Serafimidis Ioannis, Rakatzi Irini, Episkopou Vasso, Gouti Mina, Gavalas Anthony
Developmental Biology Laboratory, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Stem Cells. 2008 Jan;26(1):3-16. doi: 10.1634/stemcells.2007-0194. Epub 2007 Oct 11.
The delineation of regulatory networks involved in early endocrine pancreas specification will play a crucial role in directing the differentiation of embryonic stem cells toward the mature phenotype of beta cells for cell therapy of type 1 diabetes. The transcription factor Ngn3 is required for the specification of the endocrine lineage, but its direct targets and the scope of biological processes it regulates remain elusive. We show that stepwise differentiation of embryonic stem cells using successive in vivo patterning signals can lead to simultaneous induction of Ptf1a and Pdx1 expression. In this cellular context, Ngn3 induction results in upregulation of its known direct target genes within 12 hours. Microarray gene expression profiling at distinct time points following Ngn3 induction suggested novel and diverse roles of Ngn3 in pancreas endocrine cell specification. Induction of Ngn3 expression results in regulation of the Wnt, integrin, Notch, and transforming growth factor beta signaling pathways and changes in biological processes affecting cell motility, adhesion, the cytoskeleton, the extracellular matrix, and gene expression. Furthermore, the combination of in vivo patterning signals and inducible Ngn3 expression enhances ESC differentiation toward the pancreas endocrine lineage. This is shown by strong upregulation of endocrine lineage terminal differentiation markers and strong expression of the hormones glucagon, somatostatin, and insulin. Importantly, all insulin(+) cells are also C-peptide(+), and glucose-dependent insulin release was 10-fold higher than basal levels. These data suggest that bona fide pancreas endocrine cells have been generated and that timely induction of Ngn3 expression can play a decisive role in directing ESC differentiation toward the endocrine lineage.
阐明参与早期内分泌胰腺特化的调控网络，对于引导胚胎干细胞向成熟β细胞表型分化以用于1型糖尿病的细胞治疗将起到关键作用。转录因子Ngn3是内分泌谱系特化所必需的，但它的直接靶点以及它所调控的生物过程范围仍不清楚。我们表明，利用连续的体内模式信号对胚胎干细胞进行逐步分化，可导致同时诱导Ptf1a和Pdx1表达。在这种细胞环境中，Ngn3的诱导会导致其已知直接靶基因在12小时内上调。Ngn3诱导后不同时间点的基因芯片基因表达谱分析表明，Ngn3在胰腺内分泌细胞特化中具有新的多样作用。Ngn3表达的诱导导致Wnt、整合素、Notch和转化生长因子β信号通路的调控，以及影响细胞运动、黏附、细胞骨架、细胞外基质和基因表达的生物过程的变化。此外，体内模式信号和可诱导的Ngn3表达的组合增强了胚胎干细胞向胰腺内分泌谱系的分化。这通过内分泌谱系终末分化标志物的强烈上调以及激素胰高血糖素、生长抑素和胰岛素的强烈表达得以体现。重要的是，所有胰岛素（+）细胞也是C肽（+），并且葡萄糖依赖性胰岛素释放比基础水平高10倍。这些数据表明已产生了真正的胰腺内分泌细胞，并且适时诱导Ngn3表达在引导胚胎干细胞向内分泌谱系分化中可起到决定性作用。
