Name: differentiation of human pluripotent stem cells into insulin-producing islet clusters
Uploaded: 2023-06-23T14:00:00
Description: Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters at JoVE.com

我们非常感谢 STEMCELL Technologies、Michael Smith Health Research BC、Stem Cell Network、JDRF 和加拿大卫生研究院的支持。Jia Zhao 和 Shenghui Liang 是 Michael Smith Health Research BC 实习生奖的获得者。Mitchell J.S. Braam 是 Mitacs Accelerate Fellowship 的获得者。Diepiriye G. Iworima 是 Alexander Graham Bell 加拿大研究生奖学金和 CFUW 1989 年巴黎综合理工学院纪念奖的获得者。我们衷心感谢来自MCRI和莫纳什大学的Edouard G. Stanley博士分享Mel1 INS GFP / W 系和阿尔伯塔省糖尿病研究所胰岛核心分离和分布人类胰岛。我们还要感谢不列颠哥伦比亚大学生命科学研究所成像和流式细胞术设施的支持。 图 1 是使用 BioRender.com 创建的。

该协议基于在无饲养层条件下使用 hPSC 系，包括 H1、HUES4 PDXeG 和 Mel1 INSGFP/W。本节详细介绍了分步过程，并在代表性结果部分提供了来自Mel1 INSGFP/W 区分的支持数据。我们建议在使用此处未说明的其他 hPSC 细胞系时需要进一步优化。有关本方案中使用的所有试剂和溶液的详细信息，请参阅 材料表 。
1. 分化培养基及溶液的制备
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本文描述了一种七阶段混合方案，该方案允许在体外培养后 40 天内产生能够在葡萄糖激发时分泌胰岛素的 hPSC 胰岛。在这些多个步骤中，有效诱导最终内胚层被认为为最终分化结果设定了重要的起点 18,27,28。在制造商的方案中，建议接种密度为2.6×105 / cm 2以启动分化，并将细胞暴露于第1阶段培养基中2?...

胰腺β细胞分泌胰岛素，对血糖水平的升高做出反应。由于 1 型糖尿病 （T1D）1 中 β 细胞的自身免疫性破坏或 2 型糖尿病 （T2D）2 中 β 细胞功能障碍而缺乏足够胰岛素分泌的患者通常使用外源性胰岛素进行治疗。尽管这种疗法可以挽救生命，但它无法精确地与真正的β细胞动态分泌胰岛素所实现的血糖精确控制相媲美。因此，患者经常遭受危及生命的低血糖发作和慢性高血糖波动引起的其他并发症的后果。人类尸体胰岛的移植成功地恢复了 T1D 患者的严格血糖控制，但受到胰岛供体可用性和纯化健康胰岛进行移植的困难的限制 3,4。原则上，这一挑战可以通过使用hPSC作为替代起始材料来解决。
目前在体外从 hPSC 产生胰岛素分泌胰岛的策略通常旨在模拟体内胚胎胰腺发育的过程 5,6。这需要了解负责任的信号通路，并定时添加相应的可溶性因子，以模拟发育中的胚胎胰腺的关键阶段。胰腺程序从确定的内胚层开始，其标志是转录因子叉头盒 A2 （FOXA2） 和性别决定区 Y-box 17 （SOX17）7。最终内胚层的连续分化包括形成原始肠管，形成表达胰腺和十二指肠同源框 1 （PDX1）7,8,9 的后前肠，以及上皮扩张为共表达 PDX1 和 NK6 同源框 1 （NKX6.1）10,11 的胰腺祖细胞。
对内分泌胰岛细胞的进一步承诺伴随着促内分泌主调节因子神经原蛋白-3 （NGN3）12 的瞬时表达以及关键转录因子神经元分化 1 （NEUROD1） 和 NK2 同源框 2 （NKX2.2） 13 的稳定诱导。随后对主要的激素表达细胞进行编程，例如产生胰岛素的 β 细胞、产生胰高血糖素的 α 细胞、产生生长抑素的 delta 细胞和产生胰腺多肽的 PPY 细胞。凭借这些知识，以及广泛的高通量药物筛选研究的发现，最近的进展使 hPSC 胰岛能够产生类似于 β 细胞的细胞，能够分泌胰岛素 14,15,16,17,18,19。
已经报道了产生葡萄糖反应性 β 细胞的分步方案 6,14,18,19。基于这些研究，本方案涉及使用胰腺祖细胞试剂盒在平面培养物中产生 PDX1+/NKX6.1+ 胰腺祖细胞，然后将微孔板聚集成大小均匀的簇，并在静态 3D 悬浮培养物中使用 R 方案进一步分化为分泌胰岛素的 hPSC 胰岛。进行质量控制分析，包括流式细胞术、免疫染色和功能评估，以严格表征分化细胞。本文详细介绍了定向分化的每个步骤，并概述了体外表征方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,3&#8217;,5-Triiodo-L-thyronine (T3)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td>Thyroid hormone</td>
		</tr>
		<tr>
			<td>4% PFA solution</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Should be handled in fume hood</td>
		</tr>
		<tr>
			<td>96-Well, Ultralow Attachment, flat bottom</td>
			<td>Corning Costar (VWR)</td>
			<td>CLS3474</td>
			<td>Flat bottom; for static suspension culture in the last three stages</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>07920</td>
			<td>Dissociation reagent for Stage 4 cells</td>
		</tr>
		<tr>
			<td>Aggrewell400 plates</td>
			<td>STEMCELL Technologies</td>
			<td>34415</td>
			<td>400 &#181;m diameter microwell plates</td>
		</tr>
		<tr>
			<td>Aggrewell800 plates</td>
			<td>STEMCELL Technologies</td>
			<td>34815</td>
			<td>800 &#181;m diameter microwell plates</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-Human FOXA2 (goat IgG)</td>
			<td>R&#38;D Systems</td>
			<td>IC2400G</td>
			<td>1:100 in flow cytometry; used for assaying Stage 1 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat IgG Isotype Control</td>
			<td>R&#38;D Systems</td>
			<td>IC108G</td>
			<td>1:100 in flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Mouse anti-Human SST (mouse IgG2B)</td>
			<td>BD Sciences</td>
			<td>566032</td>
			<td>1:250 in flow cytometry; used for assaying Stage 7 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Mouse IgG2B Isotype Control</td>
			<td>R&#38;D Systems</td>
			<td>IC0041G</td>
			<td>1:500 in flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human C-peptide (mouse IgG1&#954;)</td>
			<td>BD Pharmingen</td>
			<td>565831</td>
			<td>1:2,000 in flow cytometry; used for assaying Stage 7 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human INS (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>565689</td>
			<td>1:2,000 in flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human NKX6.1 (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>563338</td>
			<td>1:33 in flow cytometry; used for assaying Stage 4 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human SOX17 (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>562594</td>
			<td>1:50 in flow cytometry; used for assaying Stage 1 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse IgG1&#954; Isotype Control</td>
			<td>BD Sciences</td>
			<td>557714</td>
			<td>1:50 in flow cytometry</td>
		</tr>
		<tr>
			<td>ALK5i II</td>
			<td>Cayman Chemicals</td>
			<td>14794</td>
			<td>TGF-beta signaling inhibitor</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>7010</td>
			<td>Microwell Rinsing Solution</td>
		</tr>
		<tr>
			<td>Assay chamber</td>
			<td>Cellvis</td>
			<td>D35-10-1-N</td>
			<td>For static GSIS and confocal imaging purposes</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1600-100</td>
			<td>For immunostaining procedure</td>
		</tr>
		<tr>
			<td>CK19 antibody</td>
			<td>DAKO</td>
			<td>M0888</td>
			<td>1:50 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>For nuclear counterstaining</td>
		</tr>
		<tr>
			<td>DMEM/F12, HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td>Matrix diluting solution</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa Fluor 555</td>
			<td>Life technologies</td>
			<td>A21432</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A21447</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Alexa Fluor 555</td>
			<td>Life technologies</td>
			<td>A31570</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A31571</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa Fluor 555</td>
			<td>Life technologies</td>
			<td>A31572</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A31573</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-sheep Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A21448</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td>Without Ca2+ and Mg2+</td>
		</tr>
		<tr>
			<td>ELISA, insulin, human</td>
			<td>Alpco</td>
			<td>80-INSHU-E01.1</td>
			<td>For human insulin measurement</td>
		</tr>
		<tr>
			<td>Fatty acid-free BSA</td>
			<td>Proliant</td>
			<td>68700</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>Fixation and Permeabilization Solution Kit</td>
			<td>BD Sciences</td>
			<td>554714</td>
			<td>Fix/Perm and 10x Perm/Wash solutions included</td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>7174</td>
			<td>For clump passaging hPSCs during maintenance culture</td>
		</tr>
		<tr>
			<td>Glucagon antibody</td>
			<td>Sigma</td>
			<td>G2654</td>
			<td>1:400 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>GLUT1 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA1-37782</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>GlutaMAX-I (100x)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td>G33-4</td>
			<td>For tissue clearing and mounting</td>
		</tr>
		<tr>
			<td>GSi XX</td>
			<td>Sigma Millipore</td>
			<td>565789</td>
			<td>Notch inhibitor</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>ITS-X (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>51500056</td>
			<td>Insulin-Transferrin-Selenium-Ethanolamine; medium supplement</td>
		</tr>
		<tr>
			<td>LDN193189&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>72147</td>
			<td>BMP antagonist</td>
		</tr>
		<tr>
			<td>MAFA antibody</td>
			<td>Abcam</td>
			<td>ab26405</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Matrigel, hESC-qualified</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-774-552</td>
			<td>Extracellular matrix for vessel surface coating</td>
		</tr>
		<tr>
			<td>MCDB131 medium</td>
			<td>Life technologies</td>
			<td>10372019</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>mTeSR1 Complete Kit</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>stem cell medium and 5x supplement included</td>
		</tr>
		<tr>
			<td>N-Cys (N-acetyl cysteine)</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>Antioxidant</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>NEUROD1 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF2746</td>
			<td>1:20 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>NKX6.1 antibody</td>
			<td>DSHB</td>
			<td>F55A12-c</td>
			<td>1:50 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Pancreatic polypeptide antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF6297</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>D8662</td>
			<td>With Ca2+ and Mg2+</td>
		</tr>
		<tr>
			<td>PDX1 antibody</td>
			<td>Abcam</td>
			<td>ab47267</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human GCG (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>565860</td>
			<td>1:2,000 in flow cytometry; used for assaying Stage 7 cells</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human NKX6.1 (mouse IgG1k)</td>
			<td>BD Sciences</td>
			<td>563023</td>
			<td>1:250 in flow cytometry</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human PDX1 (mouse IgG1k)</td>
			<td>BD Sciences</td>
			<td>562161</td>
			<td>1:200 in flow cytometry; used for assaying Stage 4 cells</td>
		</tr>
		<tr>
			<td>PE Mouse IgG1&#954; Isotype Control</td>
			<td>BD Sciences</td>
			<td>554680</td>
			<td>1:2,000 in flow cytometry</td>
		</tr>
		<tr>
			<td>PE Mouse-Human Chromogranin A (CHGA, mouse IgG1k)</td>
			<td>BD Sciences</td>
			<td>564563</td>
			<td>1:200 in flow cytometry</td>
		</tr>
		<tr>
			<td>R428&#160;</td>
			<td>Cayman Chemicals</td>
			<td>21523</td>
			<td>AXL tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>Retinoid acid, all-trans</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Light-sensitive</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer, 10x</td>
			<td>Sigma</td>
			<td>20-188</td>
			<td>For hormone extraction</td>
		</tr>
		<tr>
			<td>SANT-1</td>
			<td>Sigma</td>
			<td>S4572</td>
			<td>SHH inhibitor</td>
		</tr>
		<tr>
			<td>SLC18A1 antibody</td>
			<td>Sigma</td>
			<td>HPA063797</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Somatostatin antibody</td>
			<td>Sigma</td>
			<td>HPA019472</td>
			<td>1:100 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>STEMdiff Pancreatic Progenitor Kit</td>
			<td>STEMCELL Technologies</td>
			<td>05120</td>
			<td>Basal media and supplements included</td>
		</tr>
		<tr>
			<td>Synaptophysin antibody</td>
			<td>Novus</td>
			<td>NB120-16659</td>
			<td>1:25 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>For permeabilization</td>
		</tr>
		<tr>
			<td>Trolox&#160;</td>
			<td>Sigma Millipore</td>
			<td>648471</td>
			<td>Vitamin E analog</td>
		</tr>
		<tr>
			<td>TrypLE Enzyme Express</td>
			<td>Life technologies</td>
			<td>12604-021</td>
			<td>cell dissociation enzyme reagent for single cell passaging hPSCs</td>
		</tr>
		<tr>
			<td>Trypsin1/2/3 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF3586</td>
			<td>1:25 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>Sigma</td>
			<td>Z0251</td>
			<td>Medium supplement</td>
		</tr>
	</tbody>
</table>

differentiation of human pluripotent stem cells into insulin-producing islet clusters

人类多能干细胞 （hPSC） 分化为分泌胰岛素的 β 细胞为研究 β 细胞功能和糖尿病治疗提供了材料。然而，在获得充分模拟天然人类β细胞的干细胞衍生的β细胞方面仍然存在挑战。在先前研究的基础上，已经生成了 hPSC 衍生的胰岛细胞，以创建具有改进的分化结果和一致性的方案。这里描述的方案在第 1-4 阶段使用胰腺祖细胞试剂盒，然后在第 5-7 阶段使用根据 2014 年发表的论文修改的方案（以下简称“R 协议”）。包括使用胰祖细胞试剂盒和 400 μm 直径微孔板生成胰祖细胞簇的详细程序、用于 96 孔静态悬浮形式内分泌分化的 R 方案，以及 hPSC 衍生胰岛的 体外 表征和功能评估。完整的方案需要 1 周进行初始 hPSC 扩增，然后需要 ~5 周才能获得产生胰岛素的 hPSC 胰岛。具有基本干细胞培养技术和生物测定培训的人员可以重现该协议。

我们开发了一种混合策略，通过七个步骤将干细胞分化为分泌胰岛素的 hPSC 胰岛，该策略在平面培养的前四个阶段使用胰腺祖细胞试剂盒，然后在最后三个阶段的静态悬浮培养中以先前报道的方法6 为基础（图 1).使用该方案，确保在细胞接种后 24 小时（第 0 阶段）进行接近汇合 （90%-100%） 的培养对于启动大多数 hPSC 系的有效分化至关重要（<strong class="xfig"...

Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters at JoVE.com

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

干细胞分化为胰岛细胞为传统的糖尿病治疗和疾病建模提供了另一种解决方案。我们描述了一种详细的干细胞培养方案，该方案将商业分化试剂盒与先前验证的方法相结合，以帮助在培养皿中产生分泌胰岛素的干细胞衍生胰岛。

人类多能干细胞分化为产生胰岛素的胰岛簇

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

该方案使用静态悬浮培养策略将胰腺祖细胞分化为胰岛簇，减少细胞在振荡培养过程中所经历的应力，并大大提高分化成功率。硬件协议对初学者友好，并节省了前四个阶段的优化时间。具有基本干细胞培养技术的人员可以重现该协议，并很有可能产生功能性胰岛样簇。首先，用在冰冷的 DMEM/F12 中稀释的符合 hESC 标准的基质胶预包被培养孔，并将板置于潮湿的 37 摄氏度 5% 二氧化碳培养箱中 30 分钟。从 hPSC 培养物中吸出用过的培养基，并用 DPBS 冲洗一次。用细胞解离酶在37°C下将hPSC培养物解离到单个细胞中，持续3至5分钟。解离后，通过加入温热的 DMEM/F12 培养基冲洗细胞，并将它们转移到 15 或 50 毫升锥形管中。将细胞悬液以 300 g 离心 5 分钟，并将沉淀重悬于含有 10 微摩尔 Y-27632 的干细胞完全培养基中。然后，使用血细胞计数器进行活细胞计数。计数后，吸出稀释的基质胶，并立即在基质凝胶包被的孔上以1.6至1.8倍10至每平方厘米密度5%接种活细胞。将细胞在加湿的培养箱中孵育 24 小时。接下来，在第一阶段，第一天，准备阶段 1A 培养基。用 1A 期培养基替换用过的培养基并孵育 24 小时。第二天，用新鲜制备的1B阶段培养基替换孔中的用过的培养基。在第二阶段，第一天，制备 2A 期培养基，并用 2A 期培养基替换孔中的培养基。孵育 24 小时，然后将 2B 期培养基加入孔中;第三阶段，第一天，从孔中吸出用过的培养基，然后加入新鲜制备的第 3 阶段培养基并在加湿培养箱中孵育。在第 4 阶段，第 1 天，用新鲜制备的第 4 阶段培养基替换用过的培养基，并在加湿培养箱中孵育。在第四阶段，第五天，每孔用 500 微升抗粘附冲洗液预处理微孔。将微孔板以1， 300 G离心五分钟，并在显微镜下观察，以确保微孔中没有气泡。接下来，从孔中吸出冲洗液，并用一毫升DPBS冲洗一次。然后，向每个孔中加入一毫升聚集介质。从胰腺祖细胞培养物中取出用过的培养基后，用DPBS洗涤培养物一次，然后用解离试剂在37摄氏度下解离成单细胞10至12分钟。用温热的DMEM / F12冲洗细胞，并将它们转移到试管中以300G离心五分钟。将沉淀重悬于聚集培养基中并进行活细胞计数。每孔接种 2.4 至 360 万个细胞，并向每个孔中加入聚集培养基，以达到每孔 2 毫升的最终体积。使用 P-1000 移液器吸头，轻轻上下移液细胞以使其均匀分布。将细胞悬液以 300 g 离心 5 分钟以捕获微孔中的细胞，并在加湿培养箱中孵育 24 小时以开始聚集。聚集后，轻轻地上下移液聚集体几次，以浮动任何未聚集的细胞。一旦聚集体沉降，小心地除去含有漂浮的非聚集细胞的废培养基，而没有吸出聚集体。将新鲜的阶段 5 培养基分配到微孔板的表面上，以从微孔中去除聚集体，并将它们转移到超低附着的六孔板中。在超低附着的六孔中收集所有聚集体后，将它们重悬于第 5 阶段培养基中，以将密度调节至每 100 微升 20 个簇。然后，使用多通道移液器，将 50 微升 5 期培养基分配到超低附着 ULA 平底 96 孔板的每个孔中。用 200 微升 DPBS 填充角孔和边缘孔。每次分液前轻轻混合簇重悬液。然后，将 100 微升簇重悬液加入 ULA 平底 96 孔板的每个孔中。将培养板放在加湿培养箱的水平表面上 24 小时。第二天或第五阶段，第二天，使用多通道移液器，从每个孔中轻轻取出 90 微升用过的培养基，并用 100 微升的第 5 阶段培养基刷新。在第六阶段，第一天，每孔用 100 微升第 6 阶段培养基替换 90 微升用过的培养基。在第 7 阶段，第 1 天，取出用过的培养基，每孔加入 100 微升第 7 阶段培养基。在这项研究中，使用微孔板，一次生成数千个大小和形态均匀的胰祖细胞簇，平均聚集效率约为 74%，重要的是，PDX1 和 NKX6.1 的表达在这些簇中保持高水平聚集后。胰岛素表达细胞在内分泌簇内逐渐被诱导，如活培养物中胰岛素GFP信号随时间增加所表明的那样。在第五阶段和第七阶段之间，簇的大小从平均直径 150 微米增加到 220 微米。细胞组成的表征表明，混合方案产生的第七阶段 hPSC 胰岛主要是内分泌的，由四种主要的胰岛细胞类型组成。这种混合方案主要产生单激素胰岛细胞和少数双激素细胞。分化 β 细胞表达 NKX6.1、MAFA、NEUROD1、PDX1 和 GLUT1。此外，在第七阶段培养中诱导了 60% 胰岛素和 PDX1 双阳性细胞以及 50% C 肽和 NKX6.1 双阳性细胞。在体外，静态葡萄糖刺激的胰岛素分泌试验表明，第七阶段 hPSC 胰岛分泌高胰岛素水平以响应高葡萄糖和直接去极化。hPSC胰岛的总胰岛素含量约为尸体胰岛的一半。用于生成干细胞来源胰岛的多孔静态培养系统有助于各种原位检测，并可作为方案开发和小规模筛选研究的合适平台。为了在 96 孔板中静态培养期间尽量减少簇融合，在进行培养基更换时不要倾斜板。始终将板水平保持在水平表面上。

Research

JoVE Journal

Developmental Biology

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

人类多能干细胞的模板微图案用于探测分化命运的空间组织

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

科研

发育生物学

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

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

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

在体外人骨髓间充质干细胞分化为心肌样细胞功能

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

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...