Characterization of an In Vitro Differentiation Assay for Pancreatic-Like Cell Development from Murine Embryonic Stem Cells: Detailed Gene Expression Analysis
Characterization of an In Vitro Differentiation Assay for Pancreatic-Like Cell Development from Murine Embryonic Stem Cells: Detailed Gene Expression Analysis
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DOI：
10.1089/adt.2010.0314
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发表时间：
2011-08-01
影响因子：
1.8
通讯作者：
Ku, Hsun Teresa
中科院分区：
文献类型：
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作者：
Chen, Chialin;Chai, Jing;Ku, Hsun Teresa
胚胎干细胞（ES）技术可以作为发现治疗糖尿病等疾病的药物的平台。然而，由于难以建立可靠的ES细胞分化方法和为高通量形式创造成本有效的平板条件，因此筛选调节胰腺β细胞及其直接祖细胞的分子受到限制。一个相对简单和廉价的分化协议，允许有效的生成胰岛素表达细胞从小鼠ES细胞先前建立在我们的实验室。在这份报告中，该系统的特点是更详细地映射发育细胞阶段，为未来的筛选实验。我们的研究结果表明，顺序激活未分化的ES细胞，上胚层，定形内胚层，前肠，胰腺谱系的多个基因标记被发现遵循模仿胰腺个体发育的事件序列。表达增强型绿色荧光蛋白（由胰腺和十二指肠同源框1或胰岛素1启动子驱动）的细胞正确表达已知的β细胞谱系标志物。内胚层命运决定转录因子Sox 17在分化的非常早期阶段（第2-3天）的过表达增强了胰腺基因的表达。在胰腺和十二指肠同源框1信息存在的阶段（第10-16天），神经生成素3（一种内分泌祖细胞标记物）的过表达诱导胰高血糖素表达。MafA（胰腺成熟因子）的强制表达（第16天至第25天）导致胰岛素基因、葡萄糖转运蛋白2和葡萄糖激酶的表达增强，以及葡萄糖响应性胰岛素分泌增强。第20天的细胞植入体内导致胰腺样细胞。总之，我们的分化试验概括了胰腺发育的过程和行为，对将来筛选β细胞效应子有价值。
Embryonic stem (ES) cell technology may serve as a platform for the discovery of drugs to treat diseases such as diabetes. However, because of difficulties in establishing reliable ES cell differentiation methods and in creating cost-effective plating conditions for the high-throughput format, screening for molecules that regulate pancreatic beta cells and their immediate progenitors has been limited. A relatively simple and inexpensive differentiation protocol that allows efficient generation of insulin-expressing cells from murine ES cells was previously established in our laboratories. In this report, this system is characterized in greater detail to map developmental cell stages for future screening experiments. Our results show that sequential activation of multiple gene markers for undifferentiated ES cells, epiblast, definitive endoderm, foregut, and pancreatic lineages was found to follow the sequence of events that mimics pancreatic ontogeny. Cells that expressed enhanced green fluorescent protein, driven by pancreatic and duodenal homeobox 1 or insulin 1 promoter, correctly expressed known beta cell lineage markers. Overexpression of Sox17, an endoderm fate-determining transcription factor, at a very early stage of differentiation (days 2-3) enhanced pancreatic gene expression. Overexpression of neurogenin3, an endocrine progenitor cell marker, induced glucagon expression at stages when pancreatic and duodenal homeobox 1 message was present (days 10-16). Forced expression (between days 16 and 25) of MafA, a pancreatic maturation factor, resulted in enhanced expression of insulin genes, glucose transporter 2 and glucokinase, and glucose-responsive insulin secretion. Day 20 cells implanted in vivo resulted in pancreaticlike cells. Together, our differentiation assay recapitulates the proceedings and behaviors of pancreatic development and will be valuable for future screening of beta cell effectors.