Mature Pancreatic Β-cells Research Articles

Metformin Perturbs Pancreatic Differentiation From Human Embryonic Stem Cells.

Metformin is becoming a popular treatment before and during pregnancy, but current literature on in utero exposure to metformin lacks long-term clinical trials and mechanistic studies. Current literature on the effects of metformin on mature pancreatic β-cells highlights its dual, opposing, protective, or inhibitory effects, depending on metabolic environment. However, the impact of metformin on developing human pancreatic β-cells remains unknown. In this study, we investigated the potential effects of metformin exposure on human pancreatic β-cell development and function in vitro. In the absence of metabolic challenges such as high levels of glucose and fatty acids, metformin exposure impaired the development and function of pancreatic β-cells, with downregulation of pancreatic genes and dysfunctional mitochondrial respiration. It also affected the insulin secretion function of pancreatic β-cells. These findings call for further in-depth evaluation of the exposure of human embryonic and fetal tissue during pregnancy to metformin and its implications for long-term offspring health.

In vitro Characterization of Insulin−Producing β-Cell Spheroids

Over the years, immortalized rodent β-cell lines such as RIN, HIT, MIN, βTC, and INS-1 have been used to investigate pancreatic β-cell physiology using conventional two-dimensional (2D) culture techniques. However, physical and physiological limitations inherent to 2D cell culture necessitates confirmatory follow up studies using sentient animals. Three-dimensional (3D) culture models are gaining popularity for their recapitulation of key features of in vivo organ physiology, and thus could pose as potential surrogates for animal experiments. In this study, we aimed to develop and characterize a rat insulinoma INS-1 3D spheroid model to compare with 2D monolayers of the same cell line. Ultrastructural verification was done by transmission electron microscopy and toluidine blue staining, which showed that both 2D monolayers and 3D spheroids contained highly granulated cells with ultrastructural features synonymous with mature pancreatic β-cells, with increased prominence of these features observed in 3D spheroids. Viability, as assessed by cellular ATP quantification, size profiling and glucose utilization, showed that our spheroids remained viable for the experimental period of 30 days, compared to the limiting 5-day passage period of INS-1 monolayers. In fact, increasing ATP content together with spheroid size was observed over time, without adverse changes in glucose utilization. Additionally, β-cell function, assessed by determining insulin and amylin secretion, showed that the 3D spheroids retained glucose sensing and insulin secretory capability, that was more acute when compared to 2D monolayer cultures. Thus, we were able to successfully demonstrate that our in vitro INS-1 β-cell 3D spheroid model exhibits in vivo tissue-like structural features with extended viability and lifespan. This offers enhanced predictive capacity of the model in the study of metabolic disease, β-cell pathophysiology and the potential treatment thereof.

Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function.

Key transcription factors control the gene expression program in mature pancreatic β-cells, but their integration into regulatory networks is little understood. Here, we show that Insm1, Neurod1 and Foxa2 directly interact and together bind regulatory sequences in the genome of mature pancreatic β-cells. We used Insm1 ablation in mature β-cells in mice and found pronounced deficits in insulin secretion and gene expression. Insm1-dependent genes identified previously in developing β-cells markedly differ from the ones identified in the adult. In particular, adult mutant β-cells resemble immature β-cells of newborn mice in gene expression and functional properties. We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant β-cells. Remarkably, combinatorial binding of Insm1, Neurod1 and Foxa2 but not binding of Insm1 alone explained a significant fraction of gene expression changes. Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits. Thus, our data explain part of the mechanisms by which β-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in β-cells, and disruption of this network results in functional failure.

Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells

Embryonic pancreatic bud cells, the earliest pancreas-committed cells, generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into mature pancreatic β-cells in vivo, indicating the feasibility of hESC/iPSC-based cell therapy for diabetes. However, the key factors required for the differentiation of these cells into pancreatic bud cells are incompletely understood. The purpose of this study was to establish culture conditions that efficiently induce PDX1+NKX6.1+ pancreatic bud cells from hESCs/iPSCs. We differentiated a hESC line, KhES-3, into pancreatic lineages with a stepwise protocol recapitulating developmental process. The induction rate of PDX1+NKX6.1+ cells was correlated with cell density in adherent cultures, and markedly improved with cell aggregation cultures. The positive effects of cell aggregation cultures on the differentiation of pancreatic bud cells were reproduced in multiple hESC/iPSC lines. The human PDX1+NKX6.1+ cells developed into pancreatic epithelia after implantation into immunocompromised mice. Moreover, human C-peptide secretion into mouse bloodstream was stimulated by glucose challenges after in vivo maturation. Taken together, these results suggest that cultures with high cell density are crucial for the differentiation of pancreas-committed progenitor cells from hESCs/iPSCs. Our findings may be applicable for the development of hESC/iPSC-based cell therapy for diabetes.

FOXO1 inhibition yields functional insulin-producing cells in human gut organoid cultures.

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors, we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive, insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells, we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded shRNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells, release C-peptide in response to secretagogues, and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes.

Retinoblastoma tumor suppressor protein in pancreatic progenitors controls α- and β-cell fate

Pancreatic endocrine cells expand rapidly during embryogenesis by neogenesis and proliferation, but during adulthood, islet cells have a very slow turnover. Disruption of murine retinoblastoma tumor suppressor protein (Rb) in mature pancreatic β-cells has a limited effect on cell proliferation. Here we show that deletion of Rb during embryogenesis in islet progenitors leads to an increase in the neurogenin 3-expressing precursor cell population, which persists in the postnatal period and is associated with increased β-cell mass in adults. In contrast, Rb-deficient islet precursors, through repression of the cell fate factor aristaless related homeobox, result in decreased α-cell mass. The opposing effect on survival of Rb-deficient α- and β-cells was a result of opposing effects on p53 in these cell types. As a consequence, loss of Rb in islet precursors led to a reduced α- to β-cell ratio, leading to improved glucose homeostasis and protection against diabetes.

Pluripotent Stem Cell-Derived Pancreatic β-Cells: Potential for Regenerative Medicine in Diabetes

Diabetes mellitus, which affects 346 million people, is one of the leading causes of death worldwide. Pancreatic β-cells, existing in the islets of Langerhans, play central roles in the progression of diabetes. An efficient strategy to produce functional pancreatic β-cells is important for both transplantation therapy and disease modeling of diabetes. Human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, provide unlimited starting materials to generate differentiated cells for regenerative studies. Significant progress has been made in human embryonic/induced pluripotent stem cell differentiation in the last several years. However, efficient generation of mature pancreatic β-cells with complete functional capabilities has not yet been accomplished. Here, we review recent successes as well as the technical and theoretical challenges in the use of pluripotent stem cell-derived pancreatic β-cells for disease modeling and replacement therapy of diabetes.

NKX6.1 Promotes PDX-1-Induced Liver to Pancreatic β-Cells Reprogramming

Reprogramming adult mammalian cells is an attractive approach for generating cell-based therapies for degenerative diseases, such as diabetes. Adult human liver cells exhibit a high level of developmental plasticity and have been suggested as a potential source of pancreatic progenitor tissue. An instructive role for dominant pancreatic transcription factors in altering the hepatic developmental fate along the pancreatic lineage and function has been demonstrated. Here we analyze whether transcription factors expressed in mature pancreatic β-cells preferentially activate β-cell lineage differentiation in liver. NKX6.1 is a transcription factor uniquely expressed in β-cells of the adult pancreas, its potential role in reprogramming liver cells to pancreatic lineages has never been analyzed. Our results suggest that NKX6.1 activates immature pancreatic markers such as NGN-3 and ISL-1 but not pancreatic hormones gene expression in human liver cells. We hypothesized that its restricted capacity to activate a wide pancreatic repertoire in liver could be related to its incapacity to activate endogenous PDX-1 expression in liver cells. Indeed, the complementation of NKX6.1 by ectopic PDX-1 expression substantially and specifically promoted insulin expression and glucose regulated processed hormone secretion to a higher extent than that of PDX-1 alone, without increasing the reprogrammed cells. This may suggest a potential role for NKX6.1 in promoting PDX-1 reprogrammed cells maturation along the β-cell-like lineage. By contrast, NKX6.1 repressed PDX-1 induced proglucagon gene expression. The individual and concerted effects of pancreatic transcription factors in adult extra-pancreatic cells, is expected to facilitate developing regenerative medicine approaches for cell replacement therapy in diabetics.

TCF7L2 controls insulin gene expression and insulin secretion in mature pancreatic β-cells

Genetic studies have linked the risk of Type 2 diabetes with SNPs (single nucleotide polymorphisms) in the gene encoding the Wnt signalling-associated transcription factor, TCF7L2 (T-cell factor 7-like 2). The risk alleles have been associated with reduced glucose and GLP-1 (glucagon-like peptide 1)-stimulated insulin secretion. Recent evidence has suggested that inheritance of the at-risk T allele at SNP rs7903146 may increase the expression of TCF7L2 in adult human islets. However, the cellular mechanisms by which changes in TCF7L2 levels may affect insulin secretion are unclear. In the present paper, we describe the use of RNA silencing to investigate the role of TCF7L2 on insulin secretion and gene expression in rodent islets. We find that reduced TCF7L2 expression reduces glucose-simulated insulin secretion and insulin gene expression while slightly potentiating glucose stimulated changes in intracellular free Ca(2+) concentrations.

Transcription Factor TCF7L2 Genetic Study in the French Population

Recently, the transcription factor 7-like 2 (TCF7L2) gene has been associated with type 2 diabetes in subjects of European origin in the DeCode study. We genotyped the two most associated variants (rs7903146 and rs12255372) in 2,367 French type 2 diabetic subjects and in 2,499 control subjects. Both the T-allele of rs7903146 and the T-allele of rs12255372 significantly increase type 2 diabetes risk with an allelic odds ratio (OR) of 1.69 (95% CI 1.55-1.83) (P = 6.0 x 10(-35)) and 1.60 (1.47-1.74) (P = 7.6 x 10(-28)), respectively. In nonobese type 2 diabetic subjects (BMI <30 kg/m2, n = 1,346), the ORs increased to 1.89 (1.72-2.09) (P = 2.1 x 10(-38)) and 1.79 (1.62-1.97) (P = 5.7 x 10(-31)), respectively. The rs7903146 T at-risk allele associates with decreased BMI and earlier age at diagnosis in the type 2 diabetic subjects (P = 8.0 x 10(-3) and P = 3.8 x 10(-4), respectively), which is supported by quantitative family-based association tests. TCF7L2 is expressed in most human tissues, including mature pancreatic beta-cells, with the exception of the skeletal muscle. In the subcutaneous and omental fat from obese type 2 diabetic subjects, TCF7L2 expression significantly decreased compared with obese normoglycemic individuals. During rat fetal beta-cell differentiation, TCF7L2 expression pattern mimics the key marker NGN3 (neurogenin 3), suggesting a role in islet development. These data provide evidence that TCF7L2 is a major determinant of type 2 diabetes risk in European populations and suggests that this transcription factor plays a key role in glucose homeostasis.