INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells
INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells
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DOI：
10.1007/s00125-011-2379-y
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发表时间：
2012-03-01
期刊：
影响因子：
8.2
通讯作者：
Stanley, E. G.
中科院分区：
文献类型：
--
作者：
Micallef, S. J.;Li, X.;Stanley, E. G.
我们的目的是建立人胚胎干细胞（hESC）报告细胞系，以便于体外培养的胰岛素产生（INS+）细胞的特征。以创建报告细胞系，从而能够前瞻性地分离活的INS+细胞。使用公开的方案的hESC证明所有GFP（+）细胞共产生胰岛素，证实了报告基因的保真度。INS-GFP（+）细胞通常共同产生胰高血糖素和生长抑素，证实了先前研究的结论，即早期hESC衍生的胰岛素产生细胞是多激素的。INS（GFP/w）hESC用于开发96孔格式的旋转胚状体（EB）分化方案，其使用基于重组蛋白的完全成分确定的培养基APEL。与用其他方法产生的INS-GFP（+）细胞一样，使用自旋EB方案衍生的那些细胞表达一套胰腺相关的转录因子基因，包括ISL 1、PAX 6和NKX2.2。然而，与以前的方法相比，旋转EB方案产生的INS-GFP（+）细胞也共表达β细胞转录因子基因NKX6.1，INS（GFP/w）hESC是用于研究β细胞个体发育中早期INS+祖细胞的性质的有价值的工具，并且将促进用于产生INS+细胞的新方案的开发。分化的hESC细胞。
We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells.Differentiation of INS (GFP/w) hESCs using published protocols demonstrated that all GFP(+) cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP(+) cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS (GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP(+) cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP(+) cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells.INS (GFP/w) hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs.