Sources Of Insulin-secreting Cells Research Articles

Single-nucleus RNA-Seq reveals singular gene signatures of human ductal cells during adaptation to insulin resistance.

Adaptation to increased insulin demand is mediated by β cell proliferation and neogenesis, among other mechanisms. Although it is known that pancreatic β cells can arise from ductal progenitors, these observations have been limited mostly to the neonatal period. We have recently reported that the duct is a source of insulin-secreting cells in adult insulin-resistant states. To further explore the signaling pathways underlying the dynamic β cell reserve during insulin resistance, we undertook human islet and duct transplantations under the kidney capsule of immunodeficient NOD/SCID-γ (NSG) mouse models that were pregnant, were insulin-resistant, or had insulin resistance superimposed upon pregnancy (insulin resistance + pregnancy), followed by single-nucleus RNA-Seq (snRNA-Seq) on snap-frozen graft samples. We observed an upregulation of proliferation markers (e.g., NEAT1) and expression of islet endocrine cell markers (e.g., GCG and PPY), as well as mature β cell markers (e.g., INS), in transplanted human duct grafts in response to high insulin demand. We also noted downregulation of ductal cell identity genes (e.g., KRT19 and ONECUT2) coupled with upregulation of β cell development and insulin signaling pathways. These results indicate that subsets of ductal cells are able to gain β cell identity and reflect a form of compensation during the adaptation to insulin resistance in both physiological and pathological states.

PDX1- /NKX6.1+ progenitors derived from human pluripotent stem cells as a novel source of insulin-secreting cells.

Beta cell replacement strategies are a promising alternative for diabetes treatment. Human pluripotent stem cells (hPSCs) serve as a scalable source for producing insulin-secreting cells for transplantation therapy. We recently generated novel hPSC-derived pancreatic progenitors, expressing high levels of the transcription factor NKX6.1, in the absence of PDX1 (PDX1- /NKX6.1+ ). Herein, our aim was to characterize this novel population and assess its ability to differentiate into insulin-secreting beta cells in vitro. Three different hPSC lines were differentiated into PDX1- /NKX6.1+ progenitors, which were further differentiated into insulin-secreting cells using two different protocols. The progenitors and beta cells were extensively characterized. Transcriptome analysis was performed at different stages and compared with the profiles of various pancreatic counterparts. PDX1- /NKX6.1+ progenitors expressed high levels of nestin, a key marker of pancreatic islet-derived progenitors, in the absence of E-cadherin, similar to pancreatic mesenchymal stem cells. At progenitor stage, comparison of the two populations showed downregulation of pancreatic epithelial genes and upregulation of neuronal development genes in PDX1- /NKX6.1+ cells in comparison to the PDX1+ /NKX6.1+ cells. Interestingly, on further differentiation, PDX1- /NKX6.1+ cells generated mono-hormonal insulin+ cells and activated pancreatic key genes, such as PDX1. The transcriptome profile of PDX1- /NKX6.1+ -derived beta (3D-beta) was closely similar to those of human pancreatic islets and purified hPSC-derived beta cells. Also, the 3D-beta cells secreted C-peptide in response to increased glucose concentrations indicating their functionality. These findings provide a novel source of insulin-secreting cells that can be used for beta cell therapy for diabetes.

Short-Term Protocols to Obtain Insulin-Producing Cells from Rat Adipose Tissue: Signaling Pathways and In Vivo Effect.

Studies using mesenchymal stromal cells (MSCs) as a source of insulin-secreting cells (IPCs) are a promising path in the pursuit for diabetes therapy. Here, we investigate three short-term differentiation protocols in order to generate IPCs from autologous adipose-derived stromal cells (ADSCs) with an expressive insulin-secreting profile in vitro and in vivo, as well as the signaling pathways involved in the chosen differentiation protocols. We extracted and cultured ADSCs and differentiated them into IPCs, using three different protocols with different inductors. Afterwards, the secretory profile was analyzed and IPCs differentiated in exendin-4/activin A medium, which presented the best secretory profile, was implanted in the kidney subcapsular region of diabetic rats. All protocols induced the differentiation, but media supplemented with exendin-4/activin A or resveratrol induced the expression and secretion of insulin more efficiently, and only the exendin-4/activin-A-supplemented medium generated an insulin secretion profile more like β-cells, in response to glucose. The PI3K/Akt pathway seems to play a negative role in IPC differentiation; however, the differentiation of ADSCs with exendin-4/activin A positively modulated the p38/MAPK pathway. Resveratrol medium activated the Jak/STAT3 pathway and generated IPCs apparently less sensitive to insulin and insulin-like receptors. Finally, the implant of IPCs with the best secretory behavior caused a decrease in hyperglycemia after one-week implantation in diabetic rats. Our data provide further information regarding the generation of IPCs from ADSCs and strengthen evidence to support the use of MSCs in regenerative medicine, specially the use of exendin-4/activin A to produce rapid and effectively IPCs with significant in vivo effects.

Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line

Background: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells. Methods: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with donor pancreatic islets and EndoC-βH1, an immortalized human β cell line. Results: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iβ were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iβ basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iβ are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iβ to respond to glucose instead was more related to that of EndoC-βH1. Discussion: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human β cells.

Human induced pluripotent stem cells differentiate into insulin-producing cells able to engraft in vivo.

New sources of insulin-secreting cells are strongly required for the cure of diabetes. Recent successes in differentiating embryonic stem cells, in combination with the discovery that it is possible to derive human induced pluripotent stem cells (iPSCs) from somatic cells, have raised the possibility that patient-specific beta cells might be derived from patients through cell reprogramming and differentiation. In this study, we aimed to obtain insulin-producing cells from human iPSCs and test their ability to secrete insulin in vivo. Human iPSCs, derived from both fetal and adult fibroblasts, were differentiated in vitro into pancreas-committed cells and then transplanted into immunodeficient mice at two different stages of differentiation (posterior foregut and endocrine cells). IPSCs were shown to differentiate in insulin-producing cells in vitro, following the stages of pancreatic organogenesis. At the end of the differentiation, the production of INSULIN mRNA was highly increased and 5 ± 2.9 % of the cell population became insulin-positive. Terminally differentiated cells also produced C-peptide in vitro in both basal and stimulated conditions. In vivo, mice transplanted with pancreatic cells secreted human C-peptide in response to glucose stimulus, but transplanted cells were observed to lose insulin secretion capacity during the time. At histological evaluation, the grafts resulted to be composed of a mixed population of cells containing mature pancreatic cells, but also pluripotent and some neuronal cells. These data overall suggest that human iPSCs have the potential to generate insulin-producing cells and that these differentiated cells can engraft and secrete insulin in vivo.

Mesenchymal Stem Cell Therapy in Diabetes Mellitus: Progress and Challenges

Advanced type 2 diabetes mellitus is associated with significant morbidity and mortality due to cardiovascular, nervous, and renal complications. Attempts to cure diabetes mellitus using islet transplantation have been successful in providing a source for insulin secreting cells. However, limited donors, graft rejection, the need for continued immune suppression, and exhaustion of the donor cell pool prompted the search for a more sustained source of insulin secreting cells. Stem cell therapy is a promising alternative for islet transplantation in type 2 diabetic patients who fail to control hyperglycemia even with insulin injection. Autologous stem cell transplantation may provide the best outcome for those patients, since autologous cells are readily available and do not entail prolonged hospital stays or sustained immunotoxic therapy. Among autologous adult stem cells, mesenchymal stem cells (MSCs) therapy has been applied with varying degrees of success in both animal models and in clinical trials. This review will focus on the advantages of MSCs over other types of stem cells and the possible mechanisms by which MSCs transplant restores normoglycemia in type 2 diabetic patients. Sources of MSCs including autologous cells from diabetic patients and the use of various differentiation protocols in relation to best transplant outcome will be discussed.

Adult Pancreas Side Population Cells Expand after β Cell Injury and Are a Source of Insulin-Secreting Cells

Pancreas stem cells are a potential source of insulin-producing β cells for the therapy of diabetes. In adult tissues the ‘side population’ (SP) of cells that effluxes the DNA binding dye Hoechst 33342 through ATP-binding cassette transporters has stem cell properties. We hypothesised therefore that the SP would expand in response to β cell injury and give rise to functional β cells. SP cells were flow sorted from dissociated pancreas cells of adult mice, analysed for phenotype and cultured with growth promoting and differentiation factors before analysis for hormone expression and glucose-stimulated insulin secretion. SP cell number and colony forming potential (CFP) increased significantly in models of type diabetes, and after partial pancreatectomy, in the absence of hyperglycaemia. SP cells, ∼1% of total pancreas cells at 1 week of age, were enriched >10-fold for CFP compared to non-SP cells. Freshly isolated SP cells contained no insulin protein or RNA but expressed the homeobox transcription factor Pdx1 required for pancreas development and β cell function. Pdx1, along with surface expression of CD326 (Ep-Cam), was a marker of the colony forming and proliferation potential of SP cells. In serum-free medium with defined factors, SP cells proliferated and differentiated into islet hormone-expressing cells that secreted insulin in response to glucose. Insulin expression was maintained when tissue was transplanted within vascularised chambers into diabetic mice. SP cells in the adult pancreas expand in response to β cell injury and are a source of β cell progenitors with potential for the treatment of diabetes.

Effects of Streptozotocin on In Vitro Long-Term Cultured Human Islet Cell Monolayers

Replacement β cells may be generated from stem/progenitor cells, possibly residing within the pancreatic islet cells. We sought to investigate the possible use of human islet (HI)-derived cell monolayers as a possible source for β cells. These cells could be propagated in vitro and potentially induced to acquire glucose-stimulated insulin release (GSIR) ability. Loss of three-dimensional architecture, following monolayer establishment, resulted in either rearrangement of both pancreatic hormone expression and key transcription factors or decrease in insulin production or GSIR disappearance. We showed that cell deregulation/dedifferentiation was reversible by treating the cell monolayers, after five passages with streptozotocin (STZ), a well-known β-cell toxin. When used at subtoxic concentrations, STZ promoted differentiation of HI-derived cell monolayers and GSIR recovery. This effect allowed production of insulin-secreting cells starting from cell monolayers doubled five times in vitro, meaning a 400-fold increase in the cellular starting material. Such islet cell expansion capability in vitro might elucidate a new source of insulin-secreting cells thereby possibly overcoming the problem posed by searching for human organ donation.

Neonatal Pig Liver–Derived Progenitors for Insulin-Producing Cells: AnIn VitroStudy

Beta (beta)-cell replacement represents an attractive approach for the possible cure of type 1 insulin-dependent diabetes mellitus (IDDM). In a search for potential sources of insulin-secreting cells for IDDM substitution therapy, we have focused on the neonatal pig liver, which is putatively enriched in multipotent stem cells. We then isolated cells measuring 10 to 15 microm in diameter, identified as small cells, characterized by a high proliferation rate and positive staining for immature liver and pancreatic endocrine cell markers (i.e., insulin and pancreatic duodenal homeobox). The ability of these cells to transdifferentiate into pancreatic beta-like cells under culture conditions with exendin-4 (Ex-4) or high glucose concentration was examined. We observed that insulin secretion was not physiological in basal conditions, although it became responsive to glucose after 5 days of exposure to Ex-4. This beta-cell-like phenotype remained physiologically stable, even after stimulus withdrawal. Based on these observations, we contend that the proposed cell and tissue model might offer several advantages as a candidate for substitution cell therapy in IDDM, because the neonatal pig liver seems enriched in cells, with a mixed pancreas-liver phenotype, that are easier to purify and grow in culture and are more functional than other beta-like cells upon in vitro single short-term stimulation challenge.