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

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.

Correction of Hyperglycemia in Type 1 Diabetic Models by Transplantation of Encapsulated Insulin-producing Cells Derived from Mouse Embryo Progenitor

“…This strategy essentially describes a reproducible protocol that yielded three groups of insulin-producing cell lines from differentiation of mouse early embryo culture, an embryo-derived progenitor RoSH cell line or an ES cell-derived lineagerestricted E-RoSH cell line respectively (Li et al 2009a,b). Despite being generated from different precursors, these insulin-producing cell lines are highly similar in terms of their gene expression profile, signaling events, and insulin secretion responses to glucose stimulation (Li et al 2009a,b, Chen et al 2010. These cells are highly homogenous and expansible, and could directly address the problem of limited b-cell source for transplantation.…”

“…The mice were subject to two injections (15 mg/kg in the first and 12 . 5 mg/kg in the second on 2 days later) of STZ to induce hyperglycemia (Fujikawa et al 2005, Li et al 2009a. An aliquot of capsules containing 2 .…”

“…We have previously generated more than a dozen functional insulin-producing cell lines from progenitor cells of early gastrulating mouse embryos (Li et al 2009b) and also from mouse ES cells (Li et al 2009a). Although these cell lines were derived from different precursors, they all displayed similar properties in terms of gene expression profile and responsiveness to glucose stimulation (Li et al 2009a,b, Chen et al 2010.…”

“…Although these cell lines were derived from different precursors, they all displayed similar properties in terms of gene expression profile and responsiveness to glucose stimulation (Li et al 2009a,b, Chen et al 2010. One of the lines, mouse embryo progenitorderived insulin-producing-1 (MEPI-1), has been extensively characterized.…”

Stem Cell Therapies for Type I Diabetes

“…Moreover, the clones were able to rescue diabetic mice and did not form teratomas, demonstrating the absence of undifferentiated ESC. However, these cells continued proliferating after transplantation and showed an abnormal karyotype, suggesting an insulinoma phenotype, that may be responsible for the hyperinsulinemia and hypoglycemia observed in the animals (Li, Luo et al 2009). …”