Int. J. Dev. Biol. 53: 569 - 578 (2009)

https://doi.org/10.1387/ijdb.092856mm

Vol 53, Issue 4

Mouse ES cells over-expressing the transcription factor NeuroD1 show increased differentiation towards endocrine lineages and insulin-expressing cells

Original Article | Published: 2 March 2009

Mélanie Marchand^1,2,3,4,5, Insa S. Schroeder⁶, Suzy Markossian^1,2,3,4, Anouchka Skoudy⁷, Didier Nègre^3,4,10,11, François-Loïc Cosset^3,4,10,11, Paco Real^7,8,9, Christian Kaiser⁶, Anna M. Wobus⁶ and Pierre Savatier*^,1,2,3

¹Inserm, U846 and ²Stem Cell and Brain Research Institute, Bron, France, ³Université de Lyon and ⁴Université Lyon I, Lyon, France, ⁵Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA, ⁶In vitro Differentiation Group, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany, ⁷Cell and Molecular Biology Unit, Institut Municipal d’Investigació Mèdica (IMIM), Barcelona, Spain, ⁸Departament de Ciències Experimentals I de la Salut, Universitat Pompeu Fabra, Barcelona, Spain, ⁹Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid, Spain, ¹⁰Inserm, U758, Human Virology Department and ¹¹Ecole Normale Supérieure de Lyon, Lyon, France

Abstract

Embryonic stem (ES) cells which constitutively express the Pdx-1, Ngn-3, NeuroD1, Nkx2.2, and Nkx6.1 transcription factors were engineered by means of lentiviral vectors, following a multi-step infection procedure to successively generate ES cell lines expressing one, two, and three factors, respectively. Each ES cell line was allowed to differentiate into nestin+/Isl-1+ endocrine precursors, then into more mature pancreatic cells, and subsequently analysed for expression of Glc, Ins, and Sst, markers of alpha, beta and delta cells, respectively. Each ES cell line generated displayed a unique pattern of gene expression. The ES cell line expressing NeuroD1 displayed vastly elevated levels of Glc, Ins-1, Ins-2 and Sst, and showed an increase in Pdx-1, Pax-4, Nkx6.1, Isl-1, Glut-2 and GK transcript levels. Furthermore, immunofluorescence analysis revealed that differentiation of NeuroD1-expressing ES cells in nestin+/Isl-1+ multilineage progenitors, followed by the formation of C-peptide+/insulin+ clusters, was accelerated. Together, these results indicate that stable expression of NeuroD1 in ES cells facilitates differentiation into endocrine and insulin-producing cells.

Keywords

embryonic stem, endocrine differentiation, insulin, NeuroD1, lentiviral vector