我们非常感谢所有毛和胡实验室成员，无论是过去还是现在，都对这个项目进行了有趣的讨论和巨大贡献。我们感谢国家儿童健康临床研究中心的大力支持。本研究由国家自然科学基金（U20A20351建华毛，82200784立单胡）、浙江省自然科学基金（No.LQ22C070004力丹胡）和江苏省自然科学基金（批准号）BK20210150给王刚）。

用于本研究的 iPSC 是从商业来源获得的。将细胞用 mTeSR 培养基维持在市售的基底膜基质包被板上（参见 材料表）。 表1 包含研究中使用的所有培养基组合物。
1. 接种 iPSC 以分化和诱导后原始条纹 （PS）
<ol><li>用 2 mL DPBS 在膜基质包被的 6 孔板上洗涤 iPSC。使用移液器吸出DPBS。</li>
	<li>加入1mL市售的细胞分离溶液（参见 材料表）以分离iPSCs，并在37°C下孵育5分钟。</li>
	<li>向 iPSC 中加入 1 mL mTeSR，并确保细胞已从塑料表面抬起。</li>
	<li>在室温（RT）下以400× g 离心细胞3分钟。重悬细胞沉淀并使用血细胞计数器计数细胞数。</li>
	<li>将一系列细胞密度接种到涂有膜基质的 24 孔或 6 孔板上，并用补充有 10 μM Rho 激酶抑制剂 （Y-27632） 的 mTeSR 培养（参见 材料表）。将它们在37°C CO2 培养箱中培养过夜。 注：为了诱导 PS，CHIR99021的最佳浓度和合适的细胞密度因不同的 iPSC 细胞系而异。接种密度范围（例如，每平方厘米0.6、0.9、1.2、1.5、1.8、2.4 * 10 3个单细胞 ）和7μM-12μM的CHIR99021浓度范围。 在同一天开始每种密度和每种CHIR的分化。在 24 孔板中找到最佳浓度的 CHIR99021 和合适的细胞密度，然后在 6 孔板中扩增培养物。在这里，我们提供了一个基于6孔板的方案。</li>
	<li>第二天，吸出 mTeSR 并用 2 mL DPBS 洗涤细胞。</li>
	<li>加入 2 mL I 期培养基（含有 8-12 μM CHIR99021 的干细胞分化培养基）（表 1），称为第 0 天。</li>
	<li>将它们在37°C培养箱中培养4天，每两天更新一次I期培养基。</li>
</ol>2. 诱导肾源性中间体中胚层（IM）
<ol><li>在第 4 天，取出 I 期培养基并用 2 mL DPBS 洗涤。</li>
	<li>向细胞中加入2mL II期培养基（含有200ng / mL FGF9,1μg/ mL肝素和1μM CHIR99021的干细胞分化培养基）（表1）。</li>
	<li>将它们在37°C培养箱中培养，并每2天更新一次培养基，直到第7天。</li>
</ol>3. 在悬浮培养中产生肾脏类器官
注意：在第 0 天到第 7 天的观察期间，细胞的状态至关重要。如果细胞表现出成功的扩增并堆积而没有明显的细胞碎片，则表明中间中胚层 （IM） 的成功衍生，表明已准备好进入下一阶段。然而，如果细胞出现初始凋亡的迹象，随后出现坏死或破损，则可能表明CHIR99021浓度或细胞密度不合适。
<ol><li>在第 7 天，取出 II 期培养基并用 2 mL DPBS 洗涤细胞。</li>
	<li>向每个孔中加入1mL细胞分离溶液，并在37°C下孵育5分钟，直到细胞在相衬下折射。</li>
	<li>将 1 mL II 期培养基移液到细胞中，混合并确保细胞已从表面抬起。</li>
	<li>将细胞悬液收集在 15 mL 试管中，并在室温下以 400 x g 离心 3 分钟。</li>
	<li>除去上清液，将细胞沉淀重悬于补充有10μMRho激酶抑制剂（Y-27632）的2mL III期培养基（含有200ng/mL FGF9,1μg/ mL肝素和1μM CHIR99021 0.1%甲基纤维素（MC），0.1%聚乙烯醇（PVA）的干细胞分化培养基）（参见 材料表）。轻轻混合。</li>
	<li>将细胞悬液和种子以 1：3 的比例混合到 6 孔低粘附板中。将板放在37°C和5%CO 2的培养箱中的轨道振荡器（耐CO2，以60rpm旋转）上。</li>
	<li>24小时后，除去Rho激酶抑制剂（Y-27632）并加入III期培养基（补充含有200ng / mL FGF9,1μg/ mL肝素，1μM CHIR99021,0.1%MC，0.1%PVA的干细胞分化培养基）。培养 2 天。按照以下步骤更改悬浮培养物中的培养基：<ol><li>用无菌剪刀剪掉 1 mL 移液器的尖端。</li>
		<li>轻轻摇晃 6 孔板，将类器官排列在板的中间。</li>
		<li>用准备好的移液器将类器官吸入 15 mL 管中，并让它们静置 5 分钟。</li>
		<li>吸出上清液并将类器官留在管底部。</li>
		<li>加入新鲜培养基以重悬类器官，然后重新铺板至 6 孔低粘附板中。</li>
	</ol></li>
	<li>继续在培养箱中以 60 rpm 的速度摇动低附着力板。每两天更换一次III期培养基，直到第12天。</li>
	<li>在第 12 天，将培养基更改为 IV 期培养基（含有 0.1% MC 和 0.1% PVA 的干细胞分化培养基）。</li>
	<li>每两天更换一次培养基，直到第 25 天。类器官可以从第 12-25 天逐渐发育成成熟的肾单位结构。</li>
</ol>4.肾脏类器官的免疫荧光染色
<ol><li>将肾脏类器官收集到 15 mL 试管中，并用 2 mL PBS 洗涤。</li>
	<li>在使其凝固5-10分钟后，将肾脏类器官在新鲜制备的2%PFA中在4°C下固定20分钟。</li>
	<li>除去PFA，用含有0.3%Triton X-100（0.3%PBST）的1mL PBS洗涤类器官，在振荡器（转速不超过60rpm）上均匀摇动8分钟，静置5分钟，然后除去上清液。重复三次。 注意：类器官可以在 4°C 下储存在 0.3% PBST 中 2-4 周。</li>
	<li>将固定类器官与10%驴血清在PBST（封闭缓冲液）（参见 材料表）中在4°C下孵育过夜。</li>
	<li>在封闭缓冲液中以 1：300 的比例稀释一抗（在 材料表中列出）。</li>
	<li>将类器官与一抗在4°C下搅拌孵育过夜。 注意：确保肾小球和肾小管可以同时在一个类器官上染色。</li>
	<li>用 1 mL 0.3% PBST 洗涤类器官，在振荡器（转速不超过 60 rpm）上均匀摇动 8 分钟，静置 5 分钟，然后除去上清液。重复三次。</li>
	<li>将类器官与二抗（列在 材料表中）和DAPI在4°C下搅拌孵育过夜。然后重复步骤 4.7。 注：二抗和DAPI均在封闭液，二抗（1：400）和DAPI（1：1000）中稀释。</li>
	<li>染色后，用甲醇（25%、50%、75% 和 100% 各 5 分钟）脱水类器官，然后用苯甲醇和苯甲酸苄酯（BABB，1：2 比例）（参见 材料表）渗透 5 分钟。</li>
	<li>在玻璃底培养皿上清除清除的类器官（参见 材料表），并通过共聚焦显微镜检查它们。 注意：成像时，选择玻璃盘底部的培养皿，渗透时，将BABB直接放入培养皿中，以保持培养皿底部湿润。</li>
</ol>5. 体外 葡聚糖摄取试验
<ol><li>在第25天，将类器官与100μg/ mL荧光标记的葡聚糖（参见 材料表）在37°C和5%CO2的培养箱中孵育4小时。</li>
	<li>4小时后，切换到新鲜培养基并使用宽视场荧光显微镜捕获活细胞培养图像。</li>
</ol>

使用诱导多能干细胞悬液生产肾脏类器官

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>Takasato, 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=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature Cell Biology. 16 (1), 118-126 (2014).</li><li>Morizane, R., Lam, A. Q., Freedman, B. S., Kishi, S., Valerius, M. T., Bonventre, J. 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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=Modelling+kidney+disease+with+CRISPR-mutant+kidney+organoids+derived+from+human+pluripotent+epiblast+spheroids.">Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.</a> Nature Communications. 6 (1), 8715 (2015).</li><li>Takasato, 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=Kidney+organoids+from+human+iPS+cells+contain+multiple+lineages+and+model+human+nephrogenesis.">Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.</a> Nature. 526 (7574), 564-568 (2015).</li><li>Mugford, J. W., Sipil&#228;, P., McMahon, J. A., McMahon, A. 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作者报告没有潜在的利益冲突。

已经描述了从 iPSC 生成肾脏类器官的详细方案，包括对基础培养基、初始细胞密度和CHIR99021浓度的微小修改。在各种实验中，发现成功产生肾脏类器官的关键因素是中间中胚层 （IM） 的初始分化和第 7 天的细胞状态。此外，不同的iPSC细胞系表现出细胞增殖和分化潜力的变化，导致不同的最佳细胞密度和CHIR99021浓度5,13,14。因此...

肾脏在维持生理稳态方面起着关键作用，具体取决于其功能单位。排泄废物的肾单位可以调节体液的成分。由遗传突变或其他高危因素引起的慢性肾脏病 （CKD） 最终会发展为终末期肾病 （ESKD）1,2。ESKD显然是由于肾单位的再生能力有限。因此，需要肾脏替代疗法。人类 iPSC 的定向分化能够在体外生成患者特异性 3D 肾脏类器官，可用于研究肾脏发育、模拟患者特定疾病和进行肾毒性药物筛选 3,4。
在胚胎发育过程中，肾脏起源于中间中胚层 （IM），这与原始条纹 （PS） 不同。经典的 WNT 信号通路可能在 FGF （FGF9， FGF20） 和 BMP （通过 JNK 的 Bmp7 信号转导）的协调参与下诱导 IM 的额外分化5,6,7。它们产生肾祖细胞 （NPC） 的两个重要细胞群：输尿管芽 （UB） 和肾间充质 （MM），分别形成集合管和肾单位 8,9。每个肾单位由肾小球和肾小管段组成，例如近端和远端小管，以及 Henle10,11 的环。根据上述理论，目前发表的方案模拟信号级联和生长因子刺激以诱导肾脏类器官5,12。
在过去的几年中，已经开发了许多方案来将人类 iPSC 区分为肾脏类器官 5,6,7,12。Takasato 等 7 优化了 FGF9 替代前 CHIR（WNT 激动剂）治疗的持续时间。根据他们的方案，CHIR 暴露 4 天，然后 FGF9 暴露 3 天，是诱导 iPSC IM 的最有效方法。Transwell过滤器在其程序中用作培养形式;但是，这种方法对于初学者来说很困难。因此，Kumar et al.13 试图改变文化形式，选择暂停文化。他们在第 7 天解离贴壁细胞，以接种在低贴壁板中，以帮助它们组装成含有肾单位样结构的胚状体 （EB）。然而，这些方法的批量效应是显而易见的，尤其是在不同的iPSC中。此外，不同的文献报道了 CHIR 的浓度从 7 μM 到 12 μM 不等 5,13,14。
我们推测细胞密度和CHIR的浓度可能会影响不同iPSC中类器官的产生，这在我们的实验中已经得到了多次验证。本方案略微修改了Kumar等人的研究方法13 ，并为用户提供了分步程序。该方法的时间表和示意图如图 1所示。

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			<td>Biotin anti-human LTL (1:300)</td>
			<td>Vector Laboratories</td>
			<td>Cat #B-1325</td>
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			<td>Blood mononuclear cells hiPS-B1 (iPSc, female)</td>
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			<td>STEMCELL Technologies</td>
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			<td>STEMCELL Technologies</td>
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			<td>Coolaber</td>
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			<td>Cat #MB4516-1</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (without calcium, magnesium, phenol red)</td>
			<td>Solarbio Life Science</td>
			<td>Cat #D1040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Bath Incubator</td>
			<td>Shanghai Jingxin</td>
			<td>Cat #JX-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 488-Goat Anti-Mouse IgG (1:400)</td>
			<td>Earthox</td>
			<td>Cat #E032210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 488-Goat Anti-Rabbit IgG (1:400)</td>
			<td>Earthox</td>
			<td>Cat #E032220</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 549-Goat Anti-Mouse IgG (1:400)</td>
			<td>Earthox</td>
			<td>Cat #E032310</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 549-Goat Anti-Rabbit IgG (1:400)</td>
			<td>Earthox</td>
			<td>Cat #E032320</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 649-Goat Anti-Rabbit IgG (1:400)</td>
			<td>Earthox</td>
			<td>Cat #E032620</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental models: Cell Lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Steri-Cycle CO2 Incubator</td>
			<td>Thermo SCIENTIFIC</td>
			<td>Cat #370</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltre LDEV-Free</td>
			<td>Gibco</td>
			<td>Cat #A1413202</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Culture Dishes</td>
			<td>NEST</td>
			<td>Cat #801002</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human CUBN (1:300)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Cat #sc-20607</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Solution (Cell culture supplement)</td>
			<td>STEMCELL Technologies</td>
			<td>Cat #07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant FGF-9</td>
			<td>STEMCELL Technologies</td>
			<td>Cat #78161</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td>Cat #CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>OLYMPUS</td>
			<td>Cat #FV3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>Cat #M7027</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge</td>
			<td>HENGNUO</td>
			<td>Cat #2-4B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD31 (1:300)</td>
			<td>BD Biosciences</td>
			<td>Cat #555444</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human ECAD (1:300)</td>
			<td>BD Biosciences</td>
			<td>Cat #610182</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human Integrin beta 1 (1:300)</td>
			<td>Abcam</td>
			<td>Cat #ab30394</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human MEIS 1/2/3 (1:300)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>Cat #39795</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88 (Polyvinylalcohol 4-88)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat #81381</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 5X Supplement</td>
			<td>STEMCELL Technologies</td>
			<td>Cat #85852</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 Basal Medium</td>
			<td>STEMCELL Technologies</td>
			<td>Cat #85851</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc CryoTube Vials</td>
			<td>Thermo SCIENTIFIC</td>
			<td>Cat #377267</td>
			<td></td>
		</tr>
		<tr>
			<td>Others</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human GATA3 (1:300)</td>
			<td>Cell Signaling Technology</td>
			<td>Cat #5852S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human LRP2 (1:300)</td>
			<td>Sapphire Bioscience</td>
			<td>Cat #NBP2-39033</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human Synaptopodin (1:300)</td>
			<td>Abcam</td>
			<td>Cat #ab224491</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human WT1 (1:300)</td>
			<td>Abcam</td>
			<td>Cat #ab89901</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse PDGFR (1:300)</td>
			<td>Abcam</td>
			<td>Cat #ab32570</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Serum Albumin (rHSA)</td>
			<td>YEASEN</td>
			<td>Cat #20901ES03</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep anti-human NPHS1 (1:300)</td>
			<td>R&#38;D Systems</td>
			<td>Cat #AF4269</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff APEL 2 Medium</td>
			<td>STEMCELL Technologies</td>
			<td>Cat #05275</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Cy3 (1:400)</td>
			<td>Gene Tex</td>
			<td>Cat #GTX85902</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene (1X)</td>
			<td>Gibco</td>
			<td>Cat #15040066</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 (Dihydrochloride)</td>
			<td>STEMCELL Technologies</td>
			<td>Cat #72304</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating kidney organoids in suspension from induced pluripotent stem cells

肾脏类器官可以通过多种方法从诱导多能干细胞 （iPSC） 中产生。这些类器官在疾病建模、药物筛选和潜在的治疗应用方面具有巨大的前景。本文介绍了从 iPSC 创建肾脏类器官的分步过程，从后原始条纹 （PS） 到中间中胚层 （IM）。该方法依赖于 APEL 2 培养基，这是一种确定的、不含动物成分的培养基。它补充高浓度的 WNT 激动剂 （CHIR99021），持续 4 天，然后补充成纤维细胞生长因子 9 （FGF9）/肝素和低浓度的 CHIR99021，持续 3 天。在此过程中，重点是在 iPSC 开始时选择最佳细胞密度和CHIR99021浓度，因为这些因素对于成功生成肾脏类器官至关重要。该协议的一个重要方面是在低贴壁板中悬浮培养，允许 IM 逐渐发育成肾单位结构，包括肾小球、近端肾小管和远端肾小管结构，所有这些都以视觉上可理解的形式呈现。总体而言，该详细方案提供了一种有效且特定的技术，可从不同的iPSC中生产肾脏类器官，从而确保成功和一致的结果。

The kidney plays a critical role in maintaining physiological homeostasis, depending on its functional unit. Nephrons, which excrete waste products, can regulate the composition of body fluids. Chronic kidney disease (CKD), caused by hereditary mutations or other high-risk factors, will eventually progress to end-stage kidney disease (ESKD)1,2. ESKD is apparently due to the limited regeneration capacity of nephrons. Thus, renal replacement therapy is required. Directed differentiation of human iPSCs enables the in vitro generation of patient-specific 3D kidney organoids, which can be used to study kidney development, model patient-specific diseases, and perform nephrotoxic drug screening3,4.
During embryonic development, kidneys originate from the intermediate mesoderm (IM), which differentiates from the primitive streak (PS). The classical WNT signaling pathway may induce additional differentiation of IM with the coordinated participation of FGF (FGF9, FGF20) and BMP (Bmp7 signaling through JNK)5,6,7. They produce two important cell populations of nephric progenitor cells (NPC): the ureteral bud (UB), and the metanephric mesenchyme (MM), forming the collecting ducts and the nephron, respectively8,9. Each nephron consists of glomerular and tubular segments, such as the proximal and distal tubules, and the loop of Henle10,11. According to the theory mentioned above, currently published protocols mimic the signal cascades and growth factor stimulation to induce kidney organoids5,12.
Over the past several years, many protocols have been developed to differentiate human iPSCs into kidney organoids5,6,7,12. Takasato et al.7 optimized the duration of CHIR (WNT agonist) treatment before replacement by FGF9. According to their protocol, CHIR exposure for 4 days, followed by FGF9 for 3 days, is the most effective way to induce IM from iPSCs. Transwell filters were utilized as the culture format in their procedure; however, this method is difficult for beginners. Therefore, Kumar et al.13 tried to change the culture format and chose to suspend the culture. They dissociated the adherent cells on Day 7 for seeding in low adherent plates to help them assemble into embryoid bodies (EBs) that contain nephron-like structures. However, the batch effect of these methods was apparent, especially in different iPSCs. Additionally, different literature reported that the concentration of CHIR varied from 7 &#181;M to 12 &#181;M5,13,14.
We speculated that the concentration of cell density and the CHIR might affect the generation of organoids in different iPSCs, and this has been verified numerous times in our experiments. The present protocol has slightly modified the study method of Kumar et al.13 and provided users with a step-by-step procedure. The schedule and schematic of the approach are shown in Figure 1.

作者聚焦：优化 iPSC 分化以实现高效生产以生成肾脏类器官 

从诱导多能干细胞中产生悬浮液中的肾脏类器官

Our research presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells, using suspension cultural conditions. We emphasize the importance of initial cell density and WNT agonist concentration during the inducing phase, which will educate new organoid researchers. Currently, it is difficult to obtain a single high-density cell line for iPSCs.The batch impact was noticeable, particularly in various iPSC lines. Each batch's kidney organoids, may have a varied form and size. Several attempts are necessary to get the best initial cell density and concentration of CHIR for one iPSC line.We have established that the initial differentiation of the intermediary method is important for successful kidney organoid formation. Our protocol describes in detail, how to choose the optimal cell densities and CHIR concentrations during the process because different iPSC lines showed variances in cell proliferation and differentiation ability. Researchers can quickly generate the specific kidney organoids from their iPSCs.According to our protocol, by adjusting the cell density and the concentration of CHIR, it's undoubtedly beneficial for all our researchers to focus on their kidney disease. Our lab future research has to optimize the method of the protocol in disease models, regenerate new medicine and the drug screening. In recent five years, our lab will focus on the epigenetics and the cell topic drug development of various hereditary kidney disease, based on patient development, iPSC and the kidney organized disease models.

IM 的产生是通过使用 GSK3 抑制剂 CHIR99021 激活经典 WNT 信号转导，然后使用 FGF9/肝素来实现的。从第 0 天到第 4 天，iPSC 迅速扩增并呈现菱形或三角形。汇合度达到 90%-100%，并均匀积累至第 7 天。悬浮培养后，聚集体在第 7 天解离后自发形成肾单位结构。通过悬浮培养产生的肾脏类器官显示出管状结构，并且在聚集 18 天后很容易在明场图像中观察到（图 2 和 ?...

Watch this Scientific Journal Video about Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids at JoVE.com

该方案提供了一种使用悬浮培养条件从诱导多能干细胞（iPSC）生产肾脏类器官的全面有效的方法。本研究的主要重点在于确定初始细胞密度和 WNT 激动剂浓度，从而使对肾脏类器官研究感兴趣的研究人员受益。

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

我们的研究提出了一种全面有效的方法，利用悬浮培养条件从诱导多能干细胞生产肾脏类器官。我们强调诱导阶段初始细胞密度和 WNT 激动剂浓度的重要性，这将培养新的类器官研究人员。目前，很难获得单一的高密度iPSC细胞系。批次影响是显而易见的，特别是在各种iPSC细胞系中。每批的肾脏类器官可能具有不同的形状和大小。需要多次尝试才能获得一个 iPSC 细胞系的最佳初始细胞密度和 CHIR 浓度。我们已经确定，中间方法的初始分化对于肾脏类器官的成功形成很重要。我们的方案详细描述了如何在过程中选择最佳细胞密度和CHIR浓度，因为不同的iPSC细胞系显示出细胞增殖和分化能力的差异。研究人员可以从他们的iPSC中快速生成特定的肾脏类器官。根据我们的方案，通过调整细胞密度和CHIR浓度，毫无疑问，我们所有的研究人员都专注于他们的肾脏疾病。我们实验室未来的研究必须优化疾病模型中的方案方法，再生新药和药物筛选。近五年来，本实验室将基于患者发育、iPSC和肾脏组织疾病模型，专注于各种遗传性肾病的表观遗传学和细胞专题药物开发。

generating-kidney-organoids-suspension-from-induced-pluripotent-stem

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JoVE Journal

Developmental Biology

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

1.6K 次观看.  Zhejiang University School of Medicine. 我们的研究提出了一种全面有效的方法，利用悬浮培养条件从诱导多能干细胞生产肾脏类器官。我们强调诱导阶段初始细胞密度和 WNT 激动剂浓度的重要性，这将培养新的类器官研究人员。目前，很难获得单一的高密度iPSC细胞系。批次影响是显而易见的，特别是在各种iPSC细胞系中。每批的肾脏类器官可能具有不同的形状和大小。需要多次尝试才能获得一个 iPSC 细胞系的...