Stem Cell-derived Β-cells Research Articles

Generation of a pancreas derived hydrogel for the culture of hiPSC derived pancreatic endocrine cells

Stem cell-derived β-cells (SC-BCs) represent a potential source for curing diabetes. To date, in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation, indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here, we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards β-cells in vitro. To this aim, a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most β-cell markers, but did not improve β-cell identity. In conclusion, the study describes the production of a PanMa hydrogel, which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs.

Forward programming of hiPSCs towards beta-like cells using Ngn3, Pdx1, and MafA

Transplantation of stem cell-derived β-cells is a promising therapeutic advancement in the treatment of type 1 diabetes mellitus. A current limitation of this approach is the long differentiation timeline that generates a heterogeneous population of pancreatic endocrine cells. To address this limitation, an inducible lentiviral overexpression system of mature β-cell markers was introduced into human induced-pluripotent stem cells (hiPSCs). Following the selection of the successfully transduced hiPSCs, the cells were treated with doxycycline in the pancreatic progenitor induction medium to support their transition toward the pancreatic lineage. Cells cultured with doxycycline presented the markers of interest, NGN3, PDX1, and MAFA, after five days of culture, and glucose-stimulated insulin secretion assays demonstrated that the cells were glucose-responsive in a monolayer culture. When cultured as a spheroid, the markers of interest and insulin secretion in a static glucose-stimulated insulin secretion assay were maintained; however, insulin secretion upon consecutive glucose challenges was limited. Comparison to human fetal and adult donor tissues identified that although the hiPSC-derived spheroids present similar markers to adult insulin-producing cells, they are functionally representative of fetal development. Together, these results suggest that with optimization of the temporal expression of these markers, forward programming of hiPSCs towards insulin-producing cells could be a possible alternative for islet transplantation.

Bone marrow stem cell-derived β-cells: New issue for diabetes cell therapy

This investigation aimed to establish the promising role of insulin-producing cells (IPCs) growing from bone marrow-mesenchymal stem cells (BM-MSCs) in relieving hyperglycemia induced in rats. BM-MSCs were differentiated into IPCs using three different protocols. The efficiency of BM-MSCs differentiation into IPCs in vitro was confirmed by detecting IPCs specific gene expression (Foxa-2, PDX-1 and Ngn-3) and insulin release assay. The in vivo study design included 3 groups of male Wistar rats; negative control group, diabetic group and IPCs-transfused group (5 ×106 cells of the most functional IPCs/rat). One month after IPCs infusion, serum glucose, insulin, c-peptide and visfatin levels as well as pancreatic glucagon level were quantified. Gene expression analysis of pancreatic Foxa-2 and Sox-17, IGF-1 and FGF-10 was done. Additionally, histological investigation of pancreatic tissue sections was performed. Our data clarified that, the most functional IPCs are those generated from BM-MSCs using differentiation protocol 3 as indicated by the significant up-regulation of Foxa-2, PDX-1 and Ngn-3 gene expression levels. These findings were further emphasized by releasing of a significant amount of insulin in response to glucose load. The transplantation of the IPCs in diabetic rats elicited significant decline in serum glucose, visfatin and pancreatic glucagon levels along with significant rise in serum insulin and c-peptide levels. Moreover, it triggered significant up-regulation in the expression levels of pancreatic Foxa-2, Sox-17, IGF-1 and FGF-10 genes versus the untreated diabetic counterpart. The histopathological examination of pancreatic tissue almost assisted the biochemical and molecular genetic analyses. These results disclose that the cell therapy holds potential to develop a new cure for DM based on the capability of BM-MSCs to generate β-cell phenotype using specific protocol.

Recent trends and advances in type 1 diabetes therapeutics: A comprehensive review

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic β-cells, leading to insulin deficiency. Insulin replacement therapy is the current standard of care for T1D, but it has significant limitations. However, stem cell-based replacement therapy has the potential to restore β-cell function and achieve glycaemic control eradicating the necessity for drugs or injecting insulin externally. While significant progress has been made in preclinical studies, the clinical translation of stem cell therapy for T1D is still in its early stages. In continuation, further research is essentially required to determine the safety and efficacy of stem cell therapies and to develop strategies to prevent immune rejection of stem cell-derived β-cells. The current review highlights the current state of cellular therapies for T1D including, different types of stem cell therapies, gene therapy, immunotherapy, artificial pancreas, and cell encapsulation being investigated, and their potential for clinical translation.

Generation of induced pluripotent stem cell-derived beta-cells in blood amino acids-like medium.

Traditional cell culture media do not accurately represent the availability of the nutrients in plasma. They usually contain a supraphysiological concentration of nutrients such as glucose, amino acids, etc. These high nutrients can alter the metabolism of cultured cells and induce metabolic phenotypes that do not reflect in vivo conditions. We demonstrate that the supraphysiological levels of nutrients interfere with endodermal differentiation. Refinement of media formulations has a potential application in maturity modulation of stem cell-derived β-cells (SC-β) generation in vitro. To address these issues, we established a defined culture system to derive SC-β-cells using a blood amino acids-like medium (BALM). Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into the definitive endoderm, pancreatic progenitors, endocrine progenitors, and SC-β in BALM-based med. The differentiated cells secreted C-peptide in vitro in response to high glucose levels and expressed several pancreatic β-cell markers. In conclusion, amino acids at the physiological levels are sufficient for deriving functional SC-β cells.

Self-organized insulin-producing β-cells differentiated from human omentum-derived stem cells and their in vivo therapeutic potential

BackgroundHuman omentum-derived mesenchymal stem cells (hO-MSCs) possess great potential to differentiate into multiple lineages and have self-renewal capacity, allowing them to be utilized as patient-specific cell-based therapeutics. Although the use of various stem cell-derived β-cells has been proposed as a novel approach for treating diabetes mellitus, developing an efficient method to establish highly functional β-cells remains challenging.MethodsWe aimed to develop a novel cell culture platform that utilizes a fibroblast growth factor 2 (FGF2)-immobilized matrix to regulate the adhesion and differentiation of hO-MSCs into insulin-producing β-cells via cell–matrix/cell–cell interactions. In our study, we evaluated the in vitro differentiation potential of hO-MSCs cultured on an FGF2-immobilized matrix and a round-bottom plate (RBP). Further, the in vivo therapeutic efficacy of the β-cells transplanted into kidney capsules was evaluated using animal models with streptozotocin (STZ)-induced diabetes.ResultsOur findings demonstrated that cells cultured on an FGF2-immobilized matrix could self-organize into insulin-producing β-cell progenitors, as evident from the upregulation of pancreatic β-cell-specific markers (PDX-1, Insulin, and Glut-2). Moreover, we observed significant upregulation of heparan sulfate proteoglycan, gap junction proteins (Cx36 and Cx43), and cell adhesion molecules (E-cadherin and Ncam1) in cells cultured on the FGF2-immobilized matrix. In addition, in vivo transplantation of differentiated β-cells into animal models of STZ-induced diabetes revealed their survival and engraftment as well as glucose-sensitive production of insulin within the host microenvironment, at over 4 weeks after transplantation.ConclusionsOur findings suggest that the FGF2-immobilized matrix can support initial cell adhesion, maturation, and glucose-stimulated insulin secretion within the host microenvironment. Such a cell culture platform can offer novel strategies to obtain functional pancreatic β-cells from patient-specific cell sources, ultimately enabling better treatment for diabetes mellitus.Graphical

An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors

The biomimetic pancreatic microenvironment improves the differentiation efficiency and function of human embryonic stem cell-derived β-cells (SC-β cells). Thus, a laminin subunit alpha 2-gelatin methacrylate (LAMA2-GelMA) hybrid hydrogel as a bionics carrier for the formation and maturation of endocrine lineage was developed in our research, based on pancreas proteomics analysis of postnatal mice. Pancreatic endocrine cells cultured on the hybrid hydrogel in vitro, which was composed of 0.5 μg/mL LAMA2 protein and 4% GelMA, the expression of transcription factors (TFs), including NKX6.1, NKX6.2, and NEUROD1 were upregulated. Single-cell transcriptomics was performed after LAMA2 knockdown during the early differentiation of pancreatic progenitor (PP) cells, a marked decrease in the forkhead box protein A2 (FOXA2+)/GATA-binding factor 6 (GATA6+) cluster was detected. Also, we clarified that as a receptor of LAMA2, integrin subunit alpha 7 (ITGA7) participated in Integrin-AKT signaling transduction and influenced the protein levels of FOXA2 and PDX1. In vivo experiments showed that, PP cells encapsulated in the LAMA2-GelMA hydrogel exhibited higher serum C-peptide levels compared to the GelMA and Matrigel groups in nude mice and reversed hyperglycemia more quickly in STZ-induced diabetic nude mice. Taken together, our findings highlighted the feasibility of constructing a pancreas-specific microenvironment based on proteomics and tissue engineering for the treatment of diabetes.

Three-dimensional Vascularized β-cell Spheroid Tissue Derived From Human Induced Pluripotent Stem Cells for Subcutaneous Islet Transplantation in a Mouse Model of Type 1 Diabetes.

Islet transplantation is an effective replacement therapy for type 1 diabetes (T1D) patients. However, shortage of donor organ for allograft is obstacle for further development of the treatment. Subcutaneous transplantation with stem cell-derived β-cells might overcome this, but poor vascularity in the site is burden for success in the transplantation. We investigated the effect of subcutaneous transplantation of vascularized β-cell spheroid tissue constructed 3-dimensionally using a layer-by-layer (LbL) cell-coating technique in a T1D model mouse. We used MIN6 cells to determine optimal conditions for the coculture of β-cell spheroids, normal human dermal fibroblasts, and human umbilical vein endothelial cells, and then, under those conditions, we constructed vascularized spheroid tissue using human induced pluripotent stem cell-derived β-cells (hiPS β cells). The function of insulin secretion of the vascularized hiPS β-cell spheroid tissue was evaluated in vitro. Furthermore, the function was investigated in T1D model NOD/SCID mice subcutaneously transplanted with the tissue. In vitro, the vascularized hiPS β-cell spheroid tissue exhibited enhanced insulin secretion. The vascularized hiPS β-cell spheroid tissue also significantly decreased blood glucose levels in diabetic immunodeficient mice when transplanted subcutaneously. Furthermore, host mouse vessels were observed in the explanted vascularized hiPS β-cell spheroid tissue. Vascularized hiPS β-cell spheroid tissue decreased blood glucose levels in the diabetic mice. This therapeutic effect was suggested due to host angiogenesis in the graft. This method could lead to a promising regenerative treatment for T1D patients.

307.2: Bioprinted Immune-protective Islet-containing Tissues Successfully Regulate Blood Glucose in Rodent Models of Type 1 Diabetes

Introduction: Type 1 diabetes (T1D) is a disease characterized by elevated blood glucose due to insufficient insulin release from pancreatic β-cells. Transplantation of cadaveric islets demonstrates that cell therapy can fully reverse hyperglycemia. However, limited cell supply, immune rejection of implanted allogeneic cells, and cell survival represent major challenges. Cell encapsulation has great potential to overcome these challenges by blocking immune cell access to the grafts while allowing nutrient exchange and secreted products from the implanted cells to be delivered to the body. In this study, we use a unique microfluidic bioprinting technology to precisely control the placement of cells and biomaterials within 3D tissues with micro-architectures optimized for cellular fitness and immune protection. Methods: Living tissues consisting of fibres with a cell-containing core and immune-protective alginate-based shell were generated using Aspect Biosystems’ RX1 bioprinter technology. Core-shell fibres with reaggregated primary human pancreatic islets or embryonic stem cell-derived β-cells were tested in vitro using viability and functional (glucose-stimulated insulin secretion, GSIS) assays. Bioprinted tissues were then implanted into the IP space or omentum of streptozotocin (STZ)-induced diabetic mice and rats, respectively. Glucose homeostasis, body weight, and human C-peptide secretion were monitored for up to 3 months following implantion. Retrieved grafts were fixed and analyzed by histology (H&E, Masson’s trichrome stain) and immunohistochemistry (α-SMA, CD45) to quantify fibrotic encapsulation and immune cell infiltration. Results: Bioprinted tissues supported viability and dynamic insulin secretion of cells in vitro up to 28 days. When transplanted into immunodeficient and immunocompetent diabetic rodents, bioprinted tissues containing reaggregated human islets successfully regulated blood glucose for up to 3 months (Figure 1), although normoglycemia was only sustained in a subset of immunocompetent animals and associated with variable fibrosis. Post-retrieval viability stain, assessment of GSIS, and histology revealed high viability and functionality of implanted cells, and the absence of leukocyte infiltration through the shell. Discussion: This is the first study showing a fully 3D bioprinted tissue composed of a core/shell fibre can successfully deliver a therapeutic dose of xenogeneic cells into a diabetic animal (Figure 2). The unique features of the microfluidic technology were leveraged to bioprint an implantable and retrievable tissue patch that merges the benefits of a single fibre (cell fitness, access to nutrients, immune-protection, perm-selectivity) with those provided by a 3D structure (retrievability, ease of implant, structural integrity). The promising results obtained in rodent studies warrant further investigation to minimize fibrosis and explore the scaling-up of bioprinted tissues in large animal models of T1D.NRC-IRAP. NSERC. Stem Cell Network. Genome BC.

Navigating the Genomic Landscape of Human Adipose Stem Cell-Derived β-Cells.

Diabetes is a pandemic manifested through glucose dysregulation mediated by inadequate insulin secretion by beta cells. A beta cell replacement strategy would transform the treatment paradigm from pharmacologic glucose modulation to a genuine cure. Stem cells have emerged as a potential source for beta cell (β-cell) engineering. The detailed generation of functional β-cells from both embryonic and induced pluripotent stem cells has recently been described. Adult stem cells, including adipose derived, may also offer a therapeutic approach, but remain ill defined. In our study, we performed an in-depth assessment of insulin-producing beta cells generated from human adipose, irrespective of donor patient age, gender, and health status. Cellular transformation was confirmed using flow cytometry and single-cell imaging. Insulin secretion was observed with glucose stimulation and abrogated following palmitate exposure, a common free fatty acid implicated in human beta cell dysfunction. We used next-generation sequencing to explore gene expression changes before and after differentiation of patient-matched samples, which revealed more than 5,000 genes enriched. Adipose-derived beta cells displayed comparable gene expression to native β-cells. Pathway analysis demonstrated relevance to stem cell differentiation and pancreatic developmental processes, which are vital to cellular function, structural development, and regulation. We conclude that the functions associated with adipose derived beta cells are mediated through relevant changes in the transcriptome, which resemble those seen in native β-cell morphogenesis and maturation. Therefore, they may represent a viable option for the clinical translation of stem cell-based therapies in diabetes.

ENTPD3 Marks Mature Stem Cell-Derived β-Cells Formed by Self-Aggregation In Vitro.

Stem cell–derived β-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult β-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human β-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

163-OR: Evidence for Circadian Control of Glucose-Stimulated Insulin Secretion in Transplanted Human Stem-Cell-Derived Pancreatic ß-Cells

Cell replacement strategies utilizing stem cell-derived β-cells (SC-β cells) hold therapeutic potential for patients with diabetes. In order for cell therapy to be effective, transplanted SC-β cells must recapitulate physiological features of insulin secretion in humans. In this regard, circadian regulation of insulin release is critical for normal β cell physiology, where intrinsic β cell circadian clocks function to optimize glucose-stimulated insulin secretion (GSIS) during the active/feeding phase of the circadian cycle. Thus, to elucidate whether transplanted SC-β cells retain capacity for circadian control of insulin secretion, SC-β cells were generated using a six-step directed differentiation protocol and transplanted (n=13 independent transplants) under the kidney capsule into immunocompromised (SCID-beige) mice. Prior to transplantation, differentiated SC-β cells were confirmed to express core circadian clock genes (e.g.,BMAL1, CLOCK, PER1 and REV-ERBα). Provided this information, we next assessed in vivo glucose tolerance and corresponding insulin secretory response (using human insulin ELISA) in SCID-beige mice during the inactive/sleep and active/feeding circadian cycles. Both parameters demonstrated robust diurnal rhythmicity (p<.05, inactive vs. active) with markedly enhanced glucose tolerance and in vivo SC-β cell secretory function evident during active/feeding circadian phase in mice. Notably, among 13 independent transplantations, ~90% of mice demonstrated statistically significant GSIS in response to intraperitoneal glucose injection during active/feeding compared to ~30% during inactive/sleep circadian phase. Our results provide evidence that transplanted SC-β cells retain circadian control of insulin secretion which should be taken into consideration for assessment and design of future β cell replacement strategies. Disclosure S. K. Sen: None. S. Hassoun: None. Q. P. Peterson: Other Relationship; Self; Semma Therapeutics, Inc. A. Matveyenko: None. Funding National Institutes of Health (R01DK098468 to A.M.)

Oxygenation strategies for encapsulated islet and beta cell transplants.

Human allogeneic islet transplantation (ITx) is emerging as a promising treatment option for qualified patients with type 1 diabetes. However, widespread clinical application of allogeneic ITx is hindered by two critical barriers: the need for systemic immunosuppression and the limited supply of human islet tissue. Biocompatible, retrievable immunoisolation devices containing glucose-responsive insulin-secreting tissue may address both critical barriers by enabling the more effective and efficient use of allogeneic islets without immunosuppression in the near-term, and ultimately the use of a cell source with a virtually unlimited supply, such as human stem cell-derived β-cells or xenogeneic (porcine) islets with minimal or no immunosuppression. However, even though encapsulation methods have been developed and immunoprotection has been successfully tested in small and large animal models and to a limited extent in proof-of-concept clinical studies, the effective use of encapsulation approaches to convincingly and consistently treat diabetes in humans has yet to be demonstrated. There is increasing consensus that inadequate oxygen supply is a major factor limiting their clinical translation and routine implementation. Poor oxygenation negatively affects cell viability and β-cell function, and the problem is exacerbated with the high-density seeding required for reasonably-sized clinical encapsulation devices. Approaches for enhanced oxygen delivery to encapsulated tissues in implantable devices are therefore being actively developed and tested. This review summarizes fundamental aspects of islet microarchitecture and β-cell physiology as well as encapsulation approaches highlighting the need for adequate oxygenation; it also evaluates existing and emerging approaches for enhanced oxygen delivery to encapsulation devices, particularly with the advent of β-cell sources from stem cells that may enable the large-scale application of this approach.

Late-stage differentiation of embryonic pancreatic \u03b2-cells requires Jarid2

Jarid2 is a component of the Polycomb Repressor complex 2 (PRC2), which is responsible for genome-wide H3K27me3 deposition, in embryonic stem cells. However, Jarid2 has also been shown to exert pleiotropic PRC2-independent actions during embryogenesis. Here, we have investigated the role of Jarid2 during pancreas development. Conditional ablation of Jarid2 in pancreatic progenitors results in reduced endocrine cell area at birth due to impaired endocrine cell differentiation and reduced prenatal proliferation. Inactivation of Jarid2 in endocrine progenitors demonstrates that Jarid2 functions after endocrine specification. Furthermore, genome-wide expression analysis reveals that Jarid2 is required for the complete activation of the insulin-producing β-cell differentiation program. Jarid2-deficient pancreases exhibit impaired deposition of RNAPII-Ser5P, the initiating form of RNAPII, but no changes in H3K27me3, at the promoters of affected endocrine genes. Thus, our study identifies Jarid2 as a fine-tuner of gene expression during late stages of pancreatic endocrine cell development. These findings are relevant for generation of transplantable stem cell-derived β-cells.

Aggregation of Engineered Human β-Cells Into Pseudoislets: Insulin Secretion and Gene Expression Profile in Normoxic and Hypoxic Milieu.

Innovative treatments to cure type 1 diabetes are being actively researched. Among the different strategies, the replacement of β-cells has given promising results. Classically, islets from cadaveric donors are transplanted into diabetic patients, but recently phase I clinical trials that use stem cell-derived β-cells have been started. Such protocols require either an immunosuppressive treatment or the macroencapsulation of the β-cells. They involve cell aggregation and the exposure of the cells to hypoxia. Using an engineered human β-cell, we have addressed these two problems: a novel human β-cell line called EndoC-βH3 was cultured as single cells or aggregated clusters. EndoC-βH3 cells were also cultured at normal atmospheric oxygen tension (pO2 = 21%) or hypoxia (pO2 = 3%) in the presence or absence of modulators of the hypoxia-inducible factor 1α (HIF1α) pathway. Cell aggregation improved glucose-stimulated insulin secretion, demonstrating the benefit of cell-cell contacts. Low oxygen tension decreased β-cell viability and their sensitivity to glucose, but did not alter insulin production nor the insulin secretion capacity of the remaining cells. To investigate the role of HIF1α, we first used a HIF stabilizer at pO2 = 21%. This led to a mild decrease in cell viability, impaired glucose sensitivity, and altered insulin secretion. Finally, we used a HIF inhibitor on EndoC-βH3 pseudoislets exposed to hypoxia. Such treatment considerably decreased cell viability. In conclusion, aggregation of the EndoC-βH3 cells seems to be important to improve their function. A fraction of the EndoC-βH3 cells are resistant to hypoxia, depending on the level of activity of HIF1α. Thus, these cells represent a good human cell model for future investigations on islet cell transplantation analysis.

Corrigendum: Generation of stem cell-derived β-cells from patients with type 1 diabetes.

Nature Communications 7: Article number: 11463 (2016); Published: 10 May 2016; Updated: 4 August 2016. The source of reagents for this Article was not fully acknowledged. The Acknowledgements should have included the following: ‘We thank Professors Robin Goland and Dieter Egli (https://www.cellhub.org) for patient fibroblasts.

Generation of stem cell-derived β-cells from patients with type 1 diabetes

We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology.

Recent advances in cell replacement therapies for the treatment of type 1 diabetes.

Exogenous insulin administration is currently the only treatment available to all type 1 diabetes patients, but it is not a cure. By helping to regulate circulating blood glucose levels, it has significantly improved life expectancy, but there are still long-term complications associated with the disease that may be preventable with a treatment strategy that can provide better glycemic control. Isolated islet (or whole pancreas) transplantation, xenotransplantation, and the transplant of human pluripotent stem cell-derived β-cells provide the potential to restore endogenous insulin production and reestablish normoglycemia. Here, we discuss the latest advances in these fields, including the use of encapsulation technology, as well as some of the hurdles that still need to be overcome for these alternative therapies to become mainstream and/or progress to clinical development.

Concise Review: Mesenchymal Stem Cells for Diabetes

Mesenchymal stem cells (MSCs) have already made their mark in the young field of regenerative medicine. Easily derived from many adult tissues, their therapeutic worth has already been validated for a number of conditions. Unlike embryonic stem cells, neither their procurement nor their use is deemed controversial. Here we review the potential use of MSCs for the treatment of type 1 diabetes mellitus, a devastating chronic disease in which the insulin-producing cells of the pancreas (the β-cells) are the target of an autoimmune process. It has been hypothesized that stem cell-derived β-cells may be used to replenish the islet mass in diabetic patients, making islet transplantation (a form of cell therapy that has already proven effective at clinically restoring normoglycemia) available to millions of prospective patients. Here we review the most current advances in the design and application of protocols for the differentiation of transplantable β-cells, with a special emphasis in analyzing MSC potency according to their tissue of origin. Although no single method appears to be ripe enough for clinical trials yet, recent progress in reprogramming (a biotechnological breakthrough that relativizes the thus far insurmountable barriers between embryonal germ layers) bodes well for the rise of MSCs as a potential weapon of choice to develop personalized therapies for type 1 diabetes.

Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.