Selective effect of INGAP-PP upon mouse embryonic stem cell differentiation toward islet cells.
We evaluated the effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) upon islet beta- and non-beta cell differentiation from mouse embryonic stem (mES) cells. ES-D3 cell lines were cultured following Lumelsky's protocol with or without INGAP-PP (5 microg/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. Bcl(2) (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 Bcl(2) positively immunostained cells were affected by INGAP-PP. Accordingly, INGAP-PP would promote stem cell differentiation into a beta-like cell phenotype, simultaneously decreasing its differentiation toward non-beta-cell precursors. Therefore, INGAP-PP would be potentially useful to obtain beta-cells from stem cells for replacement therapy.
Francini
Flavio
F
CENEXA - Centro de Endocrinología Experimental y Aplicada (UNLP-CCT La Plata-CONICET, Centro Colaborador de la OPS/OMS), Facultad de Ciencias Médicas Universidad Nacional de La Plata, Argentina. f_francini@yahoo.com
Del Zotto
Héctor
H
Massa
María L
ML
Gagliardino
Juan J
JJ
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology*; D051379:Mice; D000073718:Pancreatitis-Associated Proteins; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D015415:Biomarkers; D000073718:Pancreatitis-Associated Proteins; D010455:Peptides; D011506:Proteins; C000612141:REG3A protein, human; C490253:Reg3b protein, mouse