An efficient experimental strategy for mouse ES cell differentiation and separation of a cytokeratin 19 positive population of insulin-producing cells

NAUJOK O; FRANCINI F; JÖRNS A; LENZEN S

Cell Proliferation

Año: 2008 vol. 41 p. 607 - 624

Abstract. Objectives: Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. It was the aim to develop a new differentiation protocol, which drives differentiation of ES cells towards insulinproducing cells rather than neuronal cells and to combine this with a strategy for separation of insulin-positive cells. Methods: The cytokeratin 19 promoter was used to control the expression of eYFP in mouse ES cells during their differentiation towards insulin-producing cells using a new optimized 4 stage differentiation protocol. Two cell populations, CK19+ and CK19- cells, were successfully FACS-sorted and analyzed. Results: The new differentiation protocol reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly the beta cell like character of the insulin-producing cells was strengthened, as documented by a high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This new differentiation protocol was combined with a cell sorting technique. Through this combined procedure a fraction of glucose responsive insulin-secreting CK19+ cells was obtained with a 40 fold higher insulin gene expression and 50 fold higher insulin content than CK19- cells. CK19+ cells were immunoreactive for C-peptide and showed ultrastructural characteristics of an insulin secretory cell. Conclusions: It can be concluded that differentiated CK19+ cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.Objectives: Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. It was the aim to develop a new differentiation protocol, which drives differentiation of ES cells towards insulinproducing cells rather than neuronal cells and to combine this with a strategy for separation of insulin-positive cells. Methods: The cytokeratin 19 promoter was used to control the expression of eYFP in mouse ES cells during their differentiation towards insulin-producing cells using a new optimized 4 stage differentiation protocol. Two cell populations, CK19+ and CK19- cells, were successfully FACS-sorted and analyzed. Results: The new differentiation protocol reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly the beta cell like character of the insulin-producing cells was strengthened, as documented by a high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This new differentiation protocol was combined with a cell sorting technique. Through this combined procedure a fraction of glucose responsive insulin-secreting CK19+ cells was obtained with a 40 fold higher insulin gene expression and 50 fold higher insulin content than CK19- cells. CK19+ cells were immunoreactive for C-peptide and showed ultrastructural characteristics of an insulin secretory cell. Conclusions: It can be concluded that differentiated CK19+ cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.