In vitro induction of pancreatic β-cell development from mouse embryonic stem cells

胚胎干细胞（Embryonic stem cells，ES细胞）是从受精囊胚的内细胞团中分离出来的多能性细胞。ES细胞显示出在体外分化成多种细胞谱系的能力。研究表明，ES细胞可以分化为造血细胞、神经元和心肌细胞。我们研究了ES细胞分化为胰岛素分泌胰腺β细胞的可能性。为了促进向胰岛素产生细胞的分化，我们操纵ES细胞以稳定表达IPF-1，IPF-1是一种在胰腺和胰腺β细胞发育中起关键作用的转录因子。在产生胚状体后，使产生IPF-1的ES细胞在Lumelsky等（Science 292：1389）的无血清培养基中分化，并进行了修改。与天然ES细胞衍生的分化细胞相比，产生IPF-1的ES细胞衍生的细胞产生高达5倍的免疫反应性胰岛素。我们通过免疫组织化学分析证实了免疫反应性胰岛素在大约30%的最终分化阶段的细胞中表达。我们目前正在努力优化条件，以实现更有效的分化。同时，我们还对EGFP标记的ES细胞进行了详细的研究，在这些细胞中，EGFP可以在胰岛素分泌细胞中特异性表达。在分化后，将能够通过使用这些细胞的荧光辅助细胞分选来选择胰岛素分泌细胞。

Embryonic stem cells (ES cells) are pluripotent cells derived from the inner cell mass of fertilized blastocysts. ES cells display the ability to differentiate in vitro into a variety of cell lineages. It has been shown that ES cells can be differentiated to hematopoietic cells, neurons and cardiomyocytes. We investigate the possibility to differentiate the ES cells to insulin secreting pancreatic β-cells. In order to facilitate the differentiation to the insulin producing cells, we manipulated ES cells to stably express IPF-1, a transcription factor which plays critical roles in the development of pancreas and pancreatic β-cells. After production of embryoid bodies, IPF-1 producing ES cells were rendered to differentiate in the serum free medium of Lumelsky et al (Science 292: 1389) with modification. IPF-1 producing ES cell-derived cells produced up to 5 times more immunoreactive insulin when compared with the differentiated cells derived from native ES cells. We confirmed the expression of immunoreactive insulin in approximately 30 % of the cells at final differentiation stage by immunohistochemical analysis. We are currently trying to optimize the condition for more efficient differentiation. At the same time, we have elaborated the ES cells marked by EGFP in which EGFP can be expressed specifically in the insulin-producing cells. Upon the differentiation, insulin secreting cells will be able to be selected by fluorescence-assisted cell sorting using these cells.