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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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=Stem+cell-based+therapy+for+human+diseases.">Stem cell-based therapy for human diseases.</a> Signal Transduction and Targeted Therapy. 7 (1), 272 (2022).</li><li>Jiang, B., 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=Concise+review:+Mesenchymal+stem+cells+derived+from+human+pluripotent+cells,+an+unlimited+and+quality-controllable+source+for+therapeutic+applications.">Concise review: Mesenchymal stem cells derived from human pluripotent cells, an unlimited and quality-controllable source for therapeutic applications.</a> Stem Cells. 37 (5), 572-581 (2019).</li><li>Soontararak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+(MSC)+derived+from+induced+pluripotent+stem+cells+(iPSC)+equivalent+to+adipose-derived+MSC+in+promoting+intestinal+healing+and+microbiome+normalization+in+mouse+inflammatory+bowel+disease+model.">Mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) equivalent to adipose-derived MSC in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model.</a> Stem Cells Translational Medicine. 7 (6), 456-467 (2018).</li><li>Di Baldassarre, A., Cimetta, E., Bollini, S., Gaggi, G., Ghinassi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cell+technology+and+cardiomyocyte+generation:+Progress+and+clinical+applications.">Human-induced pluripotent stem cell technology and cardiomyocyte generation: Progress and clinical applications.</a> Cells. 7 (6), 48 (2018).</li><li>Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+technology:+a+decade+of+progress.">Induced pluripotent stem cell technology: a decade of progress.</a> Nature Reviews. 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. S., Seufert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+treatment+of+diabetes+mellitus+-+Focus+on+mesenchymal+stem+cells.">Stem cells in the treatment of diabetes mellitus - Focus on mesenchymal stem cells.</a> Metabolism. 90, 1-15 (2019).</li><li>Zhou, Y., 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=One-step+derivation+of+functional+mesenchymal+stem+cells+from+human+pluripotent+stem+cells.">One-step derivation of functional mesenchymal stem cells from human pluripotent stem cells.</a> Bio-Protocol. 8 (22), e3080 (2018).</li><li>Hua, Z., 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=Low-intensity+pulsed+ultrasound+promotes+osteogenic+potential+of+iPSC-derived+MSCs+but+fails+to+simplify+the+iPSC-EB-MSC+differentiation+process.">Low-intensity pulsed ultrasound promotes osteogenic potential of iPSC-derived MSCs but fails to simplify the iPSC-EB-MSC differentiation process.</a> Frontiers in Bioengineering and Biotechnology. 10, 841778 (2022).</li><li>Dupuis, V., Oltra, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+produce+induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells:+Mesenchymal+stem+cells+from+induced+pluripotent+stem+cells.">Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells.</a> World Journal of Stem Cells. 13 (8), 1094-1111 (2021).</li><li>Zhang, 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=Aging+stem+cells.+A+Werner+syndrome+stem+cell+model+unveils+heterochromatin+alterations+as+a+driver+of+human+aging.">Aging stem cells. 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. H., 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=Modelling+Fanconi+anemia+pathogenesis+and+therapeutics+using+integration-free+patient-derived+iPSCs.">Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.</a> Nature Communications. 5, 4330 (2014).</li><li>Kubben, N., 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=Repression+of+the+antioxidant+NRF2+pathway+in+premature+aging.">Repression of the antioxidant NRF2 pathway in premature aging.</a> Cell. 165 (6), 1361-1374 (2016).</li><li>Duan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+deficiency+reprogrammes+human+neural+stem+cells+towards+a+glioblastoma+stem+cell-like+phenotype.">PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype.</a> Nature Communications. 6, 10068 (2015).</li><li>Zhang, J., 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+released+from+human+induced+pluripotent+stem+cells-derived+MSCs+facilitate+cutaneous+wound+healing+by+promoting+collagen+synthesis+and+angiogenesis.">Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis.</a> Journal of Translational Medicine. 13, 49 (2015).</li><li>Hu, G. 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。

Mesenchymal stromal cells (MSCs) are mesoderm-derived adult pluripotent stem cells1. MSCs are present in almost all connective tissues2. Since MSCs were first discovered in the 1970s and successfully isolated from bone marrow in 1987 by Friedenstein et al.3,4,5, a variety of human somatic (including fetal and adult) tissues have been used for isolating MSCs such as bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic-supporting stroma1,2,6,7. MSCs demonstrate high proliferative capabilities and plasticity to differentiate into many somatic cell lineages and could migrate to injured and inflamed tissues2,8,9. These properties make MSCs a potential candidate for regenerative medicine10. However, somatic tissue-derived MSCs (st-MSCs) are restricted by limited donation, limited cell proliferative capacity, quality variations, and biosafety concern for possible transmission of pathogens, if any, from the donors11,12.
Human induced pluripotent stem cells (hiPSCs) are derived from adult cells reprogramming with transcription factors (Oct4, Sox2, Klf4, and c-Myc), which have similar functions as embryonic stem cells13,14. They can self-renew and possess the potential of differentiating into any type of somatic cells, including MSCs. Compared with st-MSCs, iPSC-MSCs has the advantage of unlimited supply, lower cost, higher purity, convenience in quality control, easy for scale production and gene modification15,16,17.
Due to these advantages of iPSC-MSCs, a variety of methods driving MSC from iPSC have been reported. These differentiation methods have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer cultures11,18,19,20. Herein, a representative approach for each of the two methodologies was characterized. Furthermore, comparisons between two representative approaches based on time, cost, proliferative ability, expression of MSC biomarkers, and differentiation capability in vitro were also accessed.

作者聚焦：iPSC 和遗传病研究的进展 

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

The scope of our study is a mechanism, and a therapeutic strategy research of genetic disease. Our research is based on multilevel disease models, such as cell lines, iPSCs, organoids, and mice. iPSCs have a distinct advantage over other models.iPSCs from patients, and any subsequent derived cells and organoids share the same genome with the patients, which provides a bank of patient cells and models for many disease. Also, with the methods of established iPSCs from patients are quite mature. Many challenges remain in the direction of differentiating of iPSCs into specific cells and organoids, with a deepening understanding of development mechanisms in current development will occur in the field.

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

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。

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

769 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 

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

科研

生物学

MSCs Differentiation from hiPSCs via EB Formation and Monolayer Culture

To begin, culture hiPSCs in IPSC maintenance medium on a six-well tissue culture plate coated with growth factor reduced extracellular matrix gel. When the cells attain 80 to 90%v, remove the medium from the plate and wash the cells once with DPBS. Then add 700 to 800 microliters of 0.48 millimolar EDTA and incubate for one minute at room temperature.Remove the digestion solution and incubate the plate at 37 degrees Celsius for three to five minutes. When cells are digested into sheets, add two milliliters of IPSCs maintenance medium to terminate digestion. Transfer the cell suspension to a six-well low attachment plate.Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 60 RPM to form spherical embryo bodies or EBs. After 24 hours, using a past pipette, transfer the EBs to the centrifuge tube and allow it to sediment for five to 10 minutes before removing the supernatant. Add MSC differentiation medium to the tube and transfer the EBs to a six-well low attachment blade with two milliliters of MSCs differentiation medium.Culture the EBs on the shaker at 37 degrees Celsius with 5%carbon dioxide for seven days. On the eighth day, transfer the EBs to the centrifuge tube as demonstrated previously. Once the EBs are sedimented, remove the supernatant from the tube.Add MSC maintenance medium into the tube and transfer the EBs to a growth factor reduced extracellular matrix gel coated six-well plate with two milliliters of MSC maintenance medium. After cell adhesion, change the medium until the culture attains 90%confluency. Next, treat the EB derived culture with a dissociation solution at 37 degrees Celsius.When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium to terminate digestion and transfer the cell suspension to a centrifuge tube. Wash the remaining undigested cells from the wells once with DPBS. And digest again as demonstrated previously.Then centrifuge the cell suspension at 250g for five minutes. Remove the supernatant and seed the cells in a gelatin coated culture plate. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.Culture hiIPCs lines as demonstrated previously. When the cells attain 50 to 60%confluency, remove the IPSC maintenance medium from the plate. Add two milliliters of MSC maintenance medium.And culture for 14 days at 37 degrees Celsius and 5%carbon dioxide with daily medium change. For the maturation of MSCs, treat the monolayer culture with a dissociation solution at 37 degrees Celsius. When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium into the wells to terminate digestion and transfer the cell suspension to a centrifuge tube.Wash the remaining undigested cells once with DPBS and digest them as demonstrated previously. Then centrifuge the cell suspension as demonstrated previously and seed the cells on a gelatin coated culture dish. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.In the EB formation method, hiIPCs colonies exhibited a compact morphology before differentiation. After dissociation, uniform spherical EBs formed, which drew in volume over time. Subsequently, EBs transformed into adherent monolayer cells achieving 90%confluency by day 18 and acquiring a spindle shape morphology after two passages.Similarly, in the monolayer method, cells proliferated forming multilayer adherent cells. After packaging multiple times, cells matured into MSC with a typical spindle shape and swirling colony. Flow cytometry analysis revealed that hiIPCs were positive for CD90 and negative for CD34, CD45, CD105, and CD73.After differentiation, the hiIPCs driving MSCs expressed CD90, CD73, and CD105, while remaining negative for CD34 and CD45. Monolayer derived MSCs showed higher calcium deposit formation than the EB method. However, no significant differences were observed in adipogenic and chondrogenic differentiation abilities.The proliferation abilities of both methods were maintained for up to 20 passages.