A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.
A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.
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DOI：
10.1371/journal.pone.0037004
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Robins AJ
中科院分区：
文献类型：
--
作者：
Schulz TC;Young HY;Agulnick AD;Babin MJ;Baetge EE;Bang AG;Bhoumik A;Cepa I;Cesario RM;Haakmeester C;Kadoya K;Kelly JR;Kerr J;Martinson LA;McLean AB;Moorman MA;Payne JK;Richardson M;Ross KG;Sherrer ES;Song X;Wilson AZ;Brandon EP;Green CE;Kroon EJ;Kelly OG;D'Amour KA;Robins AJ
开发基于人类胚胎干细胞（hESC）的1型糖尿病治疗方法将需要将原理验证概念转化为可扩展的，受控的和受监管的细胞制造过程。我们以前已经证明，人胚胎干细胞可以定向分化为胰腺祖细胞，成熟为功能性葡萄糖反应，胰岛素分泌细胞在体内。在这项研究中，我们描述了人胚胎干细胞的扩增和建库方法以及基于悬浮液的分化系统，它们共同支持了用于生产胰腺祖细胞的集成可扩展制造工艺。该系统已针对CyT 49细胞系进行了优化。因此，已生成合格的CyT 49大规模单细胞主细胞库和工作cGMP细胞库，为生产提供几乎无限的起始资源。从这些库解冻后，我们以贴壁培养形式扩增CyT 49两周，每周扩增50-100倍。然后在动态旋转悬浮培养中将未分化的CyT 49聚集成簇，然后用四阶段方案分化培养两周。如通过检查每个分化阶段的mRNA表达和最终群体的流式细胞术所示，许多成比例的分化运行产生了高度富集胰腺细胞谱系的可再现的和确定的群体组成。在植入小鼠后产生含有葡萄糖响应性胰岛素分泌细胞的胰岛样组织。移植后4 - 5个月，成熟的新胰腺组织足以防止链脲佐菌素（STZ）诱导的高血糖症。总之，我们已经开发了一种易于处理的生产工艺，用于以适于临床进入的规模从hESC产生功能性胰腺祖细胞。
Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.
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2001 年 8 月 9 日之前提取的三种人类胚胎干细胞系的核型稳定性、基因分型、分化、无饲养维持和基因表达取样
影响因子：
4
作者：
Brimble, SN;Zeng, XM;Schulz, TC
通讯作者：
Schulz, TC
DOI：
10.1006/bbrc.2001.6145
发表时间：
2001-12-21
影响因子：
3.1
作者：
Hosokawa, M;Dolci, W;Thorens, B
通讯作者：
Thorens, B
影响因子：
46.9
作者：
Kroon, Evert;Martinson, Laura A.;Baetge, Emmanuel E.
通讯作者：
Baetge, Emmanuel E.
影响因子：
4.6
作者：
Nostro, M. Cristina;Sarangi, Farida;Keller, Gordon
通讯作者：
Keller, Gordon
DOI：
10.1016/j.bbrc.2007.03.017
发表时间：
2007-05-11
影响因子：
3.1
作者：
Clark, Gregory O.;Yochem, Robert L.;Shamblott, Michael J.
通讯作者：
Shamblott, Michael J.