Glucose-responsive Insulin-producing Cells Research Articles

Exosomes Secreted by Umbilical Cord Blood-Derived Mesenchymal Stem Cell Attenuate Diabetes in Mice.

Mesenchymal stem cell (MSC) therapy is an innovative approach in diabetes due to its capacity to modulate tissue microenvironment and regeneration of glucose-responsive insulin-producing cells. In this study, we investigated the role of MSC-derived exosomes in pancreatic regeneration and insulin secretion in mice with streptozotocin-induced diabetes. Mesenchymal stem cells (MSCs) were isolated and characterized from umbilical cord blood (UCB). Exosomes were isolated and characterized from these MSCs. Diabetes was induced in male C57Bl/6 mice by streptozotocin (STZ; 40 mg/kg body weight, i.p.) for five consecutive days. The diabetic mice were administered (i.v.) with MSC (1 × 105 umbilical cord blood MSC cells/mice/day), their derived exosomes (the MSC-Exo group that received exosomes derived from 1 × 105 MSC cells/mice/day), or the same volume of PBS. Before administration, the potency of MSCs and their exosomes was evaluated in vitro by T cell activation experiments. After day 7 of the treatments, blood samples and pancreatic tissues were collected. Histochemistry was performed to check cellular architecture and β cell regeneration. In body weight, blood glucose level, and insulin level, cell proliferation assay was done to confirm regeneration of cells after MSC and MSC-Exo treatments. Hyperglycemia was also attenuated in these mice with a concomitant increase in insulin production and an improved histological structure compared to mice in the PBS-treated group. We found increased expression of genes associated with tissue regeneration pathways, including Reg2, Reg3, and Amy2b in the pancreatic tissue of mice treated with MSC or MSC-Exo relative to PBS-treated mice. MicroRNA profiling of MSC-derived exosomes showed the presence of miRs that may facilitate pancreatic regeneration by regulating the Extl3-Reg-cyclinD1 pathway. These results demonstrate a potential therapeutic role of umbilical cord blood MSC-derived exosomes in attenuating insulin deficiency by activating pancreatic islets' regenerative abilities.

Glucosamine potentiates the differentiation of adipose-derived stem cells into glucose-responsive insulin-producing cells

BackgroundIslet transplantation might be a logical strategy to restore insulin secretion for the treatment of diabetes, however, the scarcity of donors poses an obstacle for such a treatment. As an alternative islet source, differentiation of stem cells into insulin-producing cells (IPCs) has been tried. Many protocols have been developed to improve the efficiency of differentiation of stem cells into IPCs. In this study, we investigated whether glucosamine supplementation during differentiation of human adipose-derived stem cells (hADSCs) into IPCs can improve the insulin secretory function.MethodsGlucosamine was added to the original differentiation medium at different stages of differentiation of hADSCs into IPCs for 12 days and insulin secretion was analyzed.ResultsAddition of glucosamine alone to the growth medium of hADSCs did not affect the differentiation of hADSCs to IPCs. Supplementation of the differentiation medium with glucosamine at a later stage (protocol G3) proved to have the greatest effect on IPC differentiation. Basal and glucose-stimulated insulin secretion (GSIS) was significantly increased and the expression of insulin and C-peptide was increased in differentiated IPCs as compared with that in differentiated IPCs using the conventional protocol (protocol C). In addition, the expression of beta-cell specific transcription factors such as pancreatic and duodenal homeobox1 (PDX1) and neurogenin 3 (NGN3) was also increased. Furthermore, the expression of genes related to insulin secretion, including synaptotagmin 4 (Syt4), glucokinase (Gck) and glucose transporter 2 (Glut2), was also increased.ConclusionsWe conclude that glucosamine supplementation potentiates the differentiation of hADSCs into IPCs.

Management of Type 1 diabetes mellitus with autologous mesenchymal stem cells

Type 1 diabetes mellitus (DM) is one of the most common chronic metabolic disorders among children. The condition is associated with dysregulation of the immune system resulting in loss of self-tolerance and destruction of pancreatic β-cells by autoreactive T-lymphocytes. Regulation of β-cell homeostasis is vital to maintain optimum blood glucose control as well as prevent the development of complications. Exogenous insulin and oral hypoglycemic agents do not achieve this, as they do not address the core pathology of type 1 DM. This report describes a case of a 17-year-old female patient with fluctuating blood glucose levels and high glycosylated hemoglobin, unresponsive to conventional therapy. After undergoing two sessions of cell-based therapy, within a year, optimum control of blood glucose was achieved along with negative anti-insulin antibodies. The patient was able to discontinue insulin and has not experienced any adverse effects till date. Improvement in general health was noticed, and the patient is now free from any symptoms associated with her former diabetic status. Mesenchymal stem cells (MSCs) possess immunomodulatory and pro-angiogenic properties, which may aid in arresting β-cell destruction. In addition, MSCs may preserve residual β-cell mass and facilitate endogenous β-cell regeneration. These cells are capable of differentiating into glucose-responsive insulin-producing cells, making them ideal candidates in treatment of and prevention of complications in T1DM.

Insights into Islet Differentiation and Maturation through Proteomic Characterization of a Human iPSC-Derived Pancreatic Endocrine Model.

Great progresses have been made for generating in vitro pluripotent stem cell pancreatic β-like cells. However, the maturation stage of the cells still requires in vivo maturation to recreate the environmental niche. A deeper understanding of the factors promoting maturation of the cells is of great interest for clinical applications. Label-free mass spectrometry based proteomic analysis is performed on samples from a longitudinal study of differentiation of human induced pluripotent stem cells toward glucose responsive insulin producing cells. Proteome patterns correlate with specific transcription factor gene expression levels during in vitro differentiation, showing the relevance of the technology for identification of pancreatic-specific markers. The analysis of proteomes of the implanted cells in a longitudinal study shows that the neovascularization process linked to the extracellular matrix environment is time-dependent and conditions the proper maturation of the cells in β-like cells secreting insulin in response to glucose. Proteomic profiling is valuable to qualify and better understand in vivo maturation of progenitor cells toward β-cells. This is critical for future clinical trials where in vivo maturation still needs to be improved for robustness and effectiveness of cell therapy. Novel biomarkers for predicting the efficiency of maturation represents noninvasive monitoring tools for following efficiency of the implant.

Islet Encapsulation: Physiological Possibilities and Limitations.

A logical cure for type 1 diabetes (T1D) involves replacing the lost insulin-producing cells with new ones, preferably cells from a well-characterized and unlimited source of human insulin-producing cells. This straightforward and simple solution to provide a cure for T1D is immensely attractive but entails at least two inherent and thus far unresolved hurdles: 1) provision of an unlimited source of functional human insulin-producing cells and 2) prevention of rejection without the side effects of systemic immunosuppression. Generation of transplantable insulin-producing cells from human embryonic stem cells or induced pluripotent stem cells is at present close to reality, and we are currently awaiting the first clinical studies. Focus is now directed to foster development of novel means to control the immune system to enable large-scale clinical application. Encapsulation introduces a physical barrier that prevents access of immune cells to the transplanted cells but also hinders blood vessel ingrowth. Therefore, oxygen, nutrient, and hormonal passage over the encapsulation membrane is solely dependent on diffusion over the immune barrier, contributing to delays in glucose sensing and insulin secretion kinetics. This Perspective focuses on the physiological possibilities and limitations of an encapsulation strategy to establish near-normoglycemia in subjects with T1D, assuming that glucose-responsive insulin-producing cells are available for transplantation.

Transplantation of Human Pancreatic Endoderm Cells Reverses Diabetes Post Transplantation in a Prevascularized Subcutaneous Site.

SummaryBeta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of “healthy” human donor pancreata restricts the broader application of this effective curative therapy. “β-Like” cells derived from human embryonic stem cells (hESC), with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs) in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells.

Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc.

A Comparison Between Islet And Stem Cell Transplantation For Treatment Of Type 1 Diabetes Mellitus: A Systematic Review

Introduction: The incidence of type 1 Diabetes Mellitus (T1DM) has been increasing rapidly worldwide in the past decade. The current standard treatment is exogenous insulin therapy, however, this procedure is highly associated with poor glycemic control that may lead to life-threatening hypoglycemic episodes. Cellular-based therapy for T1DM has been recently developed, making it pertinent to compare the effectiveness between two most anticipated breakthroughs: islet cell and stem cell transplantation, in order to determine which procedure is more effective. Methods: A comprehensive digital literature search was performed using PubMed and Ovid Medline for primary research studies published between Jan 2000 – Nov 2015. Relevant cohort, case-control, case series, and in vivo studies were included. The abstracts and full text of the retrieved articles were scanned for potential studies that fulfilled the inclusion criterias. The quality assessment of studies were conducted using ARRIVE, NOS, and MINORS. Results: Nineteen primary research studies met the inclusion criterias and were assessed for the review. Eleven out of 19, were considered as high-quality, while the rest were moderate-quality. The studies generally reported the insulin independence, graft functionality, and glycemic control. The insulin-independent period for islet cell tranplantation was proven to be longer compared to stem cell transplantation with better glycemic control. Stem cells were successfully differentiated into glucoseresponsive insulin-producing cells, that also released glucagon and somatostatin. Discussion: The majority of the included studies were using the same outcome measures which allow a more comprehensive comparison to be conducted. Based on the assessment, islet cell transplantation is currently better. This treatment was found to lead to significant improvements in insulin independence and glycemic control observed through insulin-free period, HbA1c, blood glucose, and C-peptide serum measurement. On the other hand, certain challenges – such as donor shortage and poor engraftment - hinders the widespread application of the treatment. Therefore, stem cell transplantation is thought to possibly be replacing islet cell transplantation in the future. Stem cells had successfully been differentiated into β-like cells that were not only producing insulin, but also glucagon and somatostatin, as well as acting in glucose-stimulated manner, imitating the physiologic mechanism of β-cells. Conclusion: It is conclusive that islet stem cell transplantation was proven to perform relatively better in terms of insulin independence and glycemic control compared to stem cell transplantation for treating T1DM. Both cellular-based treatments provided relatively better glycemic control compared to the current standard treatment, exogenous insulin therapy. All the studies have reported that both treatments lead to substantial improveent between pre-and post-transplantation periods. Stem cell transplantation was also proven to have unlimited potentials to be the future solution for T1DM. Although, there was limited studies on human subjects, but based on the current available studies, the results were quite conclusive.

Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of β-cell neogenesis.

The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation.

274. Expansion and Conversion of Patient-Derived Human Gallbladder Cells Into Insulin-Producing Pancreatic Endocrine-Like Type Cells

Islet transplantation is an efficacious therapeutic option for T1D, but, supply of donor pancreata does not meet the demand. Variable successes in generating glucose-responsive insulin-producing beta-like cells from pluripotent and differentiated cells have been reported. We report on genetic reprogramming of highly expandable patient-derived human gallbladder cells (GBCs) to produce insulin for autologous transplant. Using feeder-free and feeder-type culture methods, we expanded GBCs in vitro in the order of 10^8 total cells after 4 passages starting from 10^4 GBCs. Cultures can be maintained for at least 15 passages, albeit with slowdown in growth. Transcriptome profiling revealed GBCs to be deficient in factors important for beta-cell development, specification, and maintenance such as NEUROG3, MAFA, NKX6-1, NKX2-2, PAX4, PAX6, NEUROD1, INSM1, RFX6, GCK, and INS. MNX1 and PDX1 are moderately expressed in cultures but at significantly lower levels found in beta-cells. Viral transduction of PDX1, MAFA, and NEUROG3 (PNM) induced expression of INS at 1/10,000th compared to that of human islet cells. Transduced GBCs began to show morphological changes from a monolayer into 3D clusters as early as 2 days after transduction. Retinoic acid, GLP-1, FGF10, DAPT, T3, Alk5 inhibitor, and B27 in the reprogramming medium and the addition of PAX6 to PNM transduction highly improved both INS and NKX6-1 induction reaching 10% of islet cells. These reprogrammed GBCs also express high levels of NEUROD1, NKX2-2, RFX6, PAX4, MAFB, HOPX, SST, GHRL, CHGA, TMEM27, SYP, KCNJ11, and ABCC8 comparable to islet cells. Average transduction efficiency was 50% yielding 9% C-peptide+ cells by immunostaining. Cellular C-peptide content was equivalent to 0.3% of that of purified beta-cells. At best, we observed 5-fold increase in C-peptide concentration in response to high glucose. We further enriched for INS+ reprogrammed GBCs using a cell surface mAb (HPi1) shown to isolate human pancreatic islet cells. Immunolabeling showed that reprogramming significantly increased the percentage of Hpi1+ GBCs. RT-qPCR of Hpi1+ GBCs showed greater than 1000-fold enrichment in gene expression of INS, NKX6-1, and PCSK1 compared to Hpi1- cells. Likewise, we observed greater than 100-fold enrichment for NEUROD1, SST, GHRL, and RFX6. In addition, we saw significant increase in reprogramming efficiency by adding an isoxazole to the reprogramming medium resulting to 10-fold higher INS expression, 2.5-fold increase in the frequency of C-peptide+ cells, and significant drop in SST and GHRL expression. Reprogrammed GBCs can engraft, survive, and produce C-peptide for at least 2 weeks in under the kidney capsule, mammary fat pad, and dorsal subcutaneous fat in NSG mice. Transplant experiments in diabetic mice are ongoing. In summary, we have transdifferentiated patient-derived adult GBCs into beta-like cells ex vivo, thus showing that GBCs are potential replacement sources of autologous insulin-producing cells.

PDX-1 mRNA-induced reprogramming of mouse pancreas-derived mesenchymal stem cells into insulin-producing cells in vitro.

Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for β-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel β-cell source for the treatment of diabetes.

Mesenchymal Stem Cell Therapy in Type 1 Diabetes Mellitus and Its Main Complications: From Experimental Findings to Clinical Practice

Type 1 diabetes mellitus (T1DM) is a complex multifactorial disorder which involves a loss of self-tolerance leading to the autoimmune destruction of pancreatic β−cells. Exogenous insulin administration cannot mimic precise pancreatic β-cell regulation of glucose homeostasis, thereby leading to severe long-term complications. Pancreas or islet transplant only provides partial exogenous insulin independence and induces several adverse effects, including increased morbidity and mortality. The scientific community and diabetic patients are thus, still waiting for an effective therapy which could preserve the remaining β-cells, replenish islet mass and protect newly-generated β-cells from autoimmune destruction. Mesenchymal stem cells (MSCs) have been envisioned as a promising tool for T1DM treatment over the past few years, since they could differentiate into glucose-responsive insulin-producing cells. Their immunomodulatory and proangiogenic roles can be used to help arrest β-cell destruction, preserve residual β-cell mass, facilitate endogenous β-cell regeneration and prevent disease recurrence, thereby making them ideal candidates for the comprehensive treatment of diabetic patients. This review focuses on recent pre-clinical data supporting MSC use in regenerating β-cell mass and also in treating several T1DM-associated complications. Clinical trial results and the ongoing obstacles which must be addressed regarding the widespread use of such therapy are also discussed.

In vitro generation of glucose-responsive insulin producing cells using lentiviral based pdx-1 gene transduction of mouse (C57BL/6) mesenchymal stem cells

The objective of this study was to evaluate the potential of this type of recombinant lentivirus to generate glucose-responsive insulin producing cells in vitro. All steps of cloning were confirmed using restriction digests. After the transduction, mesenchymal stem cells gradually began to change their morphology and showed differentiation into islet like structures. RT-PCR results confirmed the expression of insulin1, insulin2 and pdx-1 in differentiated cells. Dithizone staining of mouse MSCs showed the concentration of glucose in islet like structures. ELISA analysis validated the insulin secretion of islet like structures which in the high-glucose medium (25mmol/l) was 7.44 fold higher than that secreted in the low-glucose medium (5mmol/l). Our results demonstrated that mouse mesenchymal stem cells can be differentiated into effective glucose-responsive insulin producing cells through our new recombinant lentiviral transduction of pdx-1 gene in vitro. This new lentiviral vector could be suggested as an effective candidate for using in gene therapy of type-1 diabetes.

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice

Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

Stem Cell Therapy to Cure Type 1 Diabetes: From Hype to Hope

Type 1 diabetes mellitus (T1D) is a chronic, multifactorial autoimmune disease that involves the progressive destruction of pancreatic β-cells, ultimately resulting in the loss of insulin production and secretion. The goal of clinical intervention is to prevent or arrest the onset and progression of autoimmunity, reverse β-cell destruction, and restore glycometabolic and immune homeostasis. Despite promising outcomes observed with islet transplantation and advancements in immunomodulatory therapies, the need for an effective cell replacement strategy for curing T1D still persists. Stem cell therapy offers a solution to the cited challenges of islet transplantation. While the regenerative potential of stem cells can be harnessed to make available a self-replenishing supply of glucose-responsive insulin-producing cells, their immunomodulatory properties may potentially be used to prevent, arrest, or reverse autoimmunity, ameliorate innate/alloimmune graft rejection, and prevent recurrence of the disease. Herein, we discuss the therapeutic potential of stem cells derived from a variety of sources for the cure of T1D, for example, embryonic stem cells, induced pluripotent stem cells, bone marrow-derived hematopoietic stem cells, and multipotent mesenchymal stromal cells derived from bone marrow, umbilical cord blood, and adipose tissue. The benefits of combinatorial approaches designed to ensure the successful clinical translation of stem cell therapeutic strategies, such as approaches combining effective stem cell strategies with islet transplantation, immunomodulatory drug regimens, and/or novel bioengineering techniques, are also discussed. To conclude, the application of stem cell therapy in the cure for T1D appears extremely promising.

PDX1-engineered embryonic stem cell-derived insulin producing cells regulate hyperglycemia in diabetic mice

BackgroundType 1 diabetes can be treated by the transplantation of cadaveric whole pancreata or isolated pancreatic islets. However, this form of treatment is hampered by the chronic shortage of cadaveric donors. Embryonic stem (ES) cell-derived insulin producing cells (IPCs) offer a potentially novel source of unlimited cells for transplantation to treat type 1 and possibly type 2 diabetes. However, thus far, the lack of a reliable protocol for efficient differentiation of ES cells into IPCs has hindered the clinical exploitation of these cells.MethodsTo efficiently generate IPCs using ES cells, we have developed a double transgenic ES cell line R1Pdx1AcGFP/RIP-Luc that constitutively expresses pancreatic β-cell-specific transcription factor pancreatic and duodenal homeobox gene 1 (Pdx1) as well as rat insulin promoter (RIP) driven luciferase reporter. We have established several protocols for the reproducible differentiation of ES cells into IPCs. The differentiation of ES cells into IPCs was monitored by immunostaining as well as real-time quantitative RT-PCR for pancreatic β-cell-specific markers. Pancreatic β-cell specific RIP became transcriptionally active following the differentiation of ES cells into IPCs and induced the expression of the luciferase reporter. Glucose stimulated insulin secretion by the ES cell-derived IPCs was measured by ELISA. Further, we have investigated the therapeutic efficacy of ES cell-derived IPCs to correct hyperglycemia in syngeneic streptozotocin (STZ)-treated diabetic mice. The long term fate of the transplanted IPCs co-expressing luciferase in syngeneic STZ-induced diabetic mice was monitored by real time noninvasive in vivo bioluminescence imaging (BLI).ResultsWe have recently demonstrated that spontaneous in vivo differentiation of R1Pdx1AcGFP/RIP-Luc ES cell-derived pancreatic endoderm-like cells (PELCs) into IPCs corrects hyperglycemia in diabetic mice. Here, we investigated whether R1Pdx1AcGFP/RIP-Luc ES cells can be efficiently differentiated in vitro into IPCs. Our new data suggest that R1Pdx1AcGFP/RIP-Luc ES cells efficiently differentiate into glucose responsive IPCs. The ES cell differentiation led to pancreatic lineage commitment and expression of pancreatic β cell-specific genes, including Pax4, Pax6, Ngn3, Isl1, insulin 1, insulin 2 and PC2/3. Transplantation of the IPCs under the kidney capsule led to sustained long-term correction of hyperglycemia in diabetic mice. Although these newly generated IPCs effectively rescued hyperglycemic mice, an unexpected result was teratoma formation in 1 out of 12 mice. We attribute the development of the teratoma to the presence of either non-differentiated or partially differentiated stem cells.ConclusionsOur data show the potential of Pdx1-engineered ES cells to enhance pancreatic lineage commitment and to robustly drive the differentiation of ES cells into glucose responsive IPCs. However, there is an unmet need for eliminating the partially differentiated stem cells.

Glucose-stimulated insulin secretion of various mesenchymal stem cells after insulin-producing cell differentiation

Mesenchymal stem cells (MSCs) are capable of crossing germinative layer borders and are obtainable in high numbers via in vitro cultures. Therefore, many researchers have searched for diverse sources of MSCs. Recently the generation of glucose-responsive insulin-producing cells (IPCs) from MSCs has shown immense potential for the treatment of type 1 diabetes mellitus (T1DM) due to a lack of pancreas donors. In this study, we compared the growth potency of four kinds of MSCs derived from bone marrow, Wharton's jelly, adipose tissue, and the periosteum. In addition, in vitro differentiation of these MSCs into IPCs was also investigated. After 2weeks of IPCs differentiation, we compared the expression of the insulin gene and protein using RT-qPCR and immunofluorescence staining. Only IPCs derived from periosteum-derived progenitor cells (PDPCs) showed a response to glucose concentration. Glucose stimulated insulin secretion was conclusive evidence of the potential functionality of IPCs. Therefore, PDPCs are a promising alternative stem cell source for IPCs differentiation.

Immunological Applications of Stem Cells in Type 1 Diabetes

Current approaches aiming to cure type 1 diabetes (T1D) have made a negligible number of patients insulin-independent. In this review, we revisit the role of stem cell (SC)-based applications in curing T1D. The optimal therapeutic approach for T1D should ideally preserve the remaining β-cells, restore β-cell function, and protect the replaced insulin-producing cells from autoimmunity. SCs possess immunological and regenerative properties that could be harnessed to improve the treatment of T1D; indeed, SCs may reestablish peripheral tolerance toward β-cells through reshaping of the immune response and inhibition of autoreactive T-cell function. Furthermore, SC-derived insulin-producing cells are capable of engrafting and reversing hyperglycemia in mice. Bone marrow mesenchymal SCs display a hypoimmunogenic phenotype as well as a broad range of immunomodulatory capabilities, they have been shown to cure newly diabetic nonobese diabetic (NOD) mice, and they are currently undergoing evaluation in two clinical trials. Cord blood SCs have been shown to facilitate the generation of regulatory T cells, thereby reverting hyperglycemia in NOD mice. T1D patients treated with cord blood SCs also did not show any adverse reaction in the absence of major effects on glycometabolic control. Although hematopoietic SCs rarely revert hyperglycemia in NOD mice, they exhibit profound immunomodulatory properties in humans; newly hyperglycemic T1D patients have been successfully reverted to normoglycemia with autologous nonmyeloablative hematopoietic SC transplantation. Finally, embryonic SCs also offer exciting prospects because they are able to generate glucose-responsive insulin-producing cells. Easy enthusiasm should be mitigated mainly because of the potential oncogenicity of SCs.

Are stem cells a cure for diabetes?

With the already heightened demand placed on organ donation, stem cell therapy has become a tantalizing idea to provide glucose-responsive insulin-producing cells to Type 1 diabetic patients as an alternative to islet transplantation. Multiple groups have developed varied approaches to create a population of cells with the appropriate characteristics. Both adult and embryonic stem cells have received an enormous amount of attention as possible sources of insulin-producing cells. Although adult stem cells lack the pluripotent nature of their embryonic counterparts, they appear to avoid the ethical debate that has centred around the latter. This may limit the eventual application of embryonic stem cells, which have already shown promise in early mouse models. One must also consider the potential of stem cells to form teratomas, a complication which would prove devastating in an immunologically compromised transplant recipient. The present review looks at the progress to date in both the adult and embryonic stem cells fields as potential treatments for diabetes. We also consider some of the limitations of stem cell therapy and the potential complications that may develop with their use.

Production of β‐cells from human embryonic stem cells

The making of functional pancreatic islets in renewable numbers has been a goal of stem cell biologists since early 2000. Since that time, many studies have reported successful creation of glucose-responsive pancreatic beta-cells. Not until the more recent systematic application of developmental principles to stem cell biology systems were breakthroughs achieved on directed specification of the required early developmental intermediates. The most important first step is the formation of the definitive endoderm (DE) lineage which is compulsory for production of the epithelium of the pancreas and the other important endoderm-derived organs such as the liver, intestine and lung. The formation of DE from embryonic stem cells made possible additional experimentation aimed at directing the endoderm to further specified foregut and pancreatic endoderm lineages. With these discoveries came the first production of immature pancreatic endocrine cells. Most recently, the production in vivo of glucose-responsive insulin-producing cells with the capacity to correct Steptozotocin-induced hyperglycaemia in mice has been achieved. The work leading up to this achievement, in relation to the other principle human stem cell studies conducted in this area, will be briefly described. The necessary steps and ideal characteristics of embryonic stem cell-based differentiation to pancreatic beta-cells capable of glucose stimulated insulin secretion will be underscored.