Mesenchymal stem cells: biology and clinical potential in type 1 diabetes therapy

Mesenchymal stem cells (MSCs) can be derived from adult bone marrow, fat and several foetal tissues. In vitro , MSCs have the capacity to differentiate into multiple mesodermal and non-mesodermal cell lineages. Besides, MSCs possess immunosuppressive effects by modulating the immune function of the major cell populations involved in alloantigen recognition and elimination. The intriguing biology of MSCs makes them strong candidates for cell-based therapy against various human diseases. Type 1 diabetes is caused by a cell-mediated autoimmune destruction of pancreatic β-cells. While insulin replacement remains the cornerstone treatment for type 1 diabetes, the transplantation of pancreatic islets of Langerhans provides a cure for this disorder. And yet, islet transplantation is limited by the lack of donor pancreas. Generation of insulin-producing cells (IPCs) from MSCs represents an attractive alternative. On the one hand, MSCs from pancreas, bone marrow, adipose tissue, umbilical cord blood and cord tissue have the potential to differentiate into IPCs by genetic modification and/or defined culture conditions In vitro . On the other hand, MSCs are able to serve as a cellular vehicle for the expression of human insulin gene. Moreover, protein transduction technology could offer a novel approach for generating IPCs from stem cells including MSCs. In this review, we first summarize the current knowledge on the biological characterization of MSCs. Next, we consider MSCs as surrogate β-cell source for islet transplantation, and present some basic requirements for these replacement cells. Finally, MSCs-mediated therapeutic neovascularization in type 1 diabetes is discussed.     

Differentiation of insulin-producing cells from human neural progenitor cells

BackgroundSuccess in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin.Methods and FindingsHere we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors.ConclusionProduction of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.     

Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.     

In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract

Recent reports have suggested that mesenchymal cells derived from bone marrow may differentiate into not only mesenchymal lineage cells but also other lineage cells. There is possibility for insulin-producing cells (IPCs) to be differentiated from mesenchymal cells. We used self-functional repair stimuli of stem cells by partial injury. Rat pancreatic extract (RPE) from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to rat mesenchymal cells. After the treatment of RPE, they made clusters like islet of Langerhans within a week and expressed four pancreatic endocrine hormones; insulin, glucagon, pancreatic polypeptide, and somatostatin. Moreover, IPCs released insulin in response to normal glucose challenge. Here we demonstrate that the treatment of RPE can differentiate rat mesenchymal cells into IPCs which can be a potential source for the therapy of diabetes.     

Differentiation of mesenchymal stem cells to insulin-producing cells and their impact on type 1 diabetic rats

Cell therapy is thought to be a possible approach for treatment of diabetes. Cells with the ability to differentiate into insulin-producing cells (IPCs) would provide an unlimited source of islet cells for transplantation. In this study, the differentiation capacity of rat bone-marrow-derived mesenchymal stem cells (MSCs) to IPCs and the feasibility of using them for reversal of hyperglycemia were investigated. In vitro studies indicated that treatment of cells with high glucose concentration, nicotinamide and β-mercaptoethanol resulted to differentiated cells, which had characteristics of IPCs including spherical, grape-like morphology, secretion of insulin, and being positive for dithizone. To test the in vivo function of differentiated MSCs, they were injected into the spleen of diabetic rats. It was shown that diabetic rats who received IPCs, significantly reduced the glucose level, in response to intraperitoneal glucose tolerance (IPGT) test. These results indicate that MSCs are capable of in vitro differentiation into functional IPCs, which can reverse hyperglycemia in rat model of diabetes.     

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward β-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic β cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.     

大鼠骨骼肌卫星细胞诱导分化为胰岛素生成细胞的研究

目的探讨大鼠骨骼肌卫星细胞（MDSCs）定向诱导分化为胰岛素生成细胞（IPCs）,为1型糖尿病的干细胞治疗提供一种新的研究思路。 方法通过二次酶消化法和差速贴壁培养法分离、培养大鼠MDSCs,利用不同的诱导培养液使MDSCs定向分化为IPCs,并对诱导后细胞进行形态观察,通过双硫腙染色和免疫组化染色对MDSCs-IPCs形态进行鉴定,采用Q-PCR和Western Blot方法检测MDSCs-IPCs中C-peptide和Insulin的表达,通过胰岛素释放实验检测MDSCs-IPCs的生物学功能,β细胞和MDSCs-IPCs两组间比较采用t检验。 结果MDSCs在接种4 h后开始贴壁部分细胞伸出小的突起,48 h后绝大多数细胞贴壁呈梭形、胞浆丰富、折光度高。随着培养时间的延长,细胞的梭形形状更为明显且生长迅速。免疫组化结果显示细胞表达Desmin、α-Sarcomeric Actinin、MyoD1、Myf5和PAX7。成胰诱导后MDSCs形成胰岛样的圆形细胞团,双硫腙染色呈猩红色,Insulin免疫组化染色阳性。Q-PCR结果显示MDSCs-IPCs中C-peptide和Insulin mRNA表达量分别是β细胞的0.73倍（P > 0.05）和0.79倍（P > 0.05）。胰岛素释放实验显示,5.6 mmol/L和16.7 nmlol/L葡萄糖刺激培养2 h后,β细胞和MDSCs-IPCs分泌胰岛素量分别为[（20.3±4.2）mU/L]、[（16.1±3.7）mU/L]、[（60.5±9.3）mU/L]和[（40.9±7.3）mU/L],葡萄糖可调节MDSCs-IPCs胰岛素的分泌。 结论MDSCs易于分离培养、增殖能力强,体外可诱导分化为有功能的IPCs,适合作为再生医学的种子细胞。     

FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells

Insulin-producing cells (IPCs) derived from human embryonic stem cells (hESCs) hold great potential for cell transplantation therapy in diabetes. Tremendous progress has been made in inducing differentiation of hESCs into IPCs in vitro, of which definitive endoderm (DE) protocol mimicking foetal pancreatic development has been widely used. However, immaturity of the obtained IPCs limits their further applications in treating diabetes. Forkhead box O1 (FoxO1) is involved in the differentiation and functional maintenance of murine pancreatic β cells, but its role in human β cell differentiation is under elucidation. Here, we showed that although FoxO1 expression level remained consistent, cytoplasmic phosphorylated FoxO1 protein level increased during IPC differentiation of hESCs induced by DE protocol. Lentiviral silencing of FoxO1 in pancreatic progenitors upregulated the levels of pancreatic islet differentiation-related genes and improved glucose-stimulated insulin secretion response in their progeny IPCs, whereas overexpression of FoxO1 showed the opposite effects. Notably, treatment with the FoxO1 inhibitor AS1842856 displayed similar effects with FoxO1 knockdown in pancreatic progenitors. These effects were closely associated with the mutually exclusive nucleocytoplasmic shuttling of FoxO1 and Pdx1 in the AS1842856-treated pancreatic progenitors. Our data demonstrated a promising effect of FoxO1 inhibition by the small molecule on gene expression profile during the differentiation, and in turn, on determining IPC maturation via modulating subcellular location of FoxO1 and Pdx1. Therefore, we identify a novel role of FoxO1 inhibition in promoting IPC differentiation of hESCs, which may provide clues for induction of mature β cells from hESCs and clinical applications in regenerative medicine.     

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.     

Insulin-Producing Cells Derived from Human Embryonic Stem Cells: Comparison of Definitive Endoderm- and Nestin-Positive Progenitor-Based Differentiation Strategies

Human embryonic stem cells (hESCs) are pluripotent and capable of undergoing multilineage differentiation into highly specialized cells including pancreatic islet cells. Thus, they represent a novel alternative source for targeted therapies and regenerative medicine for diabetes. Significant progress has been made in differentiating hESCs toward pancreatic lineages. One approach is based on the similarities of pancreatic β cell and neuroepithelial development. Nestin-positive cells are selected as pancreatic β cell precursors and further differentiated to secrete insulin. The other approach is based on our knowledge of developmental biology in which the differentiation protocol sequentially reproduces the individual steps that are known in normal β cell ontogenesis during fetal pancreatic development. In the present study, the hESC cell line PKU1.1 was induced to differentiate into insulin-producing cells (IPCs) using both protocols. The differentiation process was dynamically investigated and the similarities and differences between both strategies were explored. Our results show that IPCs can be successfully induced with both differentiation strategies. The resulting IPCs from both protocols shared many similar features with pancreatic islet cells, but not mature, functional β cells. However, these differently-derived IPC cell types displayed specific morphologies and different expression levels of pancreatic islet development-related markers. These data not only broaden our outlook on hESC differentiation into IPCs, but also extend the full potential of these processes for regenerative medicine in diabetes.     

Mesenchymal stem cells and differentiated insulin producing cells are new horizons for pancreatic regeneration in type I diabetes mellitus

BackgroundDiabetes mellitus has become the third human killer following cancer and cardiovascular disease. Millions of patients, often children, suffer from type 1 diabetes (T1D). Stem cells created hopes to regenerate damaged body tissues and restore their function.AimThis work aimed at clarifying and comparing the therapeutic potential of differentiated and non-differentiated mesenchymal stem cells (MSCs) as a new line of therapy for T1D.Methods40 Female albino rats divided into group I (control): 10 rats and group II (diabetic), III and IV, 10 rats in each, were injected with streptozotocin (50 mg/kg body weight). Group III (MSCs) were transplanted with bone marrow derived MSCs from male rats and group IV (IPCs) with differentiated insulin producing cells. Blood and pancreatic tissue samples were taken from all rats for biochemical and histological studies.ResultsMSCs reduced hyperglycemia in diabetic rats on day 15 while IPCs normalizes blood glucose level on day 7. Histological and morphometric analysis of pancreas of experimental diabetic rats showed improvement in MSCs-treated group but in IPCs-treated group, β-cells insulin immunoreactions were obviously returned to normal, with normal distribution of β-cells in the center and other cells at the periphery. Meanwhile, most of the pathological lesions were still detected in diabetic rats.ConclusionMSCs transplantation can reduce blood glucose level in recipient diabetic rats. IPCs initiate endogenous pancreatic regeneration by neogenesis of islets. IPCs are better than MSCs in regeneration of β-cells. So, IPCs therapy can be considered clinically to offer a hope for patients suffering from T1D.     

Pancreatic Insulin-Producing Cells Differentiated from Human Embryonic Stem Cells Correct Hyperglycemia in SCID/NOD Mice,an Animal Model of Diabetes

Background

Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes.

Methods

We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5–7×10⁶ differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels.

Results

The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature β cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P<0.0001).

Conclusions

The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for ≥8 weeks.     

Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice

Background

Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants.

Methodology/Principal Findings

In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3–4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach.

Conclusions

h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.     

Human iPS Cell-Derived Insulin Producing Cells Form Vascularized Organoids under the Kidney Capsules of Diabetic Mice

Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.