en
Scientific article
English

Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line

Published inStem cell research, vol. 2, no. 1, p. 41-55
Publication date2009
Abstract

Generating surrogate insulin-producing cells from embryonic stem cells (ESCs) through in vitro replication of successive steps during pancreatic development has been challenging . Here we describe a novel reproducible protocol to establish homogeneous and scalable insulin-producing cell lines from mouse (m) ESCs via differentiation of the previously described lineage-restricted clonal mESC-derived E-RoSH cells. Unlike their parental mESCs, E-RoSH cells expressed high levels of mesodermal and endodermal genes. Nutrient depletion in the presence of nicotinamide inhibited proliferation of E-RoSH cells and induced differentiation into heterogeneous cultures comprising vascular-like structures that produced detectable levels of insulin and C-peptide in an equimolar ratio. Limiting dilution of these cultures resulted in the isolation of eight independent insulin-producing cell lines in five experiments. All these lines were cloned and shown to be amenable to repeated cycles of freeze and thaw and to replicate for months with a doubling time of 3-4 days. Under such conditions, the cultured cells exhibited genomic, structural, biochemical, and pharmacological properties of pancreatic beta cells, including storage of an equimolar ratio of insulin and C-peptide in granules and release of the contents of these organelles through a glucose-sensitive machinery. After transplantation, these cells reversed hyperglycemia in streptozotocin-treated SCID mice and did not form teratomas.

Keywords
  • Animals
  • C-Peptide/analysis
  • Cell Culture Techniques
  • Cell Differentiation
  • Cell Lineage
  • Cell Transplantation
  • Embryonic Stem Cells/*cytology
  • Endoderm
  • Hyperglycemia/therapy
  • Insulin/analysis
  • Insulin-Secreting Cells/*cytology
  • Mesoderm
  • Mice
  • Mice, SCID
  • Treatment Outcome
Citation (ISO format)
LI, Guodong et al. Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line. In: Stem cell research, 2009, vol. 2, n° 1, p. 41–55. doi: 10.1016/j.scr.2008.07.006
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ISSN of the journal1873-5061
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