Name: 作者聚焦：β细胞与多能干细胞分化的进展和挑战
Uploaded: 2024-02-02T13:00:00
Description: Watch this Scientific Journal Video about Author Spotlight: Advancements and Challenges in β-Cells Differentiation from Pluripotent Stem Cells at JoVE.com

Ines Cherkaoui 获得了 GAR 的英国糖尿病学生奖学金 （BDA 18/0005934） 的支持，他还感谢 Wellcome Trust 颁发的研究者奖 （212625/Z/18/Z）、UKRI MRC 的项目资助 （MR/R022259/1）、英国糖尿病项目资助 （BDA16/0005485）、CRCHUM 的启动资金、加拿大创新部的 John R. Evans 领袖奖 （CFI 42649）， NIH-NIDDK （R01DK135268） 获得项目资助，CIHR、JDRF 获得团队资助 （CIHR-IRSC：0682002550;JDRF 4-SRA-2023-1182-S-N）。Camille Dion 和 Harry Leitch 博士在人类 hiPSC 生成和培养方面的帮助，伦敦 NIHR 帝国 BRC（生物医学研究中心）类器官设施。

在开始分化之前，建议确定用于实验目的的胰岛样类器官的所需数量。在 6 孔板中，汇合度超过 80% 的单个孔通常由 2-230 万个 hPSC 组成。虽然由于 hPSC 细胞系和分化效率的变化，准确预测具有挑战性，但粗略估计是初始孔数的 1.5 倍。有效的定向分化通常在六孔板中每孔产生 1.6 至 200 万个细胞，包括簇内的所有细胞，而不是仅产生胰岛素的细胞。对于 50 μm 的团簇，它可以近似包含大约 10,000 个细...

将人多能干细胞分化为胰腺 β 细胞

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hPSCs成功分化为胰腺β细胞取决于优化所选hPSCs的常规培养和传代的各个方面。这包括确保细胞系具有正常的核型，对支原体感染呈阴性，并且没有质粒或病毒载体基因组。此外，在使用hiPSCs时，重要的是要避免使用仍在进行重编程的最早传代进行试点实验。这些实验应在小规模上进行，以确定具有最佳分化潜力和最佳传代次数的hPSC系。
其他可能影响分化效率的参数包括所用?...

人多能干细胞 （hPSC） 具有分化成各种细胞类型的独特能力，使其成为人胰腺β细胞的可行替代品1。这些 hPSC 分为两种类型：源自囊胚2 的胚胎干细胞 （hESC） 和通过直接重编程体细胞产生的诱导多能细胞 （hiPSC） 3。开发将 hPSC 分化为 β 细胞的技术对基础研究和临床实践都具有重要意义 1,4。糖尿病是一种慢性疾病，影响全球 >4 亿人，是由于胰腺β细胞功能障碍或丢失导致身体无法调节血糖5.用于移植的胰岛细胞的有限可用性阻碍了糖尿病细胞替代疗法的发展 2,4,6,7。使用 hPSC 产生葡萄糖反应性胰岛素分泌细胞的能力可作为研究人类胰岛发育和功能的有用细胞模型。它还可用于在受控环境中测试糖尿病治疗的潜在候选治疗药物。此外，hPSC 有可能产生与患者基因相同的胰岛细胞，从而降低移植后免疫排斥的风险 2,4,7。
近年来，hPSC培养和分化方案的改进取得了重大进展，从而提高了产生胰腺β细胞的分化过程的效率和可重复性8,9。
以下方案概述了胰腺β细胞定向分化的基本阶段。它涉及在不同时间点对特定细胞信号通路的调节。它基于Sui L.等人10 （2018）开发的用于将hPSC生成到胰腺β细胞中的方案。该方案根据 Sui L. 等人 11 （2021） 的最新更新进行了调整，因为最新研究强调了使用阿菲二可林 （APH） 治疗增强β细胞分化的重要性。目前的方案包括在工艺的后期阶段将APH添加到培养基中。此外，与初始方案相比，在分化的早期阶段对培养基的组成进行了修改。一个显着的变化是在第 6 天添加了角质形成细胞生长因子 （KGF），并持续到第 8 天。角质形成细胞生长因子 （KGF） 从第 6 天到第 8 天引入，这与初始方案10 略有不同，其中 KGF 不包括在第 4 阶段培养基中。
产生β细胞样细胞的第一步也是必不可少的一步是将hPSC定向分化为最终内胚层（DE），这是一种原始的胚层，可产生包括胰腺在内的各种器官的上皮衬里。DE形成后，细胞分化成原始肠管，随后是后前肠命运的规范。然后前肠后发育成胰腺祖细胞，这些祖细胞有可能分化成胰腺的所有细胞类型，包括内分泌细胞和外分泌细胞。该过程的后续阶段涉及胰腺内分泌祖细胞，从而产生在朗格汉斯胰岛中发现的激素分泌细胞。最后，分化过程通过产生功能齐全的胰腺β细胞样细胞达到最后阶段 9,10。需要注意的是，该过程很复杂，通常需要优化培养条件，例如特定的生长因子和细胞外基质成分，以提高分化的效率和特异性9,10。此外，在体外从hPSCs中产生功能性β细胞样细胞仍然是一个主要挑战。正在进行的研究重点是改进分化方案并增强所得β细胞的成熟度和功能9.
在该方案中，在hPSC的培养和传代过程中使用温和的细胞解离对于维持细胞活力和多能性至关重要，从而显着提高分化为胰腺β细胞的效率。此外，每种阶段特异性培养基都按照Sui L.等人开发的方案进行了精心优化10 ，以促进与人类胰岛非常相似的簇中胰岛素分泌细胞的高产量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL TubeOne Microcentrifuge Tube</td>
			<td>Starlabs</td>
			<td>S1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Cell culture plate</td>
			<td>ThermoFisher Scientific</td>
			<td>165218</td>
			<td></td>
		</tr>
		<tr>
			<td>AggreWell 400 6-well plate&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>34425</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Glucagon&#160;</td>
			<td>Sigma-aldrich</td>
			<td>G2654-100UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Insulin&#160;</td>
			<td>Dako</td>
			<td>A0564</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NKX6.1</td>
			<td>Novus Biologicals</td>
			<td>NBP1-49672SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PDX1&#160;</td>
			<td>Abcam</td>
			<td>ab84987</td>
			<td></td>
		</tr>
		<tr>
			<td>Aphidicolin</td>
			<td>Sigma-Aldrich</td>
			<td>A4487</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, fatty acid free</td>
			<td>Sigma-Aldrich</td>
			<td>A3803-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium/Magnesium free D-PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclopamine-KAAD</td>
			<td>Calbiochem</td>
			<td>239804</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose,BioXtra</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>94046-100ML-</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM plus GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>10566016</td>
			<td>For Washing Medium 2: DMEM plus GlutaMAX 1% PS.&#160;</td>
		</tr>
		<tr>
			<td>DMEM/F-12 (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredi SuperFrost Plus Adhesion slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>20821.33</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270098</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma-Secretase Inhibitor XX</td>
			<td>Thermo Fisher Scientific</td>
			<td>J64904</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413302</td>
			<td>Geltrex 1:1 into cold DMEM/F-12 medium to provide a final dilution of 1:100.</td>
		</tr>
		<tr>
			<td>Goat Anti-Guinea pig, Alexa Fluor 555</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21435</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Guinea pig, Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150187</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse Secondary Antibody, Alexa Fluor 633</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21052</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11011</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-7 (KGF) Recombinant Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen chloride</td>
			<td>Sigma-Aldrich</td>
			<td>295426</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Agar Scientific</td>
			<td>AGG4582</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Sigma-Aldrich</td>
			<td>SML0559-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M9272-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound 118 mL</td>
			<td>Agar Scientific</td>
			<td>AGR1180</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Tablets, Phosphate Buffered Saline, Fisher BioReagents</td>
			<td>Thermo Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (PS)</td>
			<td>Thermo Fisher Scientific,</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF Protein</td>
			<td>R&#38;D Systems</td>
			<td>236-EG-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular cover glasses, 22&#215;50 mm</td>
			<td>VWR</td>
			<td>631-0137</td>
			<td></td>
		</tr>
		<tr>
			<td>RepSox (Hydrochloride)</td>
			<td>STEMCELL Technologies</td>
			<td>72394</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>61870036</td>
			<td>For Washing Medium 1: RPMI 1640 plus GlutaMAX 1% PS.</td>
		</tr>
		<tr>
			<td>Shandon Immu-mount</td>
			<td>Thermo Fisher Scientific</td>
			<td>9990402</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Endoderm&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>5110</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3349401</td>
			<td>Thaw the StemFlex Supplement overnight at 4&#176;C, transfer any residual liquid of the supplement bottle to StemFlex Basal Medium.</td>
		</tr>
		<tr>
			<td>Stemolecule All-Trans Retinoic Acid</td>
			<td>Reprocell</td>
			<td>04-0021&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroid Tormone 3 (T3)</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>ThermoFisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypL Express Enzyme (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Adherent Plate for Suspension Culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>38071</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 (Dihydrochloride)&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z4750</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized protocol for generating functional pancreatic insulin-secreting cells from human pluripotent stem cells

人类多能干细胞（hPSCs）可以分化成任何类型的细胞，使其成为人类胰腺β细胞的极好替代来源。hPSC可以是源自囊胚的胚胎干细胞（hESC），也可以是使用重编程过程直接从体细胞产生的诱导多能细胞（hiPSC）。这里提出了一个基于视频的方案，概述了hPSCs在分化和随后产生胰岛素的胰腺细胞之前的最佳培养和传代条件。该方法遵循β细胞定向分化的六阶段过程，其中 hPSC 分化为确定性内胚层 （DE）、原始肠管、后前肠命运、胰腺祖细胞、胰腺内分泌祖细胞，最终分化为胰腺β细胞。值得注意的是，这种分化方法需要 27 天才能产生人胰腺β细胞。通过两个实验评估胰岛素分泌的潜力，包括免疫染色和葡萄糖刺激的胰岛素分泌。

Human pluripotent stem cells (hPSCs) have the unique ability to differentiate into various cell types, making them a viable alternative to human pancreatic &#946;-cells1. These hPSCs are categorized into two types: embryonic stem cells (hESCs), derived from the blastocyst2, and induced pluripotent cells (hiPSCs), generated by reprogramming somatic cells directly3. The development of techniques to differentiate hPSCs into &#946;-cells, has important implications for both fundamental research and clinical practice1,4. Diabetes mellitus is a chronic disease affecting &#62;400 million people worldwide and results from the inability of the body to regulate glycemia due to malfunction or loss of pancreatic &#946;-cells5. The limited availability of pancreatic islet cells for transplantation has hindered the development of cell replacement therapies for diabetes2,4,6,7. The ability to generate glucose-responsive insulin-secreting cells using hPSCs serves as a useful cellular model for studying human islet development and function. It can also be used to test potential therapeutic candidates for diabetes treatment in a controlled environment. Moreover, hPSCs have the potential to produce pancreatic islet cells that are genetically identical to the patient, reducing the risk of immune rejection after transplantation2,4,7.
In recent years, there have been significant advancements in the refinement of hPSC culture and differentiation protocols, resulting in increased efficiency and reproducibility of the differentiation process toward generating pancreatic &#946;-cells8,9.
The following protocol outlines the essential stages of directed differentiation of pancreatic &#946;-cells. It involves the regulation of specific cell signaling pathways at distinct time points. It is based on the protocol developed by Sui L. et al.10 (2018) for the generation of hPSCs into pancreatic &#946;-cells. The protocol was adjusted to recent updates from Sui L. et al.11 (2021), as the latest research emphasizes the significance of using aphidicolin (APH) treatment to enhance the differentiation of &#946;-cells. The current protocol includes the addition of APH to the medium during the later stages of the process. Furthermore, modifications have been made to the composition of the medium during the early stages of differentiation compared to the initial protocol. A notable change is the addition of Keratinocyte Growth Factor (KGF) on Day 6 and continuing until Day 8. The keratinocyte growth factor (KGF) is introduced from day 6 to day 8, which slightly differs from the initial protocol10, where KGF was not included in the stage 4 medium.
The first and essential step in the generation of &#946;-cell-like cells is the directed differentiation of hPSCs into definitive endoderm (DE), a primitive germ layer that gives rise to the epithelial lining of various organs, including the pancreas. After the formation of DE, the cells undergo differentiation into the primitive gut tube, which is followed by the specification of the posterior foregut fate. The posterior foregut then develops into pancreatic progenitor cells, which have the potential to differentiate into all cell types of the pancreas, including the endocrine and exocrine cells. The subsequent stage in the process involves pancreatic endocrine progenitors giving rise to the hormone-secreting cells found in the islets of Langerhans. In the end, the differentiation process reaches its final stage by producing fully functional pancreatic &#946;-cell like cells9,10. It is important to note that this process is complex and often requires optimization of the culture conditions, such as specific growth factors and extracellular matrix components, to improve the efficiency and specificity of differentiation9,10. Furthermore, generating functional &#946;-cell like cells from hPSCs in vitro is still a major challenge. Ongoing research focuses on improving differentiation protocols and enhancing the maturation and function of the resulting &#946;-cells9.
In this protocol, the use of gentle cell dissociation during the culture and passage of hPSCs is essential to maintain cell viability and pluripotency, significantly improving the efficiency of differentiation into pancreatic &#946;-cells. Additionally, each stage-specific medium has been meticulously optimized following the protocol developed by Sui L. et al.10 to promote a high yield of insulin-secreting cells in clusters that closely resemble the human islet.

作者聚焦：β细胞与多能干细胞分化的进展和挑战

从人多能干细胞生成功能性胰腺胰岛素分泌细胞的优化方案

Attaining a high and consistent efficiency in the differentiation process for converting pluripotent stem cells into human beta cells is a notable challenge. This challenge is influenced by the precision and the consistency with which the experimenter follows the protocol. Moreover, the amount of beta cells obtained in each experiment is quite variable, which may result in the reduced function of beta cells across multiple experiments.In the context of high leptin beta cell research in diabetes, the availability of cadaveric eyelet donors is quite limited. Consequently, the use of stem cell differentiated beta cells is offering an unlimited and abundant supply of insulin secreting cells. Beta cell differentiation is a pivotal advancement in our field, offering numerous benefits.It allows us to explore the development and function of beta cells, leading to a deeper understanding of diabetes causes and beta cell dysfunction. We aim to use this technique for our research focusing on the genetics of diabetes. Our primary objective is to focus on mutation of certain genes that can influence beta cells and eyelet function.So this protocol will facilitate the access to human eyelet derived from pre-potent stem cells with mutations of interest.

本文中描述的方案提供了一种从 hPSC 中区分 β样细胞的高效方法10。该过程利用易于扩展的 2D 培养系统，使其能够在各种实验环境中使用，例如学习分化、小型项目和实验室以及中试测试，以评估 iPSC 细胞系的分化潜力。
表征胰岛中分化β细胞的功能特性对于深入了解葡萄糖稳态至关重要。这通常通过各种实验来实现，例如β细胞标志物和胰岛素表达的免疫?...

Watch this Scientific Journal Video about Author Spotlight: Advancements and Challenges in β-Cells Differentiation from Pluripotent Stem Cells at JoVE.com

本文介绍了一种用于β细胞样细胞定向分化和功能分析的方案。我们描述了在产生产生胰岛素的胰腺细胞之前，人类多能干细胞的最佳培养条件和传代。六阶段分化从明确的内胚层形成发展到功能性β细胞样细胞，响应葡萄糖分泌胰岛素。

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic ...

在将多能干细胞转化为人类β细胞的分化过程中获得高且一致的效率是一项显着的挑战。这一挑战受到实验者遵循协议的精度和一致性的影响。此外，在每个实验中获得的β细胞的数量变化很大，这可能导致β细胞在多个实验中的功能降低。在糖尿病中高瘦素β细胞研究的背景下，尸体孔眼供体的可用性非常有限。因此，干细胞分化的β细胞的使用提供了无限和丰富的胰岛素分泌细胞供应。β细胞分化是我们领域的一项关键进步，具有许多优势。它使我们能够探索β细胞的发育和功能，从而更深入地了解糖尿病的原因和β细胞功能障碍。我们的目标是将这种技术用于我们的研究，重点是糖尿病的遗传学。我们的主要目标是关注某些可能影响β细胞和孔眼功能的基因突变。因此，该协议将促进获得来自具有目标突变的预强效干细胞的人类孔眼。

optimized-protocol-for-generating-functional-pancreatic-insulin

Research

JoVE Journal

Biology

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.