Selective effect of INGAP-PP upon mouse embryonic stem cell differentiation toward islet cells.

FRANCINI F; DEL ZOTTO H; MASSA ML; GAGLIARDINO JJ

REGULATORY PEPTIDES

ELSEVIER SCIENCE BV

Año: 2009 vol. 153 p. 43 - 48

0167-0115

We evaluated the effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) upon islet B-and non-B cell differentiation from mouse embryonic stem (mES) cells. ES-D3 cell lines were cultured following Lumelsky´s protocol with or without INGAP-PP (5 ìg/ml) at different stages. Gene expression was quantified using qPCR. mES cells were fixed and immunostained using anti insulin-, somatostatin-, glucagon-, Pdx-1-, Ngn-3-, Nkx-6.1 and PGP9.5 specific antibodies. PCNA was used to measure replication rate. Bcl2 (immunostaining) and caspase-3 (enzyme activity and gene expression) were determined as apoptosis markers. INGAP-PP increased IAPP, Glut-2, Kir-6.2, SUR-1 and insulin gene expression, and the percentage of insulin-immunostained cells. Conversely, INGAP-PP reduced significantly glucagon and somatostatin gene expression and immunopositivity. While nestin gene expression was not affected, there was a significant reduction in the percentage of PGP9.5-immunostained cells. Pdx-1 gene expression increased by 115% in INGAP-PP treated cells, as well as the percentage of Pdx-1, Ngn-3 and Nkx-6.1 immunopositive cells. Neither caspase-3 (expression and activity) nor Bcl2 positively immunostained cells were affected by INGAP-PP. Accordingly, INGAP-PP would promote stem cell differentiation into a B-like cell phenotype, simultaneously decreasing its differentiation toward non-â-cell precursors. Therefore, INGAP-PP would be potentially useful to obtain B-cells from stem cells for replacement therapy.