 DPBS 冲洗每个 T75 中的细胞，并吸气去除。</li>
	<li>在每个 T75 烧瓶中加入 3 毫升 TrypLE 快速分离解决方案。孵化在37°C/5%CO2 5分钟。</li>
	<li>凹凸烧瓶的侧面，以获得细胞的完全脱落。在显微镜下检查。如果细胞不容易分离，在37°C/5%CO 2下再孵育2-3分钟。</li>
	<li>在每个 T75 烧瓶中加入 10 毫升 20% FBS/DMEM-F12（保持在 4 °C）。使用无菌的 10 毫升移液器将分离的细胞轻轻三次对烧瓶壁进行三次，以实现以单细胞为主的悬架。</li>
	<li>将 70μm 细胞过滤器放在 50 毫升无菌聚丙烯管上。使用无菌的 10 毫升移液器，迫使从两个 T75 烧瓶中汇集的 hESC 单细胞悬架穿过过滤器。</li>
	<li>丢弃电池过滤器，更换管盖，将池式 hESC 悬架在 1，000 x g 中离心机 2 分钟。</li>
	<li>丢弃超自然物，通过在 10 毫升 20% FBS/DMEM-F12 中轻轻重新悬念颗粒（预制在 4 °C）来清洗细胞。</li>
	<li>在 1，000 x g 下进行第二次离心，2 分钟。</li>
	<li>丢弃超自然，加入10毫升20%FBS/DMEM-F12（预制在4°C），轻轻三酯以补充细胞。将50毫升管放在冰上。此管将用于下面描述的 排序 样品 免疫染色程序。</li>
	<li>使用血细胞仪计算细胞号，使用 hESC 单细胞悬架 （步骤 2.10） 的 20 μl 进行计数。您应该期望每 T75 烧瓶的产量为 1000 万至 1500 万个电池。</li>
</ol>注意：FBS 用于协议的这一部分，以增加 FACS 后的细胞生存能力。
3. 细胞表面抗原TG30和GCTM-2的免疫荧光染色
<ol><li>将 hESC 单细胞悬架（从步骤 2.11）的 150 μl（约 100，000 个细胞）放入 6 个 1.5 毫升的微中微管中。这六个辅助报价将用于用于校准 FACS 机器上检测所需的染色控制。其余约9毫升细胞悬架将是您的排序样本，其中分离培养将成本检测TG30和GCTM-2，根据表1和2。</li>
	<li>在 20% FBS/DMEM-F12 中执行 主要抗体 的结合反应。排序样本的最终反应量应为~9毫升，控件应为200 μl。将等型控制作为主要抗体。将管子水平放置在冰上，在摇动平台上轻轻混合30分钟。</li>
	<li>离心机原抗体反应在1，000 x g为2分钟。</li>
	<li>丢弃超纳特，通过将颗粒重新悬念在 20 毫升 20% FBS/DMEM-F12（预制在 4 °C）中用于排序样本和 200 μl 用于对照组，从而清洗细胞。</li>
	<li>在 1，000 x g 下进行第二次旋转 2 分钟。</li>
	<li>在 20% FBS/DMEM-F12 中执行 二次抗体 的结合反应。排序样本的最终反应量应为 2.5 毫升，控件应为 200 μl。在此阶段准备PE防鼠标CD90.2反应管。将管子水平放置在冰上，在摇动平台上轻轻混合30分钟。</li>
</ol>注意：在此孵化期间，收集CM并准备 1/3 MEF T75 烧瓶（如第 1.2 节）。
<ol start="7"><li>离心机二次抗体反应在1，000 x g为2分钟。</li>
	<li>丢弃超自然物，通过在 20 毫升 20 毫升 20% FBS/DMEM-F12（预制在 4 °C） 中的颗粒和 200 μl 的对照组中清洗两次细胞。</li>
	<li>每次洗涤后，在 1，000 x g 处旋转 2 分钟，并将管子放在冰上。</li>
	<li>在 20% FBS/DMEM-F12 中丢弃超纳特并重新喷出细胞颗粒，并以 0.3μg/ml 的速度补充碘化物 （PI）。使用 2-3 毫升的排序样本和 300 μl 的控件。</li>
</ol>注意：不要将 PI 添加到带未染色细胞的控制管中。
<ol start="11"><li>使用 P1000 微皮，通过 35 μm 盖子细胞过滤器强制每个单元格悬架，并耦合到 5 毫升管聚苯乙烯圆形底部试管。</li>
	<li>离心机在50克/分钟，迫使通过残余细胞悬浮在过滤器盖上。</li>
	<li>通过敲击管的两侧，并放置在冰上，恢复每个单元格悬架。您的细胞现在已准备好使用 FACS。请立即出发。</li>
</ol>4. 75 厘米 2 （T75） 火焰中的 TG30/GCTM-2 子折射单元的后 FACS 培养
<ol><li>我们以前曾报告过如何为TG30/GCTM-2免疫扩散7建立一个FACS机器。该程序允许恢复可行的单 HESC，没有 MEF，并在 P4 （TG30Neg-GCTM-2Neg）的大门内， P5 （TG30低- GCTM-2低）， P6 （TG30中- GCTM-2中） 和 P7 （TG30Hi- GCTM-2喜） - 见 图 1E。设置 TG30/GCTM-2 FACS 的 FACS 机器，就像我们之前的报告中一样，以恢复接下来使用的单元子字体。</li>
	<li>在从 FACS 机器收集细胞之前，在两个 5 毫升聚苯乙烯圆形底部试管中加入 1 毫升 CM，按步骤 1.2 进行制备。放在冰上。</li>
	<li>使用 TG30/GCTM-2 FACS 从分化 P4 （TG30Neg-GCTM-2Neg）和多能 P7 （TG30Hi-GCTM-2Hi）细胞子解体中各恢复 400，000 个细胞。</li>
	<li>以 1，000 x g 的速度将收集管离心 5 分钟。</li>
	<li>吸气超人。加入 1 毫升预平衡 37 °C/5% CO2 CM，使用 P1000 微管来补充每个细胞颗粒。将每个分数的细胞播种到预平衡的 1/3 MEF T75 烧瓶上，并使用 CM（按步骤 1.2 准备）</li>
	<li>在细胞培养孵化器中，24 小时内的 37 °C/5% CO 2 培养。</li>
	<li>每天吸气CM，代之以新准备的CM（如步骤1.2），持续约两周。多能P7（TG30 Hi-GCTM-2Hi）细胞的培养在一周后显示人类干细胞状的菌落，并在9-11天内达到+80%的汇合。P4（TG30 Neg-GCTM-2Neg）细胞的培养大多生长成成纤维细胞状细胞的草坪，在11-13天内达到共融。在此阶段，如果需要，P4 和 P7 细胞培养可用于 TG30/GCTM-2 FACS 调解 P4 或 P7 子折片的连续再造（见图 2和3）。</li>
</ol>

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没什么可透露的

在临床环境中，分化体细胞类型是移植和治疗应用的最终所需产品，hPSC 是自更新和可扩展的来源，可在实验室中生成这些体细胞或其祖先。在考虑将 hESC 衍生的体细胞移植到患者中时，存在具有特拉托马形成固有潜力的残余未分化 hESC 是一种安全风险。有关此风险和与细胞治疗相关的其他风险的回顾，请参阅夏普等人。16因此，要实现这种应用，研究人员必须致力于产生目标细胞群，这些细胞群也是无多能干细胞的。为此，我们在此证明，使用我们的 TG30/GCTM-2 FACS 方法，可以监控差异化细胞群中多能细胞 （TG30 Hi-GCTM-2Hi）的存在，并获得可行的 hESC 差异化衍生物，这些衍生物可以在 FACS 后得到丰富和再培养。虽然不可能获得100%多能干细胞无样本与我们的协议的单一通行证，丰富的hESC衍生物可以重新培养，并实现连续通过100%体细胞纯度后。体细胞在FACS后扩张的证明可行性也有可能使生产出足够数量的适合潜在移植疗法的细胞。
这项试验性研究的令人鼓舞的结果促使我们使用TG30/GCTM-2协议，在一项比较研究中，与几组人类诱导多能干细胞（hiPSC）线15进行更全面的研究。在这项研究中，我们发现，使用TG30/GCTM-2 FACS协议获得的多能干细胞无干细胞样本在移植到免疫缺陷小鼠体内时不会产生畸形。因此，我们可以自信地指出，使用这种体外协议，早期分化细胞培养确定是自由多能TG30 Hi-GCTM-2Hi细胞不发展在体内的畸形。这强烈支持我们的检测的稳健性，提供一种潜在的方法，以消除在临床应用中使用 hPSC 衍生细胞之前形成畸胎瘤的风险。
相反，我们还表明，TG30/GCTM-2 FACS 检测可用于检索具有高表达这两种表面抗原（TG30Hi-GCTM-2Hi）的多能细胞群。此前的研究表明，TG30Hi-GCTM-2Hi细胞 与OCT4高表达的相关性最高，形成HESC类菌落数量最多的5，6，13，15。因此，我们推断，多能网络在 TG30 Hi-GCTM-2高表达单元中在其最大级别上激活。我们认为，使用我们的 TG30/GCTM-2 FACS 协议并检索 P7 （TG30 Hi-GCTM-2Hi）单元可以大规模净化丰富的活 hESC 培养物，作为分化测定输入。
最后，我们在这里展示了基于表面抗原TG30和GCTM-2的结合表达，我们FACS检测的潜力，用于清除和丰富 hESC。今后与HPSC定向区分协议的研究也可能对这一潜力提供信息。我们知道，在某些分析中，如果区分的 hESC 衍生物会暂时表达 TG30 （CD9） 或 GCTM-2，那么这两种抗体可能不适合或不足以在测定6中的特定时间点从差异化人群中清除多能细胞。因此，目前需要找到专门识别活hESC的新抗体，以克服与某些分化细胞类型的交叉反应的可能限制。

HPSC （hPSC） 包括人类胚胎干细胞 （hESC） 和人类诱导多能干细胞 （hIPSC）。HPSC 可以在适当的文化条件下无限期地自我更新，而不会失去被称为"多能"的能力。多能性被定义为细胞分化成基本上任何体细胞系的潜力，包括代表三个胚胎生殖细胞层各部分的细胞。hPSC 的非凡潜力为包括治疗方案在内的多种基于细胞的应用提供了一种手段。例如，有些疾病涉及细胞死亡和退化，人体细胞的正常功能受到损害，如心力衰竭、脊髓损伤、糖尿病、帕金森病、一些癌症和其他临床病理。患有这些疾病的患者可以通过移植实验室中从 hPSC 中提取的健康和功能性体细胞来治疗。然而，目前的hPSC分化协议不是100%有效，并产生差异化目标和非目标细胞类型的混合物，以及剩余的hPSC没有经过分化，而是继续自我更新1-3。在用于移植到患者体内的hPSC衍生体细胞样本中，即使存在少量的hPSC，也构成严重的临床风险，因为这些细胞因其固有性质而形成所有三个胚胎生殖层的组织，有可能 在体内 形成一种称为畸胎瘤的肿瘤。因此，只有确定没有多能干细胞的目标细胞群体才能被视为安全移植到患者身上。据报道，有几种方法可以潜在地完成差异化后残留 hPSC 的清除（请参阅波兰科和拉斯莱特4）。我们以前曾报告使用单克隆抗体TG30（CD9）和GCTM-2耦合荧光激活细胞分拣（FACS），以确定多能干细胞，并区别于在hESC线5-7培养物中处于分化早期阶段的细胞。
使用抗体检测细胞表面抗原的一个优点是，目标细胞通常在抗体结合和/或标记后可行。因此，靶细胞可以在抗体标记后采集，并在移植前进行再培养，以扩大和进一步应用。hPSC上表达的细胞表面抗原的一个警告是，它们并不仅限于多能阶段，在许多情况下在发育过程中会暂时重新表达，因此将在某些分化细胞类型中检测到。因此，如果目的是使用抗体来检测人类多能细胞，并从 hPSC 衍生细胞样本中清除它们，则选定的抗体也不应与用于移植的特定分化细胞类型的抗原发生反应。不幸的是，在活hPSC 4上检测细胞表面标记的抗体数量有限，使得选择的选择有限。此外，一些研究指出，检测一个单一的细胞表面标记不足以消除所有hPSC，建议任何试图消除所有hPSC多能亚人口应依靠使用两个或两个以上的抗体检测hPSC 9-10表达的不同表皮的方法。如上所述，只有hPSC衍生细胞，可以确定为多能干细胞无细胞群适合人类移植。通过抗体介导细胞分拣技术，一次通过一次传递，就不可能达到这种细腻程度。可能需要对分化目标细胞的丰富种群进行再培养，并随后进行细胞分类，以最终获得多能干细胞无样本。
在我们的实验室中，我们广泛鉴定了两种 hES 细胞表面抗体 TG30 （CD9） 和 GCTM-2，用于检测活多能细胞。我们的研究表明，TG30和GCTM-2的结合检测与hESC线5-7中规范多能相关基因的表达密切相关。TG30/GCTM-2 FACS免疫规划一直表明，HESC培养物构成TG30/GCTM-2表达5-7的定量连续梯度。我们任意建立了四个种群（P）细胞在这个TG30/GCTM-2梯度内：P4（TG30内格-GCTM-2内），P5（TG30低-GCTM-2低），P6（TG30中-GCTM-2中）和P7（TG30 Hi-GCTM-2 Hi）5-7。我们对这些P4、P5、P6和P7细胞群的特征表明，P6和P7子基层表示了大量多能相关基因，并在FACS 2-3后重新培养时有效地形成类似干细胞的菌落。另一方面，P4（TG30Neg-GCTM-2Neg）细胞表示大量的分化标记，并构成自发分化的细胞类型，通常发生在hESC线5-6的扩展培养中。我们决定测试我们的TG30/GCTM-2 FACS在早期分化后选择性地消除残留的hPSC的潜力，以及丰富多能干细胞种群。下文所述的协议显示了如何收集和重新培养差异化的P4（TG30Neg-GCTM-2Neg）细胞后FACS完成多能P7（TG30 Hi-GCTM-2嗨）的清除。此外，我们还解释了多能P7（TG30 Hi-GCTM-2Hi）细胞的收集和再培养，以获得多能细胞的丰富培养，随后可用作定义的输入量，以潜在地提高分化检测的效率和一致性。

<table border="1">
	<tbody>
		<tr>
			<td>Short Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Long Name </td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320082</td>
			<td>Long Name: Dulbecco&#39;s Modified Eagle Medium:Nutrient Mixture F-12</td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td>Long Name: Knockout Serum Replacement</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td>Long Name: GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td>Long Name: MEM Nonessential Amino Acids Solution</td>
		</tr>
		<tr>
			<td>Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td>Long Name: 2-Mercaptoethanol</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190250</td>
			<td>Long Name: Dulbecco&#39;s Phosphate-Buffered Saline, no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Life Technologies</td>
			<td>12604039</td>
			<td>Long Name: TrypLE Express (1x), no Phenol Red</td>
		</tr>
		<tr>
			<td>AF-488</td>
			<td>Life Technologies</td>
			<td>A21131</td>
			<td>Long Name: Alexa Fluor 488 goat anti-mouse IgG2a</td>
		</tr>
		<tr>
			<td>AF-647</td>
			<td>Life Technologies</td>
			<td>A21238</td>
			<td>Long Name: Alexa Fluor 647 goat anti-mouse IgM</td>
		</tr>
		<tr>
			<td>PE-anti IgG2a</td>
			<td>Life Technologies</td>
			<td>P21139</td>
			<td>Long Name: R-Phycoerythrin Goat Anti-Mouse IgG2a (&#947;2a) Conjugate</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>Long Name: Dulbecco&#39;s Modified Eagle Medium, high glucose</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Long Name: Penicillin-Streptomycin, Liquid</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Life Technologies</td>
			<td>15230-089.</td>
			<td>Long Name: Sterile Distilled Water</td>
		</tr>
		<tr>
			<td>PI</td>
			<td>SIGMA</td>
			<td>P4864</td>
			<td>Long Name: Propidium iodide solution</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>SIGMA</td>
			<td>12203C</td>
			<td>Long Name: Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>SIGMA</td>
			<td>G-1890</td>
			<td>Long Name: Gelatin from porcine skin</td>
		</tr>
		<tr>
			<td>Human FGF-2</td>
			<td>Millipore</td>
			<td>GF003AF-100UG</td>
			<td>Long Name: Fibroblast Growth Factor basic, human recombinant, animal-free</td>
		</tr>
		<tr>
			<td>Filter 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td>Long Name: Stericup-GP, 0.22 &#956;m, polyethersulfone, 250 ml, radio-sterilized</td>
		</tr>
		<tr>
			<td>70 &#956;m Cell Strainer</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td>Long Name: Cell strainer with 70 &#956;m Nylon mesh</td>
		</tr>
		<tr>
			<td>35 &#956;m Lid cell strainer, 5 ml tube</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td>Long Name: 5 ml polystyrene round bottom test tube, with a cell strainer cap (35 &#956;m)</td>
		</tr>
		<tr>
			<td>5 ml Polystyrene round bottom test tube</td>
			<td>Becton Dickinson</td>
			<td>352054</td>
			<td>Long Name: 5 ml polystyrene round bottom test tube, sterile</td>
		</tr>
		<tr>
			<td>IgG2a Isotype Control</td>
			<td>Becton Dickinson</td>
			<td>554126</td>
			<td>Long Name: Purified Mouse IgG2a, &#954; Isotype Control</td>
		</tr>
		<tr>
			<td>IgM Isotype Control</td>
			<td>Becton Dickinson</td>
			<td>553472</td>
			<td>Long Name: Purified Mouse IgM, &#954; Isotype Control</td>
		</tr>
		<tr>
			<td>PE-CD90.2</td>
			<td>Becton Dickinson</td>
			<td>553014</td>
			<td>Long Name: PE Rat Anti-Mouse CD90.2</td>
		</tr>
		<tr>
			<td>8-peaks calibration</td>
			<td>Becton Dickinson</td>
			<td>559123</td>
			<td>Long Name: Rainbow Calibration Particles (8 peaks)</td>
		</tr>
		<tr>
			<td>50 ml sterile Polypropylene tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>Long Name: 50 ml Polypropylene tube with conical bottom, Sterile</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td>Long Name: CELLSTAR Filter Cap Cell Culture 75 cm2 Flasks</td>
		</tr>
		<tr>
			<td>TG30</td>
			<td>Merck Millipore</td>
			<td>MAB4427</td>
			<td>Long Name: TG30 Antibody, clone TG30</td>
		</tr>
		<tr>
			<td>GCTM-2</td>
			<td>Merck Millipore</td>
			<td>MAB4346</td>
			<td>Long Name: GCTM-2 is not commercially available but TG343 and equivalent monoclonal antibody is</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. List of materials and reagent</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>FACSVantage SE with FACSDiva option</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>IX71</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 5. List of equipment.</td>
		</tr>
	</tbody>
</table>

enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies tg30 and gctm-2

人类胚胎干细胞（hESC）可以在体外无限期地自我更新，并且可以诱导适当的线索分化成潜在的所有体细胞谱系。差异化的 hESC 衍生物可能用于移植疗法，以治疗各种细胞退行性疾病。然而，hESC 分化协议通常产生差异化目标和非目标细胞类型以及残余未分化细胞的混合物。对于区分 hESC 衍生物从实验室到诊所的翻译，重要的是能够区分未分化（多能）和分化细胞，并生成分离这些群体的方法。hESC 衍生体细胞类型的安全应用只能通过多能干细胞无细胞种群来实现，因为残余 hESC 可以在移植后诱发称为畸形的肿瘤。为此，我们介绍了通过荧光激活细胞分拣 （FACS） 检测多能性相关细胞表面抗原的方法，用于使用正选择识别多能 TG30 Hi-GCTM-2Hi HESC。使用我们的 TG30/GCTM-2 FACS 方法的负选择，我们能够检测和清除处于非常早期分化（TG30Neg-GCTM-2Neg）的人群中未分化的 hESC。在进一步的研究中，使用我们的TG30/GCTM-2 FACS协议选择的分化TG30 Neg-GCTM-2Neg细胞的多能干细胞无干细胞样本，一旦移植到免疫受损的小鼠体内，就不会形成畸形，这支持了我们协议的稳健性。另一方面，TG30/GCTM-2 FACS调解的丰富多能TG30 Hi-GCTM-2Hi hESC的连续传递并不影响他们在体外自我更新的能力或其内在的多能性。因此，我们的TG30/GCTM-2 FACS方法的特性提供了一个敏感的分析，以获得高度丰富的hPSC种群作为分化检测的输入，并消除潜在的肿瘤（或残余）hESC从衍生细胞群。

hESC-衍生物是多能干细胞无可取的，可以通过从P4（TG30 Neg-GCTM-2Neg）子基连续传递细胞获得。
先前的报告已经证实，在hESC标准培养物中，亚种群的混合物共存，表现出与多能性5，6，13相关的基因的表达梯度。结合检测细胞表面抗原，如TG30（CD9）和GCTM-2，允许在多能阶段和早期分化的不同阶段对细胞的一些子集进行区分，如它们表达的干细胞标记和血统特定转录因子5，6，13所示。根据先前的报告，我们得以区分用于本研究的 hESC-MEL1 行中的 P4（TG30内格-GCTM-2内格）、P5（TG30低-GCTM-2低）、P6（TG30中-GCTM-2中）和 P7（TG30Hi-GCTM-2Hi）细胞。有了这个结果，我们开始收集分化的P4（TG30 Neg-GCTM-2内格）细胞和多能P7（TG30Hi-GCTM-2Hi）细胞，以便在下面的研究中进一步培养。
我们调查了使用消极选择方法是否可以清除细胞群中未分化的 hESC（TG30Neg-GCTM-2Neg）。我们使用了固定数量的 P4 （TG30Neg-GCTM-2Neg）细胞，这些细胞在 FACS 后连续培养 （图 2A）大约两周，并在每个通道重新镀上之前使用 TG30/GCTM-2 FACS 重新分析。结果表明，在连续传递后富含TG30 Neg-GCTM-2Neg分化细胞类型的P4-分孔，在初始通道（图2A，通道1）中少量存在P7（TG30Hi-GCTM-2Hi）细胞，在进一步传递后最终消失，成为无多能干细胞样本（图2A，通道3）。
多能 hESC 细胞的浓缩可以通过收集细胞 TG30Hi-GCTM-2Hi 细胞来实现。
根据先前使用此测定进行的观测结果，我们期望通过收集应形成 hESC 菌落的 P7 （TG30Hi-GCTM-2Hi）细胞来丰富多能 hESC。因此，我们还调查了TG30/GCTM-2 FACS介质的多能P7（TG30 Hi-GCTM-2Hi）细胞的连续传递。在这些P7培养物中，TG30/GCTM-2 FACS免疫增殖在每个通道重新镀细胞之前进行，只有P7（TG30 Hi-GCTM-2Hi）细胞被重新培养。通过这种方式，我们观察到，大多数细胞位于与多能性（P6和P7）相关的子缺陷中，表明多能细胞的丰富性也保持了分化和产生一小部分P4（TG30 Neg-GCTM-2Neg）细胞（图3A）的能力。此外，P7细胞的培养在传递过程中显示出大量类似hESC的菌落（图3B），也表明多能状态的维持。
<img alt="Figure 1" fo:content-width="5.5in" src="/files/ftp_upload/50856/50856fig1.jpg"> 图1。获得 TG30/GCTM-2 FACS 子折合的代表排序策略。（A）：分离的 hES 细胞悬架按单个细胞排序，潜在团块或双体被封闭，前向散射为高度和区域（FSC-H 和 FSC-A）。（B）：潜在的碎片通过 FSC-A 和 SSC-A 被移除，并且只有完整的细胞被进一步分类。（C）：基于碘化物 （PI） 荧光 （FSC-A 和 FL3-A） 排除不动细胞。（D）：根据鼠标 CD90.2-PE（FL2-A 和 SSC-A）的表达方式，MEF 馈线单元被负选择挡在门外。（E）：分离的可行 hESC 分数，不含 MEF 馈线，最终分为 P4（TG30内格-GCTM-2内）、P5（TG30低- GCTM-2低）、P6（TG30中-GCTM-2中）和 P7（TG30Hi-GCTM-2Hi）子人口。负门（TG30Neg-GCTM-2Neg，名为P4）是根据未受污染细胞的背景自动荧光以及同位素控制而设置的。（F）：TG30/GCTM-2流量细胞测量分析中典型的不同门控子人口百分比。<a href="https://www.jove.com/files/ftp_upload/50856/50856fig1large.jpg" target="_blank">单击此处查看较大的图。</a>
<img alt="Figure 2" fo:content-width="5.75in" src="/files/ftp_upload/50856/50856fig2.jpg"> 图2。具有代表性的自发分化P4（TG30 Neg-GCTM-2内格）细胞的连续培养物，来自hESC-MEL1线。（A）：代表 TG30/GCTM-2 FACS 图显示按 TG30 和 GCTM-2 细胞表面标记的检测表达水平区分的单元（P4、P5、P6 和 P7）的封闭子集。最初有400，000个hESC衍生物，其中显示P4（TG30 Neg-GCTM-2Neg）免疫扩散是从hESC-MEL1线的批量培养物中收集的。这些 P4 （TG30Neg-GCTM-2Neg）细胞在支持 T75 烧瓶中 hESC 续订的条件中连续通过后 FACS，直到达到共存（11-13 天）。在每段之前，TG30/GCTM-2 FACS 仅执行 P4 （TG30Neg-GCTM-2Neg）分化细胞类型进行收集和再培养。（B）：14天后，区分P4（TG30 Neg-GCTM-2Neg）细胞草坪的典型形态。（C）：零星的 hESC 样菌落在 14 天后与 P4 （TG30Neg-GCTM-2Neg）细胞的草坪交织在一起后，最有可能通过污染物 P7 （TG30 Hi-GCTM-2Hi）细胞产生。比例栏 =200 μm。<a href="https://www.jove.com/files/ftp_upload/50856/50856fig2large.jpg" target="_blank">单击此处查看较大的图。</a>
<img alt="Figure 3" fo:content-width="5.75in" src="/files/ftp_upload/50856/50856fig3.jpg"> 图3。P7 （TG30Hi-GCTM-2Hi）从 hESC 线中提取的细胞可以在不丢失其内在多能特性的情况下连续通过 FACS 后。代表 TG30/GCTM-2 FACS 免疫扩散的不同连续段落，根据 TG30 和 GCTM-2 的表达水平显示细胞的分组（P4、P5、P6 和 P7）。（A）：从 hESC-MEL1 线的批量培养物中检索出显示 P7 （TG30 Hi-GCTM-2Hi）特征的 400，000 个细胞的启动群。多能P7（TG30 Hi-GCTM-2Hi）细胞在支持在T75烧瓶中续订的条件下连续通过FACS，直到达到+80%的汇合（9-11天）。TG30/GCTM-2 FACS 免疫增殖在每个通道重新镀细胞之前进行，并且只有P7（TG30 Hi-GCTM-2Hi）细胞被重新培养。结果表明，P7（TG30Hi-GCTM-2Hi）细胞通过随后的传递来保存其多能特性， 包括保护 （TG30 Hi-GCTM-2Hi）表达，形成 hESC 样菌落（如B所示），并保持区分能力，可以从每个 FACS 梯度中一小部分差异化 P4 （TG30Neg-GCTM-2Neg）的回顾中推断出来。比例栏 =200 μm。<a href="https://www.jove.com/files/ftp_upload/50856/50856fig3large.jpg" target="_blank">单击此处查看较大的图。</a>
<table border="1"><tbody>
<tr>
<td>样本（反应量）</td>
			<td>主要抗体</td>
			<td>次要抗体</td>
			<td>PE 鼠防鼠 CD90.2 b</td>
			<td>碘化物 丙酸丙酸丙</td>
		</tr>
<tr>
<td>排序样本（初级 ABs 为 9 毫升，二级 AB 为 2.5 毫升）</td>
			<td>（TG30 +GCTM-2） a
</td>
			<td>AF 488 山羊防鼠 IgG2a + AF 647 山羊防鼠 IgM</td>
			<td>+</td>
			<td>+</td>
		</tr>
<tr>
<td>控制-1：TG30 （200μl）</td>
			<td>TG30</td>
			<td>AF 488 山羊防鼠 IgG2a</td>
			<td>-</td>
			<td>+</td>
		</tr>
<tr>
<td>控制-2：GCTM-2 （200μl）</td>
			<td>GCTM-2</td>
			<td>AF 647 山羊防鼠 IgM</td>
			<td>-</td>
			<td>+</td>
		</tr>
<tr>
<td>控制-3：防鼠器 CD90.2 （200μl）</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>+</td>
		</tr>
<tr>
<td>控制-4：植物红素（PE） （200微克）</td>
			<td>鼠标 IgG2a 等型</td>
			<td>R - 植物素山羊抗鼠标 Igg2a</td>
			<td>-</td>
			<td>+</td>
		</tr>
<tr>
<td>控制-5：鼠标免疫球蛋白异种型（200微l）</td>
			<td>鼠标IgG2a等型+IgM等型</td>
			<td>AF 488 山羊防鼠 IgG2a + AF 647 山羊防鼠 IgM</td>
			<td>-</td>
			<td>+</td>
		</tr>
<tr>
<td>控制-6：未受污染的细胞（200微升）</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
</tbody></table>表1。对样本和控件进行排序，以进行 FACS 排序。a排序样本与 TG30 和 GCTM-2 抗体同时免疫。 bPE防鼠标 CD90.2 用于识别鼠标细胞，并在 FACS14期间从 hESC 中去除 MEF。 c使用碘化物的最终浓度为 0.3μg/ml 排除不可抗病毒细胞。试剂添加 （+）， 而不是添加 （-） 到反应管。
<table border="1"><tbody>
<tr>
<td>抗体 </td>
			<td>主机</td>
			<td>伊索类型</td>
			<td>工作稀释</td>
		</tr>
<tr>
<td>TG30 （1.4 毫克/毫升）</td>
			<td>鼠</td>
			<td>伊格2a</td>
			<td>1:1,000</td>
		</tr>
<tr>
<td>GCTM-2（杂交瘤超自然）</td>
			<td>鼠</td>
			<td>伊格姆</td>
			<td>1：5 ~ 1：10 a
</td>
		</tr>
<tr>
<td>AF 488 山羊防鼠 IgG2a</td>
			<td>山羊</td>
			<td>多克隆</td>
			<td>1:500</td>
		</tr>
<tr>
<td>AF 647 山羊防鼠 IgM</td>
			<td>山羊</td>
			<td>多克隆</td>
			<td>1:500</td>
		</tr>
<tr>
<td>R - 植物素 （PE） 山羊反鼠标 Igg2a</td>
			<td>山羊</td>
			<td>多克隆</td>
			<td>1:1,000</td>
		</tr>
<tr>
<td>防鼠标 CD90.2</td>
			<td>鼠</td>
			<td>伊格2b</td>
			<td>1:100</td>
		</tr>
<tr>
<td>净化鼠标 IgG2a，κ伊索型控制</td>
			<td>鼠</td>
			<td>伊格2a</td>
			<td>1:200</td>
		</tr>
<tr>
<td>净化鼠标 IgM，κ伊索型控制</td>
			<td>鼠</td>
			<td>伊格姆</td>
			<td>1:200</td>
		</tr>
</tbody></table>表2。FACS排序的抗体细节和稀释。 a 由于 GCTM-2 在 hESC 细胞表面的表达极高，因此无法用这种抗体达到饱和度。每批 GCTM-2 杂交瘤超自然必须与标准控制或上一批进行滴定，才能在规模上与 hESC 取得类似的结果，并避免过度染色。
<table border="1"><tbody>
<tr>
<td>介质名称</td>
			<td>组成</td>
		</tr>
<tr>
<td>赫塞克/科斯尔介质</td>
			<td>杜尔贝科的改良鹰介质：营养混合物F-12 （DMEM/F-12） 辅以20%的淘汰血清置换 （KOSR）， 2 mM 谷氨酰氨基， 1% MEM 非必要氨基酸，0.1 mM 2-默卡普托乙醇，10 ng/ml 人类成纤维细胞生长因子 2 （FGF-2） 和笔/链球菌在 1x.</td>
		</tr>
<tr>
<td>梅夫介质</td>
			<td>杜尔贝科的改良鹰中，高葡萄糖 （DMEM） 补充 10% 胎儿牛血清 （FBS）， 2 mM 麸质MAX， 和笔/链球菌在 1x.</td>
		</tr>
</tbody></table>表3。文化媒体的构成。

Watch this Scientific Journal Video about Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2 at JoVE.com

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

我们描述了使用单克隆抗体TG30（CD9）和GCTM-2通过荧光活性细胞分选（FACS）联合检测细胞表面抗原，用于识别和丰富活人胚胎干细胞（hESC），使用阳性选择，以及使用负选择清除混合细胞群中的hESC。

利用单克隆抗体TG30和GCTM-2检测细胞表面抗原，促进人类胚胎干细胞的浓缩和清除

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all ...

enrichment-purging-human-embryonic-stem-cells-detection-cell-surface

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Embryonic and Induced Pluripotent Stem Cells

SCIENTISTS IN ACTION

7.7K 次观看.  CSIRO. 我们描述了使用单克隆抗体TG30（CD9）和GCTM-2通过荧光活性细胞分选（FACS）联合检测细胞表面抗原，用于识别和丰富活人胚胎干细胞（hESC），使用阳性选择，以及使用负选择清除混合细胞群中的hESC。

视频: Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

从小鼠胚胎干细胞的造血干细胞的推导

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

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

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

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

视频人类胚胎干细胞克隆生长的生物信息学分析

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

胚胎干细胞分化成少突胶质前体

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.