The generation of insulin-producing cells from embryonic stem cells--a discussion of controversial findings

The derivation of insulin-producing cells from embryonic stem (ES) cells has been controversially described. Whereas several authors showed successful differentiation of mouse ES cells into islet-like clusters, others could not confirm the results. Here, we present a detailed comparison of the various strategies used to generate pancreatic cells with respect to protocols and differentiation factors and give an explanation of the contradictory findings. It is suggested that the selection or enrichment of ES-derived nestin-positive cells should be avoided, since these cells are already committed to a neural fate before pancreatic differentiation is induced.     

A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones

Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin.     

Generation of insulin-secreting islet-like clusters from human skin fibroblasts

Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future.     

Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes

Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.     

Insulin-producing cells from human pancreatic islet-derived progenitor cells following transplantation in mice

Stem/progenitor cells hold promise for alleviating/curing type 1 diabetes due to the capacity to differentiate into functional insulin-producing cells. The current study aims to assess the differentiation potential of human pancreatic IPCs (islet-derived progenitor cells). IPCs were derived from four human donors and subjected to more than 2000-fold expansion before turning into ICCs (islet-like cell clusters). The ICCs expressed ISL-1 Glut2, PDX-1, ngn3, insulin, glucagon and somatostatin at the mRNA level and stained positive for insulin and glucagon by immunofluorescence. Following glucose challenge in vitro, C-peptide was detected in the sonicated ICCs, instead of in the conditioned medium. To examine the function of the cells in vivo, IPCs or ICCs were transplanted under the renal capsule of immunodeficient mice. One month later, 19 of 28 mice transplanted with ICCs and 4 of 14 mice with IPCs produced human C-peptide detectable in blood, indicating that the in vivo environment further facilitated the maturation of ICCs. However, among the hormone-positive mice, only 9 of 19 mice with ICCs and two of four mice with IPCs were able to secrete C-peptide in response to glucose.     

Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes

Background

There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Wharton''s jelly of the umbilical cord (HUMSCs), which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets.

Methodology and Principal Findings

HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic β-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2) in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules.

Conclusions and Significance

In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton''s Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.     

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.     

SHB and angiogenic factors promote ES cell differentiation to insulin-producing cells

The potential use of embryonic stem (ES) cells for cell therapy of diabetes requires improved methods for differentiation and isolation of insulin-producing beta-cells. The signal transduction protein SHB may be involved in both angiogenesis and beta-cell development. Here we show that cells expressing the pancreatic endodermal marker PDX-1 appear in the vicinity of vascular structures in ES cell-derived embryoid bodies (EBs) cultured in vitro. Moreover, overexpression of SHB as well as culture of EBs in presence of the angiogenic growth factors PDGF or VEGF enhanced the expression of PDX-1 and/or insulin mRNA. Finally, expression of GFP under control of the PDX-1 promoter in EBs allowed for the enrichment by FACS of cells expressing PDX-1, C-peptide, and insulin as determined by immunofluorescence. It is concluded that SHB and angiogenic factors promote the development of cells expressing PDX-1 and insulin in EBs and that such cells can be separated by FACS.     

Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets

Cellular models and culture conditions for in vitro expansion of insulin-producing cells represent a key element to develop cell therapy for diabetes. Initial evidence that human beta-cells could be expanded after undergoing a reversible epithelial-mesenchymal transition has been recently negated by genetic lineage tracing studies in mice. Here, we report that culturing human pancreatic islets in the presence of serum resulted in the emergence of a population of nestin-positive cells. These proliferating cells were mainly C-peptide negative, although in the first week in culture, proliferating cells, insulin promoter factor-1 (Ipf-1) positive, were observed. Later passages of islet-derived cells were Ipf-1 negative and displayed a mesenchymal phenotype. These human pancreatic islet-derived mesenchymal (hPIDM) cells were expanded up to 10(14) cells and were able to differentiate toward adipocytes, osteocytes and chondrocytes, similarly to mesenchymal stem/precursor cells. Interestingly, however, under serum-free conditions, hPIDM cells lost the mesenchymal phenotype, formed islet-like clusters (ILCs) and were able to produce and secrete insulin. These data suggest that, although these cells are likely to result from preexisting mesenchymal cells rather than beta-cells, hPIDM cells represent a valuable model for further developments toward future replacement therapy in diabetes.     

Reduced immunogenicity of pancreatic progenitor cells derived from first-trimester human fetal pancreas

The relatively low immunogenic and tumorigenic nature of fetal stem cells makes them attractive candidates for transplantation. Pancreatic progenitor cells (PPCs) derived from human fetal pancreas that are amenable to growth and differentiation into transplantable insulin-producing islet-like cell clusters (ICCs) have been reported recently; however, the immunological nature of these cells has yet to be characterized. We thus investigated and compared the immunogenicity of pancreatic progenitor cells and islet-like cell clusters from first- and second-trimester human fetal pancreas. Polymerase chain reaction demonstrated that pancreatic progenitor cells and islet-like cell clusters express immune-related genes of major histocompatibility complex, MHC-I and MHC-II, complement component 3 (C3), chemokine ligand (CCL19), and tumor necrosis factor super family (TNFSF10), but no expression of the co-stimulatory genes, CD80 and CD86. Interestingly, pancreatic progenitor cells showed a differential expression of MHC-I and MHC-II with advancing gestational age with a greater expression in pancreatic progenitor cells from the second trimester. Pre-incubation of the second-trimester cells with interferon-γ (IFN-γ) increased MHC molecule expression. Functional alloreactivity of pancreatic progenitor cells was investigated via mixed lymphocyte reactions (MLRs). Relative to first-trimester pancreatic progenitor cells, second-trimester pancreatic progenitor cells induced a greater extent of proliferation of peripheral blood mononuclear cells (PBMCs) and resulted in more IFN-γ production in phytohaemagllutinin-stimulated peripheral blood mononuclear cells following co-culture. Results of the study indicated that first-trimester pancreatic progenitor cells and islet-like cell clusters have a distinctively lower immunogenicity relative to second-trimester pancreatic progenitor cells, even after a pro-inflammatory cytokine challenge.     

Differentiation of human adipose-derived mesenchymal stem cell into insulin-producing cells: an in vitro study

Stem cells with the ability to differentiate into insulin-producing cells (IPCs) are becoming the most promising therapy for diabetes mellitus and reduce the major limitations of availability and allogeneic rejection of beta cell transplantations. Mesenchymal stem cells (MSCs) are pluripotent stromal cells with the ability to proliferate and differentiate into a variety of cell types including endocrine cells of the pancreas. This study sought to inspect the in vitro differentiation of human adipose-derived tissue stem cells into IPCs which could provide an abundant source of cells for the purpose of diabetic cell therapy in addition to avoid immunological rejection. Adipose-derived MSCs were obtained from liposuction aspirates and induced to differentiate into insulin-secreting cells under a three-stage protocol based on a combination of low-glucose DMEM medium, β-mercaptoethanol, and nicotinamide for pre-induction and high-glucose DMEM, β-mercaptoethanol, nicotinamide, and exendin-4 for induction stages of differentiation. Differentiation was evaluated by the analysis of morphology, dithizone staining, RT-PCR, and immunocytochemistry. Morphological changes including typical islet-like cell clusters were observed by phase-contrast microscope at the end of differentiation protocol. Based on dithizone staining, differentiated cells were positive and undifferentiated cells were not stained. Furthermore, RT-PCR results confirmed the expression of insulin, PDX1, Ngn3, PAX4, and GLUT2 in differentiated cells. Moreover, insulin production by the IPCs was confirmed by immunocytochemistry analysis. It is concluded that adipose-derived MSCs could differentiate into insulin-producing cells in vitro.     

Directed engineering of umbilical cord blood stem cells to produce C-peptide and insulin

OBJECTIVES: In this study, we investigated the potential of umbilical cord blood stem cell lineages to produce C-peptide and insulin. MATERIALS AND METHODS: Lineage negative, CD133+ and CD34+ cells were analyzed by flow cytometry to assess expression of cell division antigens. These lineages were expanded in culture and subjected to an established protocol to differentiate mouse embryonic stem cells (ESCs) toward the pancreatic phenotype. Phase contrast and fluorescence immunocytochemistry were used to characterize differentiation markers with particular emphasis on insulin and C-peptide. RESULTS: All 3 lineages expressed SSEA-4, a marker previously reported to be restricted to the ESC compartment. Phase contrast microscopy showed all three lineages recapitulated the treatment-dependent morphological changes of ESCs as well as the temporally restricted expression of nestin and vimentin during differentiation. After engineering, each isolate contained both C-peptide and insulin, a result also obtained following a much shorter protocol for ESCs. CONCLUSIONS: Since C-peptide can only be derived from de novo synthesis and processing of pre-proinsulin mRNA and protein, we conclude that these results are the first demonstration that human umbilical cord blood-derived stem cells can be engineered to engage in de novo synthesis of insulin.