Human islet-derived precursor cells can cycle between epithelial clusters and mesenchymal phenotypes.

We showed previously that undifferentiated, proliferating human islet-derived precursor cells (hIPCs) are a type of mesenchymal stem/stromal cell (MSC) that can be induced by serum deprivation to form clusters and ultimately differentiate in vitro to endocrine cells. We also demonstrated that partially differentiated hIPC clusters, when implanted under the kidney capsules of mice, continued to differentiate in vivo into hormone-producing cells. However, we noted that not all hIPC preparations yielded insulin-secreting cells in vivo and that in some animals no hormone-expressing cells were found. This suggested that the implanted cells were not always irreversibly committed to further differentiation and may even de-differentiate to a mesenchymal phenotype. In this study, we show that human cells with a mesenchymal phenotype are indeed found in the grafts of mice implanted with hIPCs in epithelial cell clusters (ECCs), which are obtained after 4-day in vitro culture of hIPCs in serum-free medium (SFM); mesenchymal cells were predominant in some grafts. We could mimic the transition of ECCs to de-differentiated mesenchymal cells in vitro by exposure to foetal bovine serum (FBS) or mouse serums, and to a significantly lesser extent to human serum. In a complementary series of experiments, we show that mouse serum and FBS are more effective stimulants of mesenchymal hIPC migration than is human serum. We found that proliferation was not needed for the transition from ECCs to de-differentiated cells because mitomycin-treated hIPCs that could not proliferate underwent a similar transition. Lastly, we show that cells exhibiting a mesenchymal phenotype can be found in grafts of adult human islets in mice. We conclude that epithelial-to-mesenchymal transition (EMT) of cells in hIPC ECCs can occur following implantation in mice. This potential for EMT of human islets or differentiated precursor cells must be considered in strategies for cell replacement therapy for diabetes.

Human islet-derived precursor cells are mesenchymal stromal cells that differentiate and mature to hormone-expressing cells in vivo.

Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin-producing cells. Previously, we described proliferative populations of human islet-derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000-fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C-peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105(+)/CD73(+)/CD90(+) cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas-derived MSC that are capable of differentiating into hormone-expressing cells. Their ability to mature into functional insulin-secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet-like cells for replacement therapy in type 1 diabetes. Disclosure of potential conflicts of interest is found at the end of this article.

The transcription factors Stat5a/b are not required for islet development but modulate pancreatic beta-cell physiology upon aging.

In insulinoma cell lines proliferation and insulin gene transcription are stimulated by growth hormone and prolactin, which convey their signals through the transcription factors Stat5a and 5b (referred to as Stat5). However, the contribution of Stat5 to the physiology of beta-cells in vivo could not be assessed directly since Stat5-null mice die perinataly. To explore the physiological role of Stat5 in the mouse, the corresponding gene locus targeted with loxP sites was inactivated in beta-cells using two lines of Cre recombinase expressing transgenic mice. While the RIP-Cre transgene is active in pancreatic beta-cells and the hypothalamus, the Pdx1-Cre transgene is active in precursor cells of the endocrine and exocrine pancreas. Mice carrying two floxed Stat5alleles and a RIP-Cre transgene developed mild obesity, were hyperglycemic and exhibited impaired glucose tolerance. Since RIP-Cre transgenic mice by themselves display some glucose intolerance, the significance of these data is unclear. In contrast, mice, in which the Stat5 locus had been deleted with the Pdx1-Cre transgene, developed functional islets and were glucose tolerant. Mild glucose intolerance occurred with age. We conclude that Stat5 is not essential for islet development but may modulate beta-cell function.

Aged transgenic mice with increased glucocorticoid sensitivity in pancreatic beta-cells develop diabetes.

Glucocorticoids are diabetogenic hormones because they decrease glucose uptake, increase hepatic glucose production, and inhibit insulin release. To study the long-term effects of increased glucocorticoid sensitivity in beta-cells, we studied transgenic mice overexpressing the rat glucocorticoid receptor targeted to the beta-cells using the rat insulin I promoter. Here we report that these mice developed hyperglycemia both in the fed and the overnight-fasted states at 12-15 months of age. Progression from impaired glucose tolerance, previously observed in the same colony at the age of 3 months, to manifest diabetes was not associated with morphological changes or increased apoptosis in the beta-cells. Instead, our current results suggest that the development of diabetes is due to augmented inhibition of insulin secretion through alpha(2)-adrenergic receptors (alpha(2)-ARs). Thus, we found a significantly higher density of alpha(2)-ARs in the islets of transgenic mice compared with controls, based on binding studies with the alpha(2)-AR agonist UK 14304. Furthermore, incubation of islets with benextramine, a selective antagonist of the alpha(2)-AR, restored insulin secretion in response to glucose in isolated islets from transgenic mice, whereas it had no effect on control islets. These results indicate that the chronic enhancement of glucocorticoid signaling in pancreatic beta-cells results in hyperglycemia and impaired glucose tolerance. This effect may involve signaling pathways that participate in the regulation of insulin secretion via the alpha(2)-AR.