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. 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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。
有兴趣调整此协议的研究人员应考虑以下关键步骤。首先，将HIPS细胞播种到层压涂层板上，以诱导间皮细胞是关键的一步。虽然建议种子密度为100，000个细胞/井，但研究人员可以根据用于研究的干细胞系的复制率调整初始细胞播种密度。如有必要，此优化将确保中皮诱导阶段有足够的细胞密度，并防止形成细胞聚合物或可能损害中皮、中间中皮或足细胞表型质量的细胞聚合体或非常大的团块（图3）。还必须注意，某些试剂（如CHIR99021）的制造商规格差异可能会影响分化细胞的质量。因此，建议测试来自不同批号或供应商和供应商的试剂的生物活性和独立实验之间的一致性。此外，重要的是要避免HIPS细胞过度暴露在分离酶中，因为这可能导致不受欢迎的分离从粘合基质和细胞在中等吸入过程中可能丢失。另一个值得注意的关键步骤是确保细胞诱导介质不受光线照射，以防止光诱发的视网膜酸灭活。
与以往的人类肾细胞分化方法相比，此处描述的方法采用化学定义的因子（包括小分子、生长因子和基底膜蛋白的特定等形，在所需的浓度下），以调节肾脏发育和足细胞功能所涉及的特定细胞信号通路。具体来说，从hiPS细胞中生成中皮细胞涉及使用小分子CHIR9902119所影响的规范Wnt信号通路的激活。已知，规范的Wnt信号通路可以调节参与组织发育和体内模式的基因的转录活性，以及细胞增殖和迁移20，21。本协议中使用的细胞诱导介质使细胞细胞从 iPS 细胞衍生的中间中皮细胞中导出。这种细胞诱导介质由Activin A、BMP7、VEGF、视网膜酸和CHIR99021组成。值得注意的是，这种足细胞诱导介质不需要未定义的血清成分，如胎儿牛血清，它可以掩盖特定因素的贡献，并限制其他细胞分化方法在组织发育和疾病的机械研究中的应用，使其不同于以前尝试生成肾细胞类型22的使用。此外，据观察，FGF2和FGF9不需要从HIPS细胞6，7，22分化肾细胞。虽然以前的方法依赖于多细胞聚集物的形成，如胚胎体和器官，但这里描述的方法产生的细胞在形态上和表达血统特定标记（包括肾上腺素和痘素）以及PAX2等祖细胞标记的下调节方面都表现出成熟的表型。预计本协议中所使用的化学成分的知识将促进今后对人类肾脏组织发育、细胞血统规范和疾病发病机制的机械研究。
这种HIPS细胞分化方法还能够产生成熟、后生和功能性足细胞，效率超过90%，无需亚种群选择、遗传操作或异种移植。这与以往的尝试明显不同，即细胞异质性水平高6、7、23限制了干细胞分化方法在转化医学应用中的应用。鉴于人类肾细胞从其他来源（如原发组织或器官）获得极为有限的挑战，这种方法也为该领域带来了重大进展。其中一些挑战包括使用依靠胚胎体或器官形成的方法时，推导效率低至1%以下。
本协议的一个限制是干细胞培养和分化步骤所需的试剂成本相对较高，这可能妨碍在某些实验室或研究小组中实施。此外，由于涉及细胞微环境中的多种因素（即可溶性、不溶性或粘合性提示）的很多优化，我们建议按照描述遵循协议。其他有兴趣使用这种细胞分化方法的人指出的一个常见错误包括使用不适当的细胞粘附矩阵，如BM基质1或其他定义不明确的动物衍生蛋白质。一旦按照协议的描述进行，可以进行进一步的实验，以检查对协议的其他修改的有效性。此外，由于人类干细胞系的内在差异，值得注意的是，分化方法的某些方面，例如暴露于分化介质或细胞分离介质的时间以及细胞播种密度，可能需要加以优化。然而，这些条件没有改变的HIPS细胞系和胚胎干细胞系测试至今。这种细胞分化方法适用于多个HIPS细胞系，包括PGP1、IMR-900-1和IISH3i-CB6以及H9胚胎干细胞系8、11。在本报告中，我们还演示了使用相同协议（图 1 和图 2）成功区分DU11 hiPS 细胞线。因此，鉴于这种方法在各种细胞系中的一致性，包括来自其他独立研究实验室（出版前）的未发表的工作的结果，我们预计这种分化协议将有助于从各种HIPS细胞系中提取肾足细胞，因此，不限于本协议中使用的细胞。
鉴于HIPS细胞能够无限期地自我更新，这种分化方法可用于为研究人员提供现成的人类肾细胞来源。这将有助于促进目前对人类肾脏生物学和疾病的理解， 并有助于开发新的体外肾脏系统，为人类肾脏功能建模和对治疗的反应。例如，最近采用并结合了微流体器官片上系统，以开发人类肾脏球状毛细管壁的功能体外模型。这种球状芯片有选择地过滤循环分子，并重新总结药物引起的肾毒性时，暴露在化疗药物，阿德里亚霉素8。将来，这些结果有可能与3D生物打印技术集成，以设计更复杂的人体肾脏体外模型。这些进展可以为评估候选药物提供新的平台，特别是那些针对由球状细胞功能障碍引起的肾脏疾病形式的候选药物。这一点尤其重要，因为动物模型的物种差异和缺乏器官水平功能通常观察到的标准组织培养方法可能会阻碍发展针对各种形式的人类肾脏疾病的靶向治疗25。因此，这种协议有一天可以提供机会，揭开人类肾脏发育所涉及的信号通路，以及疾病的发病机制。

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>&lt;strong&gt;Cells&lt;/strong&gt;</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>&lt;strong&gt;Growth Factors and Media Supplements&lt;/strong&gt;</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&amp;ndash;streptomycin, liquid (100&amp;times;)</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>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488&amp;ndash; and Alexa Fluor 594&amp;ndash;conjugated secondary antibodies</td><td>A32744; A32754; A-11076; A32790</td><td>Thermo/Life Technologies</td><td></td></tr><tr><td>Brachyury(T)</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&amp;amp;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>&lt;strong&gt;ECM Molecules&lt;/strong&gt;</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>&lt;strong&gt;Enzymes and Other Reagents&lt;/strong&gt;</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&amp;rsquo;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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;mu;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&amp;ndash;treated 12-well plates</td><td>353043</td><td>Corning</td><td></td></tr><tr><td>Tissue culture&amp;ndash;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Brachyury（图1B和图2B）的间皮细胞，然后分化成PAX2阳性中皮细胞（图2C），最后分化成成熟的肾球状细胞（图1B， 图 2D、图 3D和图 4）。中间细胞在分化协议的第2天获得，中间中皮细胞在第16天生成，成熟的细胞细胞在分化协议的第21天获得。当粘附在扁平的组织培养表面（如本实验中使用的层压涂板）时，产生的细胞体积约为 150-200 μm。HIPS细胞衍生的孔囊表现出许多足部过程般的延伸，这些扩展使用多种互补技术进行可视化，包括相对比显微镜（图1）、免疫荧光显微镜（图4）和扫描电子显微镜8，11。干细胞衍生的卵细胞也染色阳性WT115（图2D）以及血统识别标记肾素14（图4）。有趣的是，肾素的亚细胞定位主要是在足部过程和细胞细胞质，符合成熟的足细胞表型（图2D和图4A，B）。在细胞细胞生理学的发展过程中，蛋白肾素在细胞核和细胞质之间穿梭，但肾素在细胞质和足部过程中主要表现为细胞细胞细胞成熟并获得功能表型18。因此，通过使用本文所述的分化协议产生的足细胞主要在细胞质和足部过程样结构中表达肾素的观察，突出了该协议对于生成更成熟或更专业的足细胞的效用。扫描HIPS细胞衍生的足细胞的电子显微图突出了HIPS细胞衍生的足细胞在HIPS细胞衍生的足细胞中初级和二级足部过程的分支，正如我们第8组，第11组先前所报道的。由于WT1本身并不是肾多细胞的明确标记，因此强烈建议研究人员也对其细胞进行特征描述，以便同时表达多细胞特异性标记物，如多霉素和肾素。此前显示，使用这种方法衍生的绝症分化多细胞对波多辛、肾素和WT1免疫性阳性，祖细胞标记PAX28、11的表达相应减少。这些结果表明，使用本协议衍生的足细胞是发育成熟或专业化的，这是功能性人类肾球状足细胞所必需的。
在优化分化方法的条件时，观察到中间中皮细胞分离不足的情况。以密度播种的细胞大大超过12井板（在最后的卵细胞诱导步骤之前）的100，000个细胞/井的建议密度，导致大簇细胞在协议的标准时间线内缺乏成熟足细胞的预期形态表型（图3A、B）。出于这些原因，建议研究人员在试验特定下游应用所需的其他条件之前，使用本协议（图 3C，D）中建议的播种密度。这些结果共同表示使用上述化学定义的介质，在三周内将 hiPS 细胞定向分化成肾球状足细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61299/61299fig01.jpg"> 图1:HIPS细胞与细胞的分化。 （A） 将hiPS细胞定向分化成卵细胞的方法的原理表示。BMP7，骨形态遗传蛋白7:RA，视网膜酸;VEGF，血管内皮生长因子。这个数字已经从参考11修改。（B） 不同阶段的 HIPS 细胞代表图像。从左到右，图像显示了特征的人类iPS细胞群落在分离前进行分化的第0天，然后是间皮细胞在2天的分化后，然后中间间皮细胞在10天左右的分化，最后在第22天最终分化的细胞。比例尺条，所有图像的 100 μm（面板 B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61299/61299fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61299/61299fig02.jpg"> 图2:HIPS细胞及其分化衍生物的免疫荧光图像，显示分化时间轴期间特定血统标记和核反染的表达。 （A） 表示多能标记 10 月 4 日人类 iPS 细胞:（B） 表示胸膜的中皮细胞 （T）:（C） 表示 PAX2 的中间中皮细胞:和（D） 表达血统识别标记、肾素和相关标记 WT1 的 hiPS 细胞衍生的足细胞。缩放栏，所有图像 100 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61299/61299fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61299/61299fig03.jpg"> 图3:亚优播种密度对从DU11 hiPS细胞系中提取的中间间皮细胞的分化潜力的影响。 （A） 当初始细胞播种密度过高（约50万细胞/井12井板）时，细胞可以在细胞诱导阶段形成密集的聚集物（B）。（C） 建议播种密度，以防止结块（100，000个细胞/井的12井板），并微观观察（D）在足细胞分化阶段（插入）期间脚过程状结构的延伸。比例尺，A-D 200 μm;和 100 μm 的嵌入在 D.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61299/61299fig03large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61299/61299fig04.jpg"> 图4:HIPS细胞衍生的足细胞 在体外表现出形态成熟的表型。 （A） 肾素的HIPS细胞衍生孔细胞的免疫，以及（B） 显示主要和次要过程（白箭头）的足部过程的更高放大图像，以及这些特殊细胞结构中肾素的表达。秤杆，100μm （A）;50μm （B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61299/61299fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

4.5K 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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

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 ...

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 ...

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

一种从人类多能干细胞中生成肾类器官的简化方法

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

发育生物学

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 ...

使用诱导多能干细胞悬液生产肾脏类器官

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

作者聚焦：优化 iPSC 分化以实现高效生产以生成肾脏类器官 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

从诱导多能干细胞中产生悬浮液中的肾脏类器官

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease ...

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.

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

摘要

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海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

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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

从诱导多能干细胞中产生悬浮液中的肾脏类器官

从诱导多能干细胞中产生悬浮液中的肾脏类器官

从诱导多能干细胞中产生悬浮液中的肾脏类器官

由人诱导多能干细胞改造的同基因肾小球芯片