Open AccessAn efficient experimental strategy for mouse embryonic stem cell differentiation and separation of a cytokeratin‐19‐positive population of insulin‐producing cells
Author(s) -
Naujok O.,
Francini F.,
Jörns A.,
Lenzen S.
Publication year - 2008
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.2008.00541.x
Subject(s) - pdx1 , insulin , biology , embryonic stem cell , stem cell , glut2 , microbiology and biotechnology , cellular differentiation , p19 cell , population , medicine , endocrinology , adult stem cell , glucose transporter , biochemistry , islet , gene , environmental health
. Objectives : Embryonic stem cells are a potential source for insulin‐producing cells, but existing differentiation protocols are of limited efficiency. Here, the aim has been to develop a new one, which drives development of embryonic stem cells towards insulin‐producing cells rather than to neuronal cell types, and to combine this with a strategy for their separation from insulin‐negative cells. Materials and methods : The cytokeratin‐19 (CK19) promoter was used to control the expression of enhanced yellow fluorescence protein in mouse embryonic stem cells during their differentiation towards insulin‐producing cells, using a new optimized four‐stage protocol. Two cell populations, CK19 + and CK19 − cells, were successfully fluorescence sorted and analysed. Results : The new method reduced neuronal progeny and suppressed differentiation into glucagon‐ and somatostatin‐producing cells. Concomitantly, β‐cell like characteristics of insulin‐producing cells were strengthened, as documented by high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This novel protocol was combined with a cell‐sorting technique. Through the combined procedure, a fraction of glucose‐responsive insulin‐secreting CK19 + cells was obtained with 40‐fold higher insulin gene expression and 50‐fold higher insulin content than CK19 − cells. CK19 + cells were immunoreactive for C‐peptide and had ultrastructural characteristics of an insulin‐secretory cell. Conclusion : Differentiated CK19 + cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.