我们非常感谢毛和胡实验室的所有成员，无论是过去还是现在，都为这个项目进行了有趣的讨论和巨大的贡献。我们感谢国家儿童健康临床医学研究中心的大力支持。本研究得到了国家自然科学基金（U20A20351毛建华，82200784立丹胡）、浙江省自然科学基金（No.LQ22C070004 Lidan 胡）。

1. hiPSCs维持
<ol><li>hiPSC的解冻<ol><li>从液氮中取出细胞，在37°C水浴中快速解冻细胞。将解冻细胞转移到用 3 mL iPSC 维持培养基制备的 15 mL 管中（材料表）。轻轻混合培养基。</li>
		<li>以300 ×g 离心5分钟。除去上清液，轻轻将细胞重悬于含有 10 μM Y-27632 的 1 mL iPSC 维持培养基中（上下移液细胞 2-3 次）。</li>
		<li>将细胞悬浮液转移到涂有生长因子降低 （GFR） -细胞外基质凝胶 （1:100） 和 2 mL iPSC 维持培养基的 6 孔组织培养板中，预先添加 10 μM Y-27632（约 4 x 104 个细胞/cm2）。</li>
		<li>将细胞在37°C，5%CO2 下培养5-6天（80%-90%汇合度），每天更换iPSC维持培养基。</li>
	</ol></li>
	<li>hiPSC的通过<ol><li>从 6 孔板中取出 iPSC 维持培养基。用DPBS洗涤hiPSC一次。</li>
		<li>加入 700-800 μL 0.48 mM EDTA 溶液，在室温 （RT） 下孵育 1 分钟，然后取出消化溶液。继续在37°C温度下孵育3-5分钟。</li>
		<li>当细胞消化成片状（不要将细胞消化成单细胞）时，加入 1 mL 含有 10 μM Y-27632 的 iPSC 维持培养基以终止消化。小心地上下移液细胞 2-3 次。</li>
		<li>将细胞悬液转移到涂有生长因子减少 （GFR） -细胞外基质凝胶 （1:100） 和 2 mL iPSC 维持培养基的 6 孔组织培养板中，预先添加 10 μM Y-27632。 注:传代比范围为1:6至1:20（约4×104 个细胞/cm2），平均聚集大小约为50-200μm。</li>
		<li>将细胞培养至80%-90%汇合（5-6天），在37°C，5%CO2 下每天更换iPSC维持培养基。</li>
	</ol></li>
</ol>2. 通过EB形成从hiPSCs分化
注:该方法源自先前文献21,22,23,24。该方法的概述如图1所示。该方法的特点总结于表1。
<ol><li>培养基的制备<ol><li>通过添加 1% （v/v） GlutaMAX、10% （v/v） FBS、10 ng/mL FGF2 和 5 ng/mL TGFβ 的 α-MEM 来制备 MSC 分化培养基。</li>
		<li>通过向 α-MEM 补充 1% （v/v） GlutMAX、10% （v/v） FBS 和 1 ng/mL FGF2 来制备 MSC 维持培养基。</li>
	</ol></li>
	<li>hiPSCs的制备<ol><li>在37°C，5%CO2 直至80%-90%汇合度的6孔组织培养板上，在涂有生长因子还原（GFR） - 细胞外基质凝胶（1:100）的iPSC维持培养基中培养hiPSCs系（解冻后至少传代2-3次）。</li>
	</ol></li>
	<li>第 0 天:EB 形成<ol><li>从 6 孔板中取出 iPSC 维持培养基。用DPBS洗涤hiPSC一次。</li>
		<li>加入 700-800 μL 0.48 mM EDTA 溶液，在室温下孵育 1 分钟，然后取出消化溶液。继续在37°C温度下孵育3-5分钟。</li>
		<li>当细胞消化成片状（不要将细胞消化成单细胞）时，加入 2 mL iPSCs 维持培养基（含有 10 μM Rock 抑制剂和 1:100 GFR-细胞外基质凝胶）以终止消化。</li>
		<li>将细胞转移到 6 孔低附着板上。以 2 孔组织培养板与 1 孔低附着板的比例接种细胞。将细胞在37°C，5%CO2 的振荡器（60rpm / min）中孵育24小时以形成球形EB。</li>
	</ol></li>
	<li>第 1 天至第 7 天:EB 分化<ol><li>用巴斯德移液管将EB转移到离心管中（不破坏EB），并让EB在室温下自然沉淀5-10分钟。然后，除去上清液。</li>
		<li>将 EB 转移到装有 2 mL MSC 分化培养基的 6 孔低连接板中。在37°C，5%CO2 的振荡器上培养EB7天，培养基更换一次。</li>
	</ol></li>
	<li>第 8 天至第 17 天:EB 接种<ol><li>用巴斯德移液管将EB转移到离心管中（不破坏EB），让EB自然沉淀5-10分钟。然后，除去上清液。</li>
		<li>将 EB 转移到含有 2 mL MSC 维持培养基的 GFR-细胞外基质凝胶包被的 6 孔板中。培养至90%汇合（~10天），细胞粘附后定期更换培养基。</li>
	</ol></li>
	<li>第 18 天至第 27 天:间充质干细胞的成熟和扩增<ol><li>用37°C解离溶液处理EB衍生的培养物5-10分钟（消化时间因存在不同的细胞类型而变化）。</li>
		<li>当部分细胞消化成单细胞时，加入 2 mL MSC 维持培养基以终止消化并将细胞悬液转移到离心管中。用DPBS洗涤剩余的未消化细胞一次，然后再次消化。重复几次，直到所有细胞都消化成单个细胞。将细胞悬浮液以250× g 离心5分钟，然后除去上清液。</li>
		<li>将细胞接种在明胶包被的培养板（约2×105 个细胞/ cm2）上。在MSC维持培养基中培养至90%汇合，定期更换培养基。此时将这一代细胞指定为传代 0 （P0）。</li>
		<li>为了使MSC纯度和成熟，在大约6天内继续以1:3的分裂比传代细胞两次。大多数细胞呈现成纤维细胞样形态（纺锤形）。</li>
	</ol></li>
</ol>3. 通过单层培养从hiPSCs分化MSCss
注:该方法源自先前文献25,26,27,28。此方法的概述如图 1 所示。该方法的特点总结于表1。
<ol><li>培养基的制备<ol><li>通过在 α-MEM 中补充 1% （v/v） GlutMAX、10% （v/v） FBS 和 1 ng/mL FGF2 来制备 MSC 维持培养基。</li>
	</ol></li>
	<li>hiPSCs的制备<ol><li>在6孔组织培养板上，在涂有生长因子减少（GFR）-细胞外基质凝胶（1:100）的6孔组织培养板上，在37°C，5%CO2 直至50%-60%汇合度的6孔组织培养板上培养hiPSCs系（解冻后至少传代2-3次）。</li>
	</ol></li>
	<li>第 0 天至第 13 天:通过直接单层培养物进行分化<ol><li>从 6 孔板中取出 iPSC 维持培养基。</li>
		<li>直接加入2mLMSC维持培养基，并在37°C，5%CO 2下培养14天， 培养基每天更换。</li>
	</ol></li>
	<li>第 14 天至第 35 天:间充质干细胞通过重复传代成熟<ol><li>用解离溶液在37°C处理单层培养物5-10分钟（消化时间因存在不同的细胞类型而变化）。</li>
		<li>当细胞消化成单细胞时，加入 2 mL MSC 维持培养基以终止消化并将细胞悬液转移到离心管中。用DPBS洗涤剩余的未消化细胞一次，然后再次消化。重复几次，直到所有细胞都消化成单个细胞。将细胞悬浮液以250× g 离心5分钟，然后除去上清液。</li>
		<li>将细胞接种在明胶包被的培养皿（约2×105 个细胞/ cm2）上。在MSC维持培养基中培养至90%汇合，并定期更换培养基。此时将这一代细胞指定为传代 0 （P0）。</li>
		<li>为了使MSC纯度和成熟，在大约18天内继续以1:3的分裂比传代细胞6次。大多数细胞呈现成纤维细胞样形态（纺锤形）。</li>
	</ol></li>
</ol>4. 通过流式细胞术对hiPSC驱动的MSC进行表面抗原分析
注:与骨髓来源的 MSC 的表面抗原类似，hiPSC 驱动 MSC 表达 CD105、CD73 和 CD90，但不表达 CD45、CD3429。此外，hiPSCs可用作阴性对照细胞。通过流式细胞术对hiPSC驱动的MSC和hiPSCs进行表面抗原分析如 图2所示。
<ol><li>用37°C的解离溶液处理hiPSC驱动的MSC2-3分钟。用0.48mM EDTA解离试剂处理hiPSC，并在室温下孵育1分钟，然后取出解离试剂。继续在37°C温度下孵育3-5分钟。</li>
	<li>当细胞消化成单细胞时，加入培养基（MSC维持培养基到MSCs，iPSC维持培养基到iPSCs）以终止消化。</li>
	<li>将细胞悬浮液以350× g 离心5分钟，然后除去上清液。</li>
	<li>用冷DPBS洗涤细胞一次，以350× g 离心5分钟，然后除去上清液。</li>
	<li>以 10 x 106 个细胞/mL 的速度计数并重悬于冷的 10% FBS-DPBS （v/v） 溶液中，并将 100 μL/管的细胞悬液（1 x 106 个细胞/管）分配到 1.5 mL 离心管中。</li>
	<li>将 5 μL 人 Fc 受体阻断溶液加入 100 μL 细胞悬液中。在室温下孵育 5-10 分钟以进行非特异性封闭。</li>
	<li>以350× g 离心5分钟，除去上清液。用冷的2%FBS-DPBS（v / v）溶液洗涤细胞两次。</li>
	<li>将细胞重悬于冷的 100 μL 2% FBS-DPBS （v/v） 溶液中。</li>
	<li>向 100 μL 细胞中加入 5 μL（1 次测试）抗 CD34-FITC 和 5 μL（1 次测试）抗 CD45-APC 悬吊。向另一个 100 μL 细胞悬浮管中加入 5 μL（1 次测试）抗 CD73-APC、5 μL（1 次测试）抗 CD90-FITC 和 5 μL（1 次测试）抗 CD105-PE。向第三个 100 μL 细胞悬浮管中加入 5 μL 人 IgG1 同型对照 FITC、人 IgG1 同型对照 PE 和人 IgG1 同型对照 APC。在黑暗中在冰上孵育 15-20 分钟。同时，准备单染色珠。</li>
	<li>用冷的2%FBS-DPBS（v / v）溶液洗涤细胞两次。</li>
	<li>加入 300 μL 冷的 2% FBS-DPBS （v/v） 溶液以重悬细胞，并通过 200 目筛网过滤器过滤。</li>
	<li>使用流式细胞术分析样品。分析用同种型对照抗体染色的细胞悬浮液并设置门。然后分析用靶抗体染色的细胞悬液。</li>
</ol>5. hiPSC驱动间充质干细胞的成骨分化
注:驱动 hiPSC 的 MSC 具有成骨分化潜力（图 3A、B）。成骨分化的方案如下。
<ol><li>通过用 10% FBS、100 nM 地塞米松、10 mM β-甘油磷酸盐和 100 μM 抗坏血酸补充 α-MEM 来制备成骨诱导培养基。</li>
	<li>将 1 x 105 个 MSC 接种到明胶包被的 48 孔板中并培养，直至其 60%-70% 汇合。</li>
	<li>取出培养基并加入 0.3 mL 成骨诱导培养基。在37°C，5%CO 2下在成骨诱导培养基中培养细胞14天， 培养基每3天更换一次。</li>
	<li>取出培养基并用 DPBS 洗涤一次。</li>
	<li>加入 0.3 mL 4% PFA，并在室温下孵育 30 分钟。</li>
	<li>去除 PFA。在室温下用 0.3 mL DPBS 冲洗细胞 10 分钟，并重复 3 次。</li>
	<li>取出 DPBS，加入 0.2 mL 茜素红染色液，并在室温下孵育 30 分钟。</li>
	<li>取出染色溶液，在室温下用0.3mL DPBS冲洗细胞10分钟，重复3次。</li>
	<li>在光学显微镜下观察钙沉积的染色。</li>
</ol>6. hiPSC驱动MSC的成脂分化
注:hiPSC驱动的MSC具有成脂分化潜力（图3C，D）。成脂分化的方案如下。
<ol><li>通过用 10% FBS、1 μM 地塞米松、1 μM IBMX、10 μg/mL 胰岛素和 100 μM 吲哚美辛补充 α-MEM 来制备成脂肪诱导培养基。通过用 10% FBS 和 10 μg/mL 胰岛素补充 α-MEM 来制备成脂肪维持培养基。</li>
	<li>将 1 x 105 个 MSC 接种到明胶包被的 48 孔板中并培养，直到细胞密度达到 90%。</li>
	<li>取出培养基并加入 0.3 mL 成脂诱导培养基。继续在37°C，5%CO2下在成脂诱导培养基中培养细胞4天。</li>
	<li>取出培养基并加入 0.3 mL 成脂维持培养基。继续在成脂维持培养基中在37°C，5%CO2下培养细胞3天。</li>
	<li>重复步骤6.3和6.4 2-3次，直到脂肪细胞分化和成熟。</li>
	<li>取出培养基并用 DPBS 洗涤一次。</li>
	<li>加入 0.3 mL 4% PFA，并在室温下孵育 10 分钟。取出 PFA 并用 DPBS 冲洗 2 次。</li>
	<li>按照制造商的说明应用 Oli Red O 染色剂，并在光学显微镜下观察脂滴。</li>
</ol>7. hiPSC驱动MSC的软骨分化
注:hiPSC驱动的MSC具有软骨生成分化潜力（图3E，F）。软骨分化的方案如下。
<ol><li>通过用 10% FBS、100 nM 地塞米松、1% 胰岛素-转铁蛋白硒 （ITS）、10 μM 抗坏血酸、1 mM 丙酮酸钠、50 μg/mL 脯氨酸、0.02 nM 转化生长因子 β3 （TGFβ3） 和 0.5 μg/mL 骨形态发生蛋白 6 （BMP-6） 补充 α-MEM 来制备软骨母细胞诱导培养基。</li>
	<li>收集 2.5 x 105 个驱动 hiPSC 的 MSC，并以 150 x g 离心细胞 5 分钟，然后除去上清液。将细胞悬浮在 1 mL α-MEM 中，然后以 150 x g 离心 5 分钟。</li>
	<li>除去上清液，将细胞悬浮在 0.5 mL 软骨诱导培养基中。将细胞以150× g 离心5分钟。</li>
	<li>直接拧开盖子，在37°C，5%CO2下每3天更换一次软骨诱导培养基培养21天。在软骨分化期间，每天轻弹动离心管以悬浮软骨形成沉淀（不破坏软骨沉淀）。</li>
	<li>取出上清液，用 0.5 mL PBS 洗涤两次。</li>
	<li>加入 0.3 mL 4% PFA 并固定 24 小时。</li>
	<li>石蜡包埋软骨形成颗粒，然后切片（3μm）。用甲苯胺蓝（1%）染色切片30分钟。</li>
	<li>在光学显微镜下观察细胞外软骨细胞基质。</li>
</ol>

表征 hiPSC 驱动 MSC 中的表面抗原和增殖能力

<ol>
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Drug Discovery. 16 (2), 115-130 (2017).</li><li>Levy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shattering+barriers+toward+clinically+meaningful+MSC+therapies.">Shattering barriers toward clinically meaningful MSC therapies.</a> Science Advances. 6 (30), eaba6884 (2020).</li><li>Zhao, C., Ikeya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+applications+of+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells.">Generation and applications of induced pluripotent stem cell-derived mesenchymal stem cells.</a> Stem Cells International. 2018, 9601623 (2018).</li><li>Path, G., Perakakis, N., Mantzoros, C. 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A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.</a> Science. 348 (6239), 1160-1163 (2015).</li><li>Liu, G. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+secreted+by+human-induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells+attenuate+limb+ischemia+by+promoting+angiogenesis+in+mice.">Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.</a> Stem Cell Research &amp; Therapy. 6 (1), 10 (2015).</li><li>Kang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+human+induced+pluripotent+stem+cells+retain+adequate+osteogenicity+and+chondrogenicity+but+less+adipogenicity.">Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.</a> Stem Cell Research &amp; Therapy. 6 (1), 144 (2015).</li><li>Wang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mesenchymal+stem+cells+from+human+induced+pluripotent+stem+cells+results+in+downregulation+of+c-Myc+and+DNA+replication+pathways+with+immunomodulation+toward+CD4+and+CD8+cells.">Differentiation of mesenchymal stem cells from human induced pluripotent stem cells results in downregulation of c-Myc and DNA replication pathways with immunomodulation toward CD4 and CD8 cells.</a> Stem Cells. 36 (6), 903-914 (2018).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Kim, S., Kim, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem-like+cells+for+producing+extracellular+vesicles.">Generation of mesenchymal stem-like cells for producing extracellular vesicles.</a> World Journal of Stem Cells. 11 (5), 270-280 (2019).</li></ol>

作者没有什么可透露的。

在该协议中，检查了将hiPSCs分化为MSCs的两种代表性方法20,21,22,23,24,25,26,27,28,30。两种方法都能够从hiPSCs中衍生MSCs。通过细胞形态（<strong class="...

间充质基质细胞 （MSC） 是中胚层来源的成体多能干细胞1。间充质干细胞存在于几乎所有结缔组织中2.自 1970 年代首次发现 MSC 并于 1987 年由 Friedenstein 等人成功从骨髓中分离出来 3,4,5 以来，各种人类体细胞（包括胎儿和成人）组织已被用于分离 MSC，例如骨骼、软骨、肌腱、肌肉、脂肪组织和造血支持基质 1,2,6,7.间充质干细胞表现出高增殖能力和可塑性，可分化为许多体细胞谱系，并可迁移到受伤和发炎的组织 2,8,9。这些特性使间充质干细胞成为再生医学的潜在候选者10.然而，体细胞组织来源的 MSC （st-MSCs） 受到有限的捐赠、有限的细胞增殖能力、质量差异以及可能从捐赠者传播病原体（如果有的话）的生物安全问题的限制11,12。
人诱导多能干细胞 （hiPSC） 来源于具有转录因子（Oct4、Sox2、Klf4 和 c-Myc）的成体细胞重编程，其功能与胚胎干细胞相似13,14。与st-MSCs相比，iPSC-MSCs具有无限供应、成本更低、纯度更高、质量控制方便、易于规模化生产和基因修饰等优点15,16,17。
由于 iPSC-MSC 的这些优势，已经报道了多种从 iPSC 驱动 MSC 的方法。这些分化方法以两种培养方法为中心:（1）胚状体（EBs）的形成和（2）使用单层培养物11,18,19,20。在此，对两种方法中的每一种都具有代表性的方法进行了表征。此外，还访问了两种基于时间、成本、增殖能力、MSC生物标志物表达和体外分化能力的代表性方法之间的比较。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alizarin red staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0148S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD105 (PE)</td>
			<td>Biolegend</td>
			<td>323206</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD34 (FITC)</td>
			<td>Biolegend</td>
			<td>343503</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD45 (APC)</td>
			<td>Biolegend</td>
			<td>304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD73( APC)</td>
			<td>Biolegend</td>
			<td>344006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD90 (FITC)</td>
			<td>Biolegend</td>
			<td>328108</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Solarbio</td>
			<td>A8100</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-6</td>
			<td>Novoprotein</td>
			<td>C012</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide level shaker</td>
			<td>Crystal</td>
			<td>CO-06UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation Beads</td>
			<td>BioLegend</td>
			<td>424601</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>STEMCELL Technology</td>
			<td>07959</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160; medium</td>
			<td>Servicebio</td>
			<td>G4610</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HAKATA</td>
			<td>HS-FBS-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Stemcell</td>
			<td>78003.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control APC</td>
			<td>BioLegend</td>
			<td>403505</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control FITC</td>
			<td>BioLegend</td>
			<td>403507</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control PE</td>
			<td>BioLegend</td>
			<td>403503</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#946;1</td>
			<td>Stemcell</td>
			<td>78067</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX&#160;</td>
			<td>BioLegend</td>
			<td>422301</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Solarbio</td>
			<td>SI9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>P3376</td>
			<td></td>
		</tr>
		<tr>
			<td>iPSC maintenance medium</td>
			<td>STEMCELL Technology</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Media Supplement</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0341-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced</td>
			<td>BD Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Oli Red O staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0158S</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Solarbio</td>
			<td>P0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;3</td>
			<td>Novoprotein</td>
			<td>CJ44</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue staining kit</td>
			<td>Solarbio</td>
			<td>G2543</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme(1x)&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 6 Well Plate</td>
			<td>Costar</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stemcell</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Hyclone</td>
			<td>SH30265</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Solarbio</td>
			<td>G8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparison of two representative methods for differentiation of human induced pluripotent stem cells into mesenchymal stromal cells

间充质基质细胞（MSCs）是成体多能干细胞，在再生医学中得到了广泛的应用。由于体细胞组织来源的 MSC 受到有限捐赠、质量差异和生物安全性的限制，过去 10 年中，从人类诱导多能干细胞 （hiPSC） 中产生 MSC 的努力大幅增加。过去和最近将hiPSCs分化为MSCs的努力都集中在两种培养方法上:（1）胚状体（EBs）的形成和（2）单层培养的使用。该协议描述了从hiPSCs中提取MSC的这两种代表性方法。每种方法都有其优点和缺点，包括时间、成本、细胞增殖能力、MSC标志物的表达及其 体外分化能力。该协议表明，这两种方法都可以从hiPSCs中获得成熟且功能性的MSC。单层法的特点是成本更低、操作更简单、更容易成骨分化，而EB法的特点是时间消耗更短。

按照方案（图1A），通过EB形成和单层培养方法将hiPSC分化为MSC。在分化过程中，细胞表现出不同的代表性形态（图1B，C）。
如 图1B所示，hiPSCs菌落在分化前表现出典型的致密形态，具有由紧密堆积的细胞组成的清晰边界。hiPSCs在振荡器上解离和培养24小时后形成的均匀球形EB。在MSCs分化培养基中?...

Watch this Scientific Journal Video about Advancements in iPSCs and Genetic Disease Research at JoVE.com

作者聚焦:iPSC 和遗传病研究的进展 

该协议描述并比较了将hiPSC分化为间充质基质细胞（MSCs）的两种代表性方法。单层法的特点是成本更低，操作更简单，更容易成骨分化。胚状体（EBs）方法的特点是时间消耗更短。

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

我们的研究范围是遗传病的机制和治疗策略研究。我们的研究基于多层次疾病模型，如细胞系、iPSC、类器官和小鼠。与其他模型相比，iPSC具有明显的优势。来自患者的 iPSC 以及任何后续衍生的细胞和类器官与患者共享相同的基因组，这为许多疾病提供了患者细胞库和模型。此外，与患者建立的iPSCs的方法相当成熟。在将iPSCs分化为特定细胞和类器官的方向上仍然存在许多挑战，随着对发育机制的深入理解，该领域将发生当前发展。

comparison-two-representative-methods-for-differentiation-human

Research

JoVE Journal

Biology

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

923 次观看.  The Children's Hospital, Zhejiang University School of Medicine. 我们的研究范围是遗传病的机制和治疗策略研究。我们的研究基于多层次疾病模型，如细胞系、iPSC、类器官和小鼠。与其他模型相比，iPSC具有明显的优势。来自患者的 iPSC 以及任何后续衍生的细胞和类器官与患者共享相同的基因组，这为许多疾病提供了患者细胞库和模型。此外，与患者建立的iPSCs的方法相当成熟。在将iPSCs分化为特定细胞和类器官的方向上仍然存在许?...

视频: 作者聚焦:iPSC 和遗传病研究的进展 

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

科研

生物学

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成:从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.