这项工作得到了杜克大学普拉特工程学院、杜克医学院肾脏病学系、杜克大学医学系主席研究奖和伯劳斯·威康基金PDEP职业过渡 特别 奖的支持，该奖授予S.M。M.B得到了国家科学基金会研究生研究金计划的支持。我们感谢Bursac实验室慷慨地为我们提供了DU11干细胞系，以及杜克大学的Varghese实验室暂时与我们的团队共享他们的组织培养设施。本出版物是献给麻省理工学院诺华化学教授劳拉·基斯林教授的，以庆祝她的60岁生日 。

1. 试剂的制备
<ol><li>稀释解冻5倍高PS细胞培养介质（CCM）补充在HIPS细胞培养基质介质获得1倍溶液的HIPS CCM。 注： 冷冻 5 倍高音响 CCM 补充剂需要一个缓慢的解冻过程， 理想情况下在 4 °C 过夜.1x hiPS CCM 的 Aliquot 可在 -20 °C 下存储长达 6 个月。</li>
	<li>准备地下室膜 （BM） 矩阵 1 涂层板用于 hiPS 细胞培养：解冻 BM 矩阵 1 在 4 °C 的冰上过夜。解冻后，根据制造商的建议，准备具有适当稀释因子的阿利报价。 注：通常，在 50 mL 圆锥管中，在 25 mL 的冷 DMEM/F12 中，然后进行彻底混合，以完全溶解 BM 矩阵 1 并避免形成残晶。</li>
	<li>将 BM 矩阵 1 溶液的 1 mL 转移到 6 井板的每个井中，并在 37 °C 下以 1-2 h 或 4 °C 孵育，至少 24 小时，用石蜡薄膜包裹。BM 矩阵 1 涂层板可在 4 °C 下存储长达 2 周。</li>
	<li>制备地下室膜 （BM） 基质 2 - 涂层板：在 9 mL 无菌蒸馏水中稀释适量的 BM 基质 2，以准备 5 μg/mL 的最终浓度。在 12 井板的每个井中加入 700 μL 的 BM 矩阵 2 溶液，并在室温下孵育板 2 小时或在 4 °C 下过夜。</li>
	<li>准备 100 μg/mL 库存解决方案，BMP7、Activin A 和 VEGF 各如下：在含有 0.1% （wt/vol） BSA 的无菌蒸馏水中重组 BMP7，并在含有 0.1% （wt/vol） BSA 的无菌 PBS 中分别重组 Activin A 和 VEGF。为了避免频繁的冻结-解冻周期，从每个库存溶液中准备 100 μL 的 aliquot，并在 -20 °C 下存储长达 6 个月。</li>
	<li>在 3.079 mL 的无菌蒸馏水中溶解 10 毫克 Y27632，准备 10 mM 库存溶液 27632 日元。Aliquot 100 μL 从股票和存储在 -20 °C 长达 6 个月。</li>
	<li>溶解 2 毫克的 CHIR99021 在 143.4 μL 的无菌 DMSO 中，以准备 30 mM 库存溶液。准备 5 μL 的 aliquot，并在 -20 °C 下存放长达 1 个月（或根据制造商的建议）。</li>
	<li>溶解10毫克全跨 视网膜酸在3.33 mL无菌DMSO。准备 500 μL 的 aliquot，并在 -20 °C 下存储长达 6 个月。</li>
</ol>2. 文化媒体的准备
<ol><li>准备中皮分化介质，通过重组相应的库存解决方案，最终浓度为 100 ng/mL Activin A、3 μM CHIR99021、10 μM Y27632 和 1x B27 无血清补充剂，在适当的 DMEM/12 卷中加入谷氨酰胺补充剂。 注：介质分化介质应在分化步骤之前以适合实验规模的体积（通常，50 mL 的介质足以满足两个 12 井板）进行新准备。</li>
	<li>准备中间中皮分化介质，通过重组相应的库存解决方案，最终浓度为 100 ng/mL BMP7、3 μM CHIR99021 和 1x B27 无血清补充剂，在 DMEM/F12 中加入谷氨酰胺补充剂。 注：如果需要，介质可以补充1%（vol/vol）青霉素-链霉素。使用DMEM/F12与谷氨酰胺补充剂调整介质的体积。这种介质可以大批量准备：但是，建议存储在较小的小孔（例如，50 mL 圆锥管中的 45 mL）中，以避免重复冻结- thaw 循环。此介质可在 -20 °C 下存储长达 3 个月，并在使用前在 4 °C 过夜解冻。</li>
	<li>通过重组到最终浓度 100 ng/mL BMP7 来准备囊细胞感应介质， 100 ng/mL 活性素 A， 50 ng/mL VEGF， 3 μM CHIR99021， 1x B27 无血清补充剂， 和 0.1 μM 全跨视网膜酸在 DMEM/F12 与谷氨酰胺补充剂.保护介质免受光线照射（例如，用铝箔纸包装容器）。 注：此介质可大批量准备，并在-20 °C的黑暗中储存长达3个月。冷冻的阿利库特应在使用前在 4 °C 下解冻过夜。</li>
	<li>在 DMEM/F12 中加入 10% （vol/vol） 热灭活 FBS 并在无菌条件下过滤，准备 25 mL 的试用素中和解决方案。</li>
	<li>对于干细胞衍生的足细胞的分化后维护，根据制造商的指南，通过在基础介质中添加补充剂，准备完整的介质，并在 4 °C 下存储长达两周。</li>
</ol>3. 使用 hiPS 细胞培养介质的无喂食高音细胞培养
<ol><li>从预涂层板中吸出BM矩阵1的剩余溶液，用1至2mL的加热DMEM/F12清洗油井3次。</li>
	<li>从 hiPS 细胞中吸气用 HIPS CCM，用加热的 DMEM/F12 冲洗细胞 3 次。加入1mL的热细胞分离溶液，在37°C下孵育1分钟，帮助分离细胞。在组织培养显微镜下对细胞进行目视检查，确保细胞群落的边缘出现圆润，然后快速将细胞分离溶液从细胞中吸气（确保细胞群落仍然附着在板上，尽管松散）。用 DMEM/F12 轻轻冲洗细胞一次，以去除细胞分离溶液。</li>
	<li>将 3 mL 的 HIPS CCM 添加到使用细胞分离溶液处理的 hiPS 细胞（在 6 井板的每个井中）。使用细胞提升器刮菌落，并轻轻地将移液器细胞悬架向上和向下移出松散粘附的细胞。彻底清洗板，以确保所有细胞都收获。 注意：建议使用 5 mL 移液器以避免细胞上出现过量的剪切。</li>
	<li>将 0.5 mL 的电池悬架转移到每口新 BM 矩阵 1 涂层 6 井板的每个井中，每口井含有 2 mL 的 HIPS CCM。以图八的方式移动板，在井内分配细胞群落，并在 5% CO2 孵化器中以 37 °C 孵育。每天刷新介质，直到细胞准备好通过或用于分化实验（约70%的汇流）。 注：使用 hiPS CCM（无 ROCK 抑制剂）在无馈线条件下，iPSC 常规传递的理想菌落大小在 200-500 μm 之间。如果细胞在治疗过程中与分离酶或缓冲器个性化，hiPS CCM 可以补充 ROCK 抑制剂（例如 10 μM Y27632），以提高细胞的生存能力。</li>
</ol>4. 将 HIPS 细胞分化为间皮细胞（天 0-2）
<ol><li>虽然HIPS细胞培养处于指数增长阶段（大约在传递后4天内培养，约70%的汇合），视觉检查在菌落边缘内和周围自发分化细胞的存在。如有必要，不恰当地刮掉区分区域。</li>
	<li>从 hiPS 细胞中吸气高音红素 CCM，并用温暖的 DMEM/F12 冲洗细胞 3 次。在 37 °C 下用 1 mL 的无酶细胞分离缓冲器孵育细胞 10 分钟，并在显微镜下检查分离情况。由于不同 HIPS 细胞系之间的固有差异，因此必须确定给定细胞系的细胞分离缓冲器的实际孵化时间。</li>
	<li>用细胞升降机轻轻刮打井，以移出松散粘附的细胞，并将细胞悬浮转移到 15 mL 圆锥管中，然后上下多次管道以个性化 HIPS 细胞。<ol>
<li>在室温下，将电池悬架与温暖的 DMEM/F12 和离心机一起将电池悬架带到 15 mL 体积中，在 290 x g 下进行 5 分钟。</li>
		<li>轻轻地吸气超纳特，用温暖的DMEM/F12重新吸纳细胞，进行另一轮离心，以去除残余的BM矩阵1和分离缓冲元件。</li>
	</ol>
</li>
	<li>如上文所述，在 1 mL 的中皮感应介质中吸气超高分子和再吸血细胞。使用血细胞计或卷子计数器计数器计数细胞总数，以确定实现 1 x 105 细胞/mL 浓度所需的介质分化介质的适当体积。</li>
	<li>从BM矩阵2涂层板中吸气ECM溶液，用温暖的DMEM/F12冲洗盘子两次。通过管道轻轻混合好几下 HIPS 细胞悬架。将 1 mL 的电池悬架转移到 BM 矩阵 2 涂层的 12 井板的每个井，然后轻轻摇动板，以更均匀地分配细胞。</li>
	<li>在 5% CO2 孵化器中将板孵化在 37 °C 下。第二天刷新介质感应介质。 注：2天后，HIPS细胞衍生的中皮细胞将准备好进行中间间皮诱导。</li>
</ol>5. 将HIPS细胞衍生的中皮细胞分化为中间间皮（第2-16天）
<ol><li>在分化协议的第 2 天，吸气介质感应介质，并补充每井中间介质介质 1 mL。</li>
	<li>每天刷新介质，以保持代谢活性细胞的生长因子和小分子的准确阈值。如果细胞大量生长和介质营养物质的快速消耗（通过介质的发黄来表示），中间间皮分化介质的体积可以增加到12井板每井1.3mL。</li>
	<li>培养细胞额外的14天，以获得中间中皮细胞。到第16天，这些细胞可以冷冻保存，供以后使用。</li>
</ol>6. 将HIPS细胞衍生的中间间皮细胞分化成卵细胞（第16至21天）
<ol><li>用温暖的DMEM/F12冲洗中间中皮细胞，然后以每口0.05mL的0.05%试金-EDTA在37°C下孵育细胞3分钟。进行目视检查，以确保细胞开始分离。</li>
	<li>使用细胞提升器刮细胞，并多次使用 1，000 μL 移液器尖端将细胞悬浮液移液器，以获得个性化（或小块）细胞。 注：在此阶段，确保细胞完全分离，因为聚合的细胞可能无法在协议的时间表内获得最终分化表型。</li>
	<li>每口试丁香中和溶液添加约2ml，以阻止试丁石的活动。</li>
	<li>使用 DMEM/F12 将细胞转移到 50 mL 圆锥管中，将体积调至 50 mL，然后在室温下在 201 x g 下离心电池悬架 5 分钟。</li>
	<li>在细胞诱导介质中吸气超高细胞和再吸血细胞。为了获得最佳结果，请确保最终播种密度约为 100，000 个细胞/井的 12 个井板。将细胞悬浮件添加到BM矩阵2涂层板中，轻轻摇动板，以帮助更均匀地分配细胞。</li>
	<li>在 37 °C 和 5% CO2 下孵育细胞，每天刷新介质长达 5 天，以便在第 21 天获得卵细胞。 注：通过使用完整的带卵细胞维护介质的介质，可在培养中再保持 2-4 周。一旦进入细胞维护介质，细胞可以每隔一天喂一次，可用于后续研究或下游分析。</li>
</ol>

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I., 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=Monitoring+and+robust+induction+of+nephrogenic+intermediate+mesoderm+from+human+pluripotent+stem+cells.">Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.</a> Nature Communications. 4, 1367 (2013).</li><li>Reya, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+in+stem+cells+and+cancer.">Wnt signalling in stem cells and cancer.</a> Nature. 434, 843-850 (2005).</li><li>Clevers, H., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt&#946;-Catenin+Signaling+and+Disease.">Wnt&#946;-Catenin Signaling and Disease.</a> Cell. 149, 1192-1205 (2012).</li><li>Ciampi, 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=Generation+of+functional+podocytes+from+human+induced+pluripotent+stem+cells.">Generation of functional podocytes from human induced pluripotent stem cells.</a> Stem Cell Research. 17, 130-139 (2016).</li><li>Fuente Mora, C., 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+Podocyte+and+Proximal+Tubule-Like+Cells+from+a+Mouse+Kidney-Derived+Stem+Cell+Line.">Differentiation of Podocyte and Proximal Tubule-Like Cells from a Mouse Kidney-Derived Stem Cell Line.</a> Stem Cells and Development. 21, 296-307 (2011).</li><li>Chuah, J. K. C., Zink, D. <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-derived+kidney+cells+and+organoids:+Recent+breakthroughs+and+emerging+applications.">Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.</a> Biotechnology Advances. 35, 150-167 (2017).</li><li>Becker, G. J., Hewitson, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+chronic+kidney+disease:+useful+but+not+perfect.">Animal models of chronic kidney disease: useful but not perfect.</a> Nephrology Dialysis Transplantation. 28, 2432-2438 (2013).</li></ol>

S.M.是一位申请专利的作者，该专利是从多能干细胞中产生肾细胞的方法（美国专利申请14/950859）。其余作者宣称他们没有相互竞争的利益。

在这份报告中，我们描述了从HIPS细胞中生成肾球状细胞的一个协议。HIPS细胞衍生的足细胞表现出形态和分子特征与成熟的肾多细胞表型13相关。在以前的出版物中，我们表明，HIPS细胞衍生的足细胞可以模仿肾球菌的结构和选择性过滤功能，当与球状微血管内皮细胞在一个敷衍的微流体器官片上设备8，11共同培养。
<p class="jove_content...

人类多能干细胞培养的进步，准备通过为研究人员提供可再生的、可扩展的生物材料来源，从而彻底改变再生医学、疾病建模和药物筛选。此策略对于衍生出专业和功能性细胞类型特别有用，否则将难以获得。人类诱导的多能干细胞（hiPS）细胞2，3，4，5特别有吸引力，因为它们的体细胞起源和它们代表个性化医学的潜力。然而，由于频繁使用定义不明确的培养条件，导致效率低下，非特定生成异质细胞群6，7，开发方法从HIPS细胞中获取其他细胞血统仍然具有挑战性。
这里介绍的是一种在化学定义条件下从具有特异性和高效率的HIPS细胞中提取成熟肾细胞的方法。通过考虑细胞微环境中多种因素的作用，制定了干细胞分化策略，包括优化细胞培养介质中呈现的可溶性因子以及不溶性因素，如细胞外基质组件或粘合基板。鉴于特格林信号在细胞发育和功能中的重要性，初步检查了细胞表面的特格林受体的表达。β1集成物不仅在hiPS细胞中，而且在它们的衍生物中，包括中皮细胞8，9，10中表达。随后的实验证实，与 β1 集成物（包括层压素 511 或层压素 511-E8 片段）结合使用时，与下面描述的可溶性感应介质结合使用时，支持 hiPS 细胞的粘附和分化到 podocyt 中。
细胞谱系承诺的诱导首先确认，在具有Activin A、CHIR99021和Y27632岩石抑制剂的介质存在的情况下，在层压涂层表面培养了两天的HIPS细胞可以分化成表达早期间皮标记HAND1、鹅科和胸针8、11的细胞。用骨形态遗传蛋白7（BMP-7）和CHIR99021补充的中等体质细胞治疗14天，使表达肾病的中间间皮细胞得以衍生 原体细胞标记威尔姆的肿瘤1（WT1），奇跳过相关蛋白质1（OSR1）8，11，和配对盒基因2蛋白质（PAX2）12。为了获得成熟的肾球状细胞，中间间皮细胞经过45天的治疗，其新介质包括BMP-7、Activin A、血管内皮生长因子（VEGF）、全跨视网膜酸和CHIR99021。流细胞学和免疫染色用于确认>90%的细胞表现出成熟肾细胞8、11、13的分子、形态和功能特征。这些特点包括发展初级和二级足部工艺：足细胞谱系特异性基因的表达，包括SYNPO、PODXL、MAF、EFNB28和蛋白质的表达，包括波多辛、肾素和WT1 14、15、16。此外，还发现，通过使用商用介质8，11，在培养中可保持长达四周的体外培养物，从而在下游实验的时机上提供额外的灵活性。有关用于确定 hiPS-podo 细胞纯度的流细胞学面板的更多信息，请参阅我们之前的出版物11。

<table>
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DU11 human iPS cells</td>
			<td></td>
			<td></td>
			<td>The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.</td>
		</tr>
		<tr>
			<td>Growth Factors and Media Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (500 mg)</td>
			<td>72262</td>
			<td>Stem Cell Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 serum-free supplement</td>
			<td>17504044</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>04-0004</td>
			<td>Stemgent</td>
			<td>May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Complete Medium Kit with CultureBoost-R</td>
			<td>4Z0-500-R</td>
			<td>Cell Systems</td>
			<td>Podocyte maintenance media</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>12634028</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX supplement</td>
			<td>10565042</td>
			<td>Thermo/Life Technologies</td>
			<td>DMEM/F12 with glutamine supplement</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>10082147</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin A</td>
			<td>PHC9564</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP7</td>
			<td>PHC9544</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF</td>
			<td>PHC9394</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>05850</td>
			<td>Stem Cell Technologies</td>
			<td>hiPS cell culture media (CCM)</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin, liquid (100&#215;)</td>
			<td>15140-163</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 ROCK inhibitor</td>
			<td>1254</td>
			<td>Tocris</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211; and Alexa Fluor 594&#8211;conjugated secondary antibodies</td>
			<td>A32744; A32754; A-11076; A32790</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Brachyury(T&#65289;</td>
			<td>ab20680</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin</td>
			<td>GP-N2</td>
			<td>Progen</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>AF1759</td>
			<td>R&#38;D Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX2</td>
			<td>71-6000</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>WT1</td>
			<td>MAB4234</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td>
			<td>N-892012</td>
			<td>Iwai North America</td>
			<td>Basement membrane (BM) matrix 2</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified matrix, 5-mL vial</td>
			<td>354277</td>
			<td>BD Biosciences</td>
			<td>Basement membrane (BM) matrix 1. May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Enzymes and Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A1110501</td>
			<td>Thermo/Life Technologies</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>A9418</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D2438</td>
			<td>Sigma-Aldrich</td>
			<td>DMSO is toxic. Should be handled in chemical safety hood</td>
		</tr>
		<tr>
			<td>Enzyme-free cell dissociation buffer, Hank&#8217;s balanced salt</td>
			<td>13150016</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% (vol/vol), biotechnology grade</td>
			<td>97065-058</td>
			<td>VWR</td>
			<td>Ethanol is flammable and toxic</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>431097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>28906</td>
			<td>Thermo/Life Technologies</td>
			<td>PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>14190-250</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Distilled Water</td>
			<td>15230162</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>97062-208</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%</td>
			<td>25300-120</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipettes, individually wrapped</td>
			<td>29442-462</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>B99516</td>
			<td>Beckman Coulter</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 15 mL</td>
			<td>352097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 50 mL</td>
			<td>352098</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes</td>
			<td>3395465</td>
			<td>Thermo/Life Technologies</td>
			<td>For storing frozen aliquots</td>
		</tr>
		<tr>
			<td>EVOS M7000</td>
			<td>AMF7000</td>
			<td>Thermo/Life Technologies</td>
			<td>Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>100503-092</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>51030403</td>
			<td>Thermo/Life Technologies</td>
			<td>For the routine culture and maintenace of iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Inverted Zeiss Axio Observer equipped with AxioCam 503 camera</td>
			<td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation</td>
		</tr>
		<tr>
			<td>Kimberly-Clark nitrile gloves</td>
			<td>40101-346</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, large</td>
			<td>21905-049</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, small</td>
			<td>21905-026</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 precision barrier pipette tips</td>
			<td>P1096-FR</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 barrier pipette tips</td>
			<td>P1125</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 barrier pipette tips</td>
			<td>P1126</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 barrier pipette tips</td>
			<td>P1121</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 barrier pipette tips</td>
			<td>P1122</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL, individually wrapped</td>
			<td>356551</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL, individually wrapped</td>
			<td>356525</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL, individually wrapped</td>
			<td>356543</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip, 0.22 &#956;m, PES</td>
			<td>SCGP00525</td>
			<td>EMD Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes</td>
			<td>1138W14</td>
			<td>Thomas Scientific</td>
			<td>For aliquoting growth factors</td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 12-well plates</td>
			<td>353043</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated six-well plates</td>
			<td>353046</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR white techuni lab coat</td>
			<td>10141-342</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-beveled cell lifter</td>
			<td>3008</td>
			<td>Corning</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions

肾脏疾病影响全球10%以上的人口，并花费数十亿美元的联邦开支。最严重的肾脏疾病和最终的末期肾衰竭通常是由球状细胞的损伤引起的，球状细胞是高度专业化的上皮细胞，与内皮细胞和球状基底膜一起发挥作用，形成肾脏的过滤屏障。肾医学的进步因初级组织供应有限和缺乏强大的人类功能性肾细胞（如足细胞）的衍生方法而受到阻碍。从干细胞等可再生能源中提取足细胞的能力有助于增进目前对人类肾脏发育和疾病机制的理解，并为治疗发现提供新的工具。本协议的目标是开发一种方法，从具有高效率和特异性的人类诱导多能茎（hiPS）细胞中提取成熟、后粒细胞，并在化学定义条件下。这种方法产生的HIPS细胞衍生的足细胞表达血统特异性标记（包括肾上腺素、波多辛和威尔姆肿瘤1），并表现出与成熟和功能性足细胞相关的特殊形态特征（包括初级和二次足部过程）。有趣的是，这些特殊特征在该领域广泛使用的不朽的足细胞系中明显缺乏，这表明此处描述的协议产生的人类肾细胞比通常用于研究人类肾脏生物学的现有足细胞系具有更成熟的表型。

本协议的目的是证明成熟的人类卵母细胞可以在化学定义的条件下从HIPS细胞中提取。这份手稿中提供的数据是使用DU11 hiPS细胞线17生成的，该线首先被测试，并发现没有支原体。还进行了染色体分析，发现这些细胞在肌体上是正常的。从未分化的 DU11 HIPS 细胞开始， 本报告概述的分化策略（图1）首先将干细胞（图2A）分化为表达Brachyu...

Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

这里介绍了一个化学定义的协议，用于从诱导多能干细胞中提取具有高效率（>90%）的人类肾细胞，并且独立于遗传操作或亚人口选择。此协议在 26 天内生成所需的细胞类型，可用于肾毒性测试和疾病建模。

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

guided-differentiation-mature-kidney-podocytes-from-human-induced

Research

JoVE Journal

Bioengineering

4.4K 次观看.  Duke University. 这里介绍了一个化学定义的协议，用于从诱导多能干细胞中提取具有高效率（>90%）的人类肾细胞，并且独立于遗传操作或亚人口选择。此协议在 26 天内生成所需的细胞类型，可用于肾毒性测试和疾病建模。

视频: Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

由人诱导多能干细胞改造的同基因肾小球芯片

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

从多能干细胞生成人体血管类器官

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...