Islets from human donors with higher but not lower hemoglobin A1c levels respond to gastrin treatment in vitro.

Gastrin is a peptide hormone, which in combination with other factors such as TGFα, EGF or GLP-1, is capable of increasing beta cell mass and lowering blood glucose levels in adult diabetic mice. In humans, administration of a bolus of gastrin alone induces insulin secretion suggesting that gastrin may target islet cells. However, whether gastrin alone is sufficient to exert an effect on isolated human islets has been controversial and the mechanism remained poorly understood. Therefore, in this study we started to examine the effects of gastrin alone on cultured adult human islets. Treatment of isolated human islets with gastrin I for 48 h resulted in increased expression of insulin, glucagon and somatostatin transcripts. These increases were significantly correlated with the levels of donor hemoglobin A1c (HbA1c) but not BMI or age. In addition, gastrin treatment resulted in increased expression of PDX1, NKX6.1, NKX2.2, MNX1 and HHEX in islets from donors with HbA1c greater than 42 mmol/mol. The addition of YM022, an antagonist of the gastrin receptor cholecystokinin B receptor (CCKBR), together with gastrin eliminated these effects, verifying that the effects of gastrin are mediated through CCKBR.CCKBR is expressed in somatostatin-expressing delta cells in islets from all donors. However, in the islets from donors with higher HbA1c (greater than 42 mmol/mol [6.0%]), cells triple-positive for CCKBR, somatostatin and insulin were detected, suggesting a de-differentiation or trans-differentiation of endocrine cells. Our results demonstrate a direct effect of gastrin on human islets from prediabetic or diabetic individuals that is mediated through CCKBR+ cells. Further, our data imply that gastrin may be a potential treatment for diabetic patients.

Factors affecting Salmonella-based combination immunotherapy for prevention of type 1 diabetes in non-obese diabetic mice.

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFß, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFß or PPI+TGFß+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFß+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.

MiR-375 promotes redifferentiation of adult human ß cells expanded in vitro.

In-vitro expansion of ß cells from adult human pancreatic islets could provide abundant cells for cell replacement therapy of diabetes. However, proliferation of ß-cell-derived (BCD) cells is associated with dedifferentiation. Here we analyzed changes in microRNAs (miRNAs) during BCD cell dedifferentiation and identified miR-375 as one of the miRNAs greatly downregulated. We hypothesized that restoration of miR-375 expression in expanded BCD cells may contribute to their redifferentiation. Our findings demonstrate that overexpression of miR-375 alone leads to activation of ß-cell gene expression, reduced cell proliferation, and a switch from N-cadherin to E-cadherin expression, which characterizes mesenchymal-epithelial transition. These effects, which are reproducible in cells derived from multiple human donors, are likely mediated by repression of PDPK1 transcripts and indirect downregulation of GSK3 activity. These findings support an important role of miR-375 in regulation of human ß-cell phenotype, and suggest that miR-375 upregulation may facilitate the generation of functional insulin-producing cells following ex-vivo expansion of human islet cells.

Redifferentiation of adult human ß cells expanded in vitro by inhibition of the WNT pathway.

In vitro expansion of adult human islet ß cells is an attractive solution for the shortage of tissue for cell replacement therapy of type 1 diabetes. Using a lineage tracing approach we have demonstrated that ß-cell-derived (BCD) cells rapidly dedifferentiate in culture and can proliferate for up to 16 population doublings. Dedifferentiation is associated with changes resembling epithelial-mesenchymal transition (EMT). The WNT pathway has been shown to induce EMT and plays key roles in regulating replication and differentiation in many cell types. Here we show that BCD cell dedifferentiation is associated with ß-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of ß-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of ß-cell gene expression. Furthermore, inhibition of ß-catenin expression synergized with redifferentiation induced by a combination of soluble factors, as judged by an increase in the number of C-peptide-positive cells. Simultaneous inhibition of the WNT and NOTCH pathways also resulted in a synergistic effect on redifferentiation. These findings, which were reproducible in cells derived from multiple human donors, suggest that inhibition of the WNT pathway may contribute to a therapeutically applicable way for generation of functional insulin-producing cells following ex-vivo expansion.

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

BACKGROUND: Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells. METHODOLOGY/PRINCIPAL FINDING: Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.