Name: comparison of two representative methods for differentiation of human induced pluripotent stem cells into mesenchymal stromal cells
Uploaded: 2023-10-20T13:50:00.000+00:00
Duration: 6 min 24 s
Description: Watch this Scientific Journal Video about Advancements in iPSCs and Genetic Disease Research at JoVE.com

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

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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><tbody><tr><td>Alizarin red staining kit</td><td>Beyotime&amp;nbsp;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&amp;nbsp;Biotechnology</td><td>ST1254</td><td></td></tr><tr><td>DMEM/F12&amp;nbsp; 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-&amp;beta;1</td><td>Stemcell</td><td>78067</td><td></td></tr><tr><td>Human TruStain FcX&amp;nbsp;</td><td>BioLegend</td><td>422301</td><td></td></tr><tr><td>IBMX</td><td>Beyotime&amp;nbsp;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&amp;nbsp;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&amp;nbsp;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&amp;nbsp;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&amp;beta;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)&amp;nbsp;</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>&amp;alpha;-MEM</td><td>Hyclone</td><td>SH30265</td><td></td></tr><tr><td>&amp;beta;-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

957 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 

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

人类多能干细胞的模板微图案用于探测分化命运的空间组织

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

科研

发育生物学

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

用DMSO对人多能干细胞的暂时性治疗促进分化

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

将人 iPSC 稳健分化为纯脂肪细胞群以研究脂肪细胞相关疾病

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

破骨细胞与人诱导多能干细胞的分化和表征

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

生物学

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

人诱导多能干细胞及其神经和神经胶质衍生物中多种线粒体参数的流式细胞术分析

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...