Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo

NAOJUK O; FRANCINI F; PICTON S; BAILEY CJ; LENZEN S; JÖRNS A

DIABETES/METABOLISM RESEARCH AND REVIEWS.

JOHN WILEY & SONS LTD

Año: 2009 vol. 25 p. 464 - 476

1520-7552

Background A comparative morphological analysis of in vitro differentiated and in vivo implanted mouse ES cells towards insulin-producing cells has not been studied before. The impact of the in vivo environment on behavior of ES cells after implantation is of major importance for a potential cell replacement therapy of type 1 diabetes mellitus. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new 4 stage differentiation protocol were analysed before and after implantation for gene expression by in situ RT-PCR, protein expression by immunohistochemistry, and by ultrastructural analysis. Results In comparison with undifferentiated and nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the new 4 stage protocol increased substantially under in vivo conditions. The cells, grown in a tissue-like structure, exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, IAPP, the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a new 4 stage protocol enabled further significant maturation for beta the cell specific markers insulin and the co-stored IAPP as well as the glucose recognition structures GLUT2 glucose transporter and glucokinase. In contrast, a further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions Thus a sufficient degree of in vitro differentiation, with the development of organelles for the protein synthesis apparatus, is an essential prerequisite for further substantial maturation in a beta cell specific way under tissue-like conditions in vivo, supported by cell-cell contacts and vascularisation.