Into Insulin-producing Cells Research Articles

Vitamin D supplementation affects bone marrow-derived mesenchymal stem cells differentiation into insulin-producing cells.

Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.

The effect of diabetes mellitus on differentiation of mesenchymal stem cells into insulin-producing cells

BackgroundDiabetes mellitus (DM) is a global epidemic with increasing incidences. DM is a metabolic disease associated with chronic hyperglycemia. Aside from conventional treatments, there is no clinically approved cure for DM up till now. Differentiating mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) is a promising approach for curing DM. Our study was conducted to investigate the effect of DM on MSCs differentiation into IPCs in vivo and in vitro.MethodsWe isolated adipose-derived mesenchymal stem cells (Ad-MSCs) from the epididymal fat of normal and STZ-induced diabetic Sprague–Dawley male rats. Afterwards, the in vitro differentiation of normal-Ad-MSCs (N-Ad-MSCs) and diabetic-Ad-MSCs (DM-Ad-MSCs) into IPCs was compared morphologically then through determining the gene expression of β-cell markers including neurogenin-3 (Ngn-3), homeobox protein (Nkx6.1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin-1 (Ins-1) and eventually, through performing glucose-stimulated insulin secretion test (GSIS). Finally, the therapeutic potential of N-Ad-MSCs and DM-Ad-MSCs transplantation was compared in vivo in STZ-induced diabetic animals.ResultsOur results showed no significant difference in the characteristics of N-Ad-MSCs and DM-Ad-MSCs. However, we demonstrated a significant difference in their abilities to differentiate into IPCs in vitro morphologically in addition to β-cell markers expression, and functional assessment via GSIS test. Furthermore, the abilities of both Ad-MSCs to control hyperglycemia in diabetic rats in vivo was assessed through measuring fasting blood glucose (FBGs), body weight (BW), histopathological examination of both pancreas and liver and immunoexpression of insulin in pancreata of study groups.ConclusionOur findings reveal the effectiveness of N-Ad-MSCs in differentiating into IPCs in vitro and controlling the hyperglycemia of STZ-induced diabetic rats in vivo compared to DM-Ad-MSCs.Graphical

Silencing of forkhead box protein O-1 (FOXO-1) enhances insulin-producing cell generation from adipose mesenchymal stem cells for diabetes therapy

AimsGeneration of mature β-cells from MSCs has been a challenge in the field of stem cell therapy of diabetes. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) have made their mark in regenerative medicine, and provide several advantages compared to other MSCs sources. Forkhead box protein O-1 (FOXO-1) is an important transcription factor for normal development of β-cells, yet its over expression in β-cells may cause glucose intolerance. In this study, we isolated, characterized Ad-MSCs from rat epididymal fat pads, differentiated these MSCs into insulin producing cells (IPCs) and studied the role of FOXO-1 in such differentiation. Materials and methodsWe examined the expression of FOXO-1 and its nuclear cytoplasmic localization in the generated IPCs. Afterwards we knocked down FOXO-1 using siRNA targeting FOXO-1 (siFOXO-1). The differentiated siFOXO-1 IPCs were compared to non-targeting siRNA (siNT) IPCs regarding expression of β-cell markers by qRT-PCR and western blotting, dithizone (DTZ) staining and glucose stimulated insulin secretion (GSIS). Key findingsIsolated Ad-MSCs exhibited all characteristics of MSCs and can generate IPCs. FOXO-1 was initially elevated during differentiation followed by a decline towards end of differentiation. FOXO-1 was dephosphorylated and localized to the nucleus upon differentiation into IPCs. Knock down of FOXO-1 improved the expression of β-cell markers in final differentiated IPCs, improved DTZ uptake and showed increased insulin secretion upon challenging with increased glucose concentration. SignificanceThese results portray FOXO-1 as a hindering factor of generation of IPCs whose down-regulation can generate more mature IPCs for MSCs therapy of diabetes mellitus.

Dysregulated lncRNAs regulate human umbilical cord mesenchymal stem cell differentiation into insulin-producing cells by forming a regulatory network with mRNAs

ObjectiveIn recent years, cell therapy has emerged as a new research direction in the treatment of diabetes. However, the underlying molecular mechanisms of mesenchymal stem cell (MSC) differentiation necessary to form such treatment have not been clarified.MethodsIn this study, human umbilical cord mesenchymal stem cells (HUC-MSCs) isolated from newborns were progressively induced into insulin-producing cells (IPCs) using small molecules. HUC-MSC (S0) and four induced stage (S1–S4) samples were prepared. We then performed transcriptome sequencing experiments to obtain the dynamic expression profiles of both mRNAs and long noncoding RNAs (lncRNAs).ResultsWe found that the number of differentially expressed lncRNAs and mRNAs trended downwards during differentiation. Gene Ontology (GO) analysis showed that the target genes of differentially expressed lncRNAs were associated with translation, cell adhesion, and cell connection. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the NF-KB signalling pathway, MAPK signalling pathway, HIPPO signalling pathway, PI3K–Akt signalling pathway, and p53 signalling pathway were enriched in these differentially expressed lncRNA-targeting genes. We also found that the coexpression of the lncRNA CTBP1-AS2 with PROX1 and the lncRNAs AC009014.3 and GS1-72M22.1 with JARID2 mRNA was related to the development of pancreatic beta cells. Moreover, the coexpression of the lncRNAs: XLOC_ 050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14- AS1, RP11-148K1.12, and CTD2020K17.3 with p53, regulated insulin secretion by pancreatic beta cells.ConclusionIn this study, HUC-MSCs combined with small molecule compounds were successfully induced into IPCs. Differentially expressed lncRNAs may regulate the insulin secretion of pancreatic beta cells by regulating multiple signalling pathways. The lncRNAs AC009014.3, Gs1-72m21.1, and CTBP1-AS2 may be involved in the development of pancreatic beta cells, and the lncRNAs: XLOC_050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14-AS1, RP11-148K1.12, and CTD2020K17.3 may be involved in regulating the insulin secretion of pancreatic beta cells, thus providing a lncRNA catalogue for future research regarding the mechanism of the transdifferentiation of HUC-MSCs into IPCs. It also provides a new theoretical basis for the transplantation of insulin-producing cells into diabetic patients in the future.Graphical

Simultaneous Administration of Berberine and Transplantation of Endometrial Stem Cell-Derived Insulin Precursor Cells on a Nanofibrous Scaffold to Treat Diabetes Mellitus in Mice.

Today, significant success has been achieved in treating diabetes with cell therapy derived from various sources of stem and progenitors. The replacement of beta cells is one of the new diabetes treatment methods. To this end, the production of pancreatic beta precursors in cell culture has created an important research field for diabetes treatment. Endometrial stem cells were isolated using an enzymatic method, and after their identity was confirmed using a flow cytometry and differentiation potential assay, the isolated cells were cultured on an electrospun PCL/CS scaffold. Endometrial cells were differentiated into insulin-producing cells (IPCs), and gene expression was analyzed using the qRT-PCR and immunofluorescence to confirm the creation of IPCs. Then, IPCs on the scaffold along with berberine were applied to 5 groups of diabetic mice, and after 6 weeks, insulin, blood glucose, and weight of the animals were measured. The findings revealed that pancreatic markers were significantly expressed in IPCs compared to control cells. In addition, when compared to the control group and scaffolds, the receiving group of IPCs on scaffolds had a significant improvement (p ≤ 0.0015), and this improvement increased with the addition of berberine (decrease in blood sugar (133 mg/dL), and an increase in weight (5/39 g) and insulin (2.29 MIU/L). Thus, tissue engineering is a promising new strategy for treating diabetes and can be used in the future for cell therapy and suitable drugs for diabetic patients.

Transplantation of insulin-producing cells derived from human MSCs to treat diabetes in a non-human primate model.

Islet cell transplantation is an emerging therapy in the treatment of diabetes mellitus. Differentiation of islet cells from mesenchymal stem cells (MSCs) is a potential solution to the challenge of insufficient donor sources. This study investigated whether human umbilical cord-derived MSCs could effectively differentiate into insulin-producing cells (IPCs) and evaluated the therapeutic efficacy of IPCs in treating diabetes. IPCs were induced from MSCs by a two-step protocol. IPC expression products were evaluated by western blot and real-time PCR. IPC insulin secretion was evaluated by ELISA. The viability of IPCs was measured by FDA/PI and dithizone staining. The non-human primate tree shrew was used as a diabetes model. After a single STZ induction into a diabetes model, a single intraportal transplantation of IPCs, MSCs, or normal saline was performed (n = 6 per group). Blood glucose was monitored for 3 weeks, then the animals were euthanized and the distribution of IPCs in the liver was examined pathologically. After about 3 weeks of in vitro induction, IPCs formed microspheres of 100-200 μm, with >95% viable cells that were dithizone stain positive. IPCs expressed islet-related genes and proteins and secreted high levels of insulin whether stimulated by low or high levels of glucose. After transplantation of IPCs into diabetic tree shrews, blood glucose levels decreased rapidly to near normal and were significantly lower than the MSC or saline groups for 3 weeks thereafter. We present the novel discovery that IPCs derived from human umbilical cord MSCs exert a therapeutic effect in a non-human primate model of diabetes. This study provides a preliminary experimental basis for the use of autologous MSC-derived IPCs in the treatment of human diabetes.

The role of the adipose tissue-derived mesenchymal stem cells enriched with melatonin in pancreatic cellular regeneration.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs) were proved to differentiate into insulin-producing cells (IPCs), but the amount of insulin secreted was relatively low compared to the insulin secreted by mature pancreatic islets. Enrichment of MSCs culture with melatonin (MT) was found to promote cartilage matrix synthesis, osteogenic and neuronal differentiation. Therefore, the present study was conducted to evaluate the potential role of MT pre-treated AD-MSCs in enhancing the treatment and regeneration of the islet cells of Langerhans in rats with diabetes induced by streptozotocin (STZ). Forty adult male Sprague Dawley albino rats were divided equally into groups; group I (control group), group II (STZ group), group III (STZ + AD-MSCs) and group IV (STZ+MT pre-treated AD-MSCs). Biochemical studies were implemented including measurements of the body weight, fasting blood glucose and serum insulin levels, Interleukin 17 (IL-17) and IL-10. Samples of the pancreas were taken and prepared for light, fluorescent microscopic examination, proliferating cell nuclear antigen and caspase-3 immunohistochemical studies and histomorphometric analysis. The present study confirmed the regenerative and therapeutic effects of AD-MSCs on the pancreatic cells. Concomitant supply of MT to the culture of AD-MSCs, in group IV, was shown to retain the normal architecture of the islet cells of Langerhans. They appeared well-defined and lightly stained, surrounded by classical pancreatic acini and contained a large number of islet cells with vesicular nuclei and prominent nucleoli. Improvement of all the biochemical parameters, in the same group, was demonstrated by increased body weight and serum insulin levels with a decrease in the fasting blood glucose levels. Significant decrease in the pro-inflammatory cytokine; IL-17 and increase in the anti-inflammatory cytokine; IL-10, compared to the STZ group, were also discovered. Significant increase in the proliferating cell nuclear antigen proliferation index, decrease in caspase-3 and increase in PKH26 labelled MSCs area per cent was recorded in the group of AD-MSCs enriched with MT compared to the group of AD-MSCs without MT. The present study confirmed the potential therapeutic and protective role of MT pre-treated AD-MSCs against the STZ-induced pancreatic islet cells damage. Further studies are recommended to investigate the efficacy of MT and AD-MSCs over longer experimental durations.

Differentiation of the mesenchymal stem cells to pancreatic β-like cells in alginate/trimethyl chitosan/alginate microcapsules

Cell therapy is one of the proposed treatments for diabetes. Cell encapsulation and differentiation inside the biodegradable polymers overcome the limitations such as islet deficiency and the host immune responses. This study was set to encapsulate the mesenchymal stem cells (MSCs) and differentiate them into insulin-producing cells (IPCs). Human bone marrow-mesenchymal stem cells (hBM-MSCs) were encapsulated in alginate/trimethyl chitosan/alginate (Alg/TMC/Alg) coating. At first, morphology and swelling properties of the cell-free microcapsules were investigated. Next, a three-step protocol was used in the presence of exendin-4 and nicotinamide to differentiate hBM-MSCs into IPCs. Viability of the encapsulated cells was investigated using MTT assay. The differentiated cells were analyzed using a real-time RT-PCR to investigate Glut-2, Insulin, Pdx-1, Ngn-3, nestin, and Isl-1 gene expression. The results revealed that differentiation of the encapsulated cells was higher than non-encapsulated cells. Also, dithizone staining in two-dimensional (2D) environment showed the differentiated cell clusters. In summary, here, hBM-MSCs after encapsulation in Alg/TMC/Alg microcapsules, as a new design, were differentiated properly in the presence of exendin-4 and nicotinamide as main inducers. A three-dimensional (3D)matrix is more similar to the native ECM in the body and prepares higher cell-cell contacts.

TAZ promotes PDX1-mediated insulinogenesis.

Transcriptional co-activator with PDZ-binding motif (TAZ) is a key mediator of the Hippo signaling pathway and regulates structural and functional homeostasis in various tissues. TAZ activation is associated with the development of pancreatic cancer in humans, but it is unclear whether TAZ directly affects the structure and function of the pancreas. So we sought to identify the TAZ function in the normal pancreas. TAZ defect caused structural changes in the pancreas, particularly islet cell shrinkage and decreased insulin production and β-cell markers expression, leading to hyperglycemia. Interestingly, TAZ physically interacted with the pancreatic and duodenal homeobox 1 (PDX1), a key insulin transcription factor, through the N-terminal domain of TAZ and the homeodomain of PDX1. TAZ deficiency decreased the DNA-binding and transcriptional activity of PDX1, whereas TAZ overexpression promoted PDX1 activity and increased insulin production even in a low glucose environment. Indeed, high glucose increased insulin production by turning off the Hippo pathway and inducing TAZ activation in pancreatic β-cells. Ectopic TAZ overexpression along with PDX1 activation was sufficient to produce insulin in non-β-cells. TAZ deficiency impaired the mesenchymal stem cell differentiation into insulin-producing cells (IPCs), whereas TAZ recovery restored normal IPCs differentiation. Compared to WT control, body weight increased in TAZ-deficient mice with age and even more with a high-fat diet (HFD). TAZ deficiency significantly exacerbated HFD-induced glucose intolerance and insulin resistance. Therefore, TAZ deficiency impaired pancreatic insulin production, causing hyperglycemia and exacerbating HFD-induced insulin resistance, indicating that TAZ may have a beneficial effect in treating insulin deficiency in diabetes.

A One-Step, Monolayer Culture and Chemical-Based Approach to Generate Insulin-Producing Cells From Human Adipose-Derived Stem Cells to Mitigate Hyperglycemia in STZ-Induced Diabetic Rats

The global population of individuals afflicted with diabetes mellitus has been increasing year by year, and this disease poses a serious threat to human health as well as the economies worldwide. Pancreatic or islet transplantations provide one of the most effective and long-term therapies available to treat diabetes, but the scarcity and quality of pancreatic islets limit their use in treatments. Here, we report the development of a one-step, monolayer culture, and chemical-based protocol that efficiently mediates the differentiation of human adipose-derived stem cells (hADSCs) into insulin-producing cells (IPCs). Our data indicate that hADSCs in monolayer culture that are allowed to differentiate into IPCs are superior to those in suspension cultures with respect to insulin secretion capacity (213-fold increase), cell viability (93.5 ± 3.27% vs. 41.67 ± 13.17%), and response to glucose stimulation. Moreover, the expression of genes associated with pancreatic lineage specification, such as PDX1, ISL1, and INS (encoding insulin), were expressed at significantly higher levels during our differentiation protocol (6-fold for PDX1 and ISL1, 11.5-fold for INS). Importantly, in vivo studies demonstrated that transplantation with IPCs significantly mitigated hyperglycemia in streptozotocin-induced diabetic rats. Our results indicate that this one-step, rapid protocol increases the efficiency of IPC generation and that the chemical-based approach for IPC induction may reduce safety concerns associated with the use of IPCs for clinical applications, thereby providing a safe and effective cell-based treatment for diabetes.

The Roles of Different Multigene Combinations of Pdx1, Ngn3, Sox9, Pax4, and Nkx2.2 in the Reprogramming of Canine ADSCs Into IPCs

Adipose-derived mesenchymal stem cells (ADSCs) are ideal sources for the treatment of diabetes, and the differentiation of ADSCs into insulin-producing cells (IPCs) through transfection of exogenous regulatory genes in vitro has been studied in depth. The differentiation of ADSCs is strictly regulated by a variety of transcription factors such as Pdx1, Ngn3, Pax4, Nkx2.2, and Sox9. However, whether these genes can coordinately regulate the differentiation of ADSCs into IPCs is still unknown. In this study, five multigene coexpressing adenovirus vectors (pAdTrack-Pdx1-Ngn3-AdEasy, pAdTrack-Pdx1-Ngn3-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Pax4-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Nkx2.2-Sox9-AdEasy, and pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) were constructed, and then the stocks of the packaged adenoviruses were used to infect the canine ADSCs (cADSCs). Based on results of morphological observation, dithizone staining, sugar-stimulated insulin secretion test, cellular insulin immunofluorescence assays, and the detection of pancreatic β-cell development–related genes in the induced cells, the best induction combination (pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) was identified after comparative screening. This study provides a theoretical reference and an experimental basis for further research on stem cell replacement therapy for diabetes.

Exosomes from β-Cells Promote Differentiation of Induced Pluripotent Stem Cells into Insulin-Producing Cells Through microRNA-Dependent Mechanisms.

ObjectiveExosomes have emerged as potential tools for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells (IPCs). Exosomal microRNAs are receiving increasing attention in this process. Here, we aimed at investigating the role of exosomes derived from a murine pancreatic β-cell line and identifying signature exosomal miRNAs on iPSCs differentiation.MethodsExosomes were isolated from MIN6 cells and identified with TEM, NTA and Western blot. PKH67 tracer and transwell assay were used to confirm exosome delivery into iPSCs. qRT-PCR was applied to detect key pancreatic transcription gene expression and exosome-derived miRNA expression. Insulin secretion was determined using FCM and immunofluorescence. The specific exosomal miRNAs were determined via RNA-interference of Ago2. The therapeutic effect of 21 day-exosome-induced IPCs was validated in T1D mice induced by STZ.ResultsiPSCs cultured in medium containing exosomes showed sustained higher expression of MAFA, Insulin1, Insulin2, Isl1, Neuroud1, Nkx6.1 and NGN3 compared to control iPSCs. In FCM analysis, approximately 52.7% of the differentiated cells displayed insulin expression at the middle stage. Consistent with the gene expression data, immunofluorescence assays showed that Nkx6.1 and insulin expression in iPSCs were significantly upregulated. Intriguingly, the expression of pancreatic markers and insulin was significantly decreased in iPSCs cultured with siAgo2 exosomes. Transplantation of 21 day-induced IPCs intoT1D mice efficiently enhanced glucose tolerance and partially controlled hyperglycemia. The therapeutic effect was significantly attenuated in T1D mice that received iPSCs cultured with siAgo2 exosomes. Of the seven exosomal microRNAs selected for validation, miR-706, miR-709, miR-466c-5p, and miR-423-5p showed dynamic expression during 21 days in culture.ConclusionThese data indicate that differentiation of exosome-induced iPSCs into functional cells is crucially dependent on the specific miRNAs encased within exosomes, whose functional analysis is likely to provide insight into novel regulatory mechanisms governing iPSCs differentiation into IPCs.

Direct Reprogramming of Mice Skin Fibroblasts into Insulin-Producing Cells In Vitro.

Transdifferentiation means mature cell conversion into other mature cells. Ethical issues, epigenetic failure, or teratoma development are found in cellular reprogramming strategies. Thus, new methods are needed. This study aimed to develop a new novel formula of chemical molecules and growth factors that differentiate skin fibroblasts into insulin-producing cells (IPCs). Newborn mice fibroblasts differentiated using four induction methods into IPCs to search for the best method. Fibroblasts, stem cells, and pancreatic markers were identified using an immunocytochemistry (ICC) assay. Insulin was measured using ELISA and dithizone (DTZ) assays. The skin fibroblasts were induced successfully into IPCs. The best method to obtain IPCs was indicated by measuring insulin concentration in differentiated cell supernatant from all induced cells by the four methods. The protein expression of the pancreatic markers of induced cells increased with time, as indicated by the ICC assay. OCT3/4 increased on day 9, after which the expression tended to decrease. DTZ-positive clusters were observed on day 16. Secreted insulin of differentiated cells was injected in streptozotocin-induced diabetic mice, which decreased blood glucose levels after injection. This study indicated an efficient new chemical method for transdifferentiating skin fibroblasts into functional IPCs, which is a promising method for diabetes mellitus therapy.

Novel Functional Genes Involved in Transdifferentiation of Canine ADMSCs Into Insulin-Producing Cells, as Determined by Absolute Quantitative Transcriptome Sequencing Analysis.

The transdifferentiation of adipose-derived mesenchymal stem cells (ADMSCs) into insulin-producing cells (IPCs) is a potential resource for the treatment of diabetes. However, the changes of genes and metabolic pathways on the transdifferentiation of ADMSCs into IPCs are largely unknown. In this study, the transdifferentiation of canine ADMSCs into IPCs was completed using five types of procedures. Absolute Quantitative Transcriptome Sequencing Analysis was performed at different stages of the optimal procedure. A total of 60,151 transcripts were obtained. Differentially expressed genes (DEGs) were divided into five groups: IPC1 vs. ADSC (1169 upregulated genes and 1377 downregulated genes), IPC2 vs. IPC1 (1323 upregulated genes and 803 downregulated genes), IPC3 vs. IPC2 (722 upregulated genes and 680 downregulated genes), IPC4 vs. IPC3 (539 upregulated genes and 1561 downregulated genes), and Beta_cell vs. IPC4 (2816 upregulated genes and 4571 downregulated genes). The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs revealed that many genes and signaling pathways that are essential for transdifferentiation. Hnf1B, Dll1, Pbx1, Rfx3, and Foxa1 were screened out, and the functions of five genes were verified further by overexpression and silence. Foxa1, Pbx1, and Rfx3 exhibited significant effects, can be used as specific key regulatory factors in the transdifferentiation of ADMSCs into IPCs. This study provides a foundation for future work to understand the mechanisms of the transdifferentiation of ADMSCs into IPCs and acquire IPCs with high maturity.

Mesenchymal Stem Cells: An Excellent Candidate for the Treatment of Diabetes Mellitus

Mesenchymal stem cells (MSCs) are adult stem cells (ASCs) known for repairing damaged cells, exerting anti-inflammatory responses and producing immunoregulatory effects that can be significantly induced into insulin-producing cells (IPCs), providing an inexhaustible supply of functional β cells for cell replacement therapy and disease modeling for diabetes. MSC therapy may be the most promising strategy for diabetes mellitus because of these significant merits. In this paper, we focused on MSC therapy for diabetes.

MiR-375 Promotes Pancreatic Differentiation In Vitro by Affecting Different Target Genes at Different Stages.

Human embryonic stem cells (hESCs) possess the ability to differentiate into insulin-producing cells (IPCs), which can be used to treat type I diabetes. miR-375 is an essential transcriptional regulator in the development and maturation of the pancreas. In this study, we optimized a protocol to differentiate hESCs into IPCs and successfully obtained IPCs. Then, we performed overexpression and inhibition experiments of miR-375 on cells at different stages of differentiation and performed RNA-seq. The results showed that the expression of miR-375 fluctuated during hESC differentiation and was affected by miR-375 mimics and inhibitors. miR-375 influences global gene expression and the target genes of miR-375. The overexpression of miR-375 can cause changes in multiple signaling pathways during pancreatic development. miR-375 is a major participant in the differentiation of pancreatic β-cells through different target genes at different stages. This study provides new ideas for further investigation of how microRNAs affect cell fate and gene transcription.

Insulin Producing Cells from Adipose Mesenchymal Stromal Origin as Implant Strategy in Diabetic Rats

Type 1 diabetes mellitus (DM1) is an autoimmune disease characterized by the destruction of insulin-producing pancreatic β cells. Among the current experimental therapies, we highlight the use of transdifferentiated mesenchymal stromal cells (MSCs) into insulin-producing cells (IPCs). Here, we studied the strategy of implanting IPCs differentiated from adipose tissue MSCs in the subcutaneous region (SC) of rats to investigate their protection against hyperglycemia induced by streptozotocin. The first phase of this study (in vitro) was to characterize adipose-derived stromal cells (ADSCs) and to evaluate the protocol for their differentiation in IPCs. The second phase was to assess the functionality of IPCs in vivo three and eight weeks after implant. The cells were then implanted in SC after one-week DM1 model induction (Process: 30626). The implant improved hyperglycemia and reduced the serum content of advanced glycation end products (AGEs) in diabetic rats at three weeks, but this effect was not observed after the longer period of eight weeks, showing a transient effect. Serum C-peptide was not detected in the SC group, neither after three nor eight weeks of DM1 model. Regarding animal body weight, both diabetic and implanted rats maintained their weight over time, while the Sham group presented the natural increase related to the normal growth of the animal. Together, these data are promising but also need improvement in the effectiveness of the therapy, with future studies attempting implantation of a larger number of cells or multiple implants, since the subcutaneous access is not very invasive and easy to perform.

Generation of Insulin-Producing Cells from Canine Adipose Tissue-Derived Mesenchymal Stem Cells.

The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide μ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.

Genome-Wide Analysis Reveals Changes in Long Noncoding RNAs in the Differentiation of Canine BMSCs into Insulin-Producing Cells.

Long noncoding RNAs (lncRNAs) have been extensively explored over the past decade, including mice and humans. However, their impact on the transdifferentiation of canine bone marrow mesenchymal stem cells (cBMSCs) into insulin-producing cells (IPCs) is largely unknown. In this study, we used a three-step induction procedure to induce cBMSCs into IPCs, and samples (two biological replicates each) were obtained after each step; the samples consisted of “BMSCs” (B), “stage 1” (S1), “stage 2” (S2), “stage 3” (S3), and “islets” (I). After sequencing, 15,091 lncRNAs were identified, and we screened 110, 41, 23, and 686 differentially expressed lncRNAs (padjusted < 0.05) in B vs. S1, S1 vs. S2, S2 vs. S3, and I vs. S3 pairwise comparisons, respectively. In lncRNA target prediction, there were 166,623 colocalized targets and 2,976,362 correlated targets. Gene Ontology (GO) analysis showed that binding represented the main molecular functions of both the cis- and trans-modes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the insulin signaling pathway, Rap1 signaling pathway, tight junctions, MAPK signaling pathway, and cell cycle were enriched for these relative genes. The expression of lncRNAs was verified using qRT-PCR. This study provides a lncRNA catalog for future research concerning the mechanism of the transdifferentiation of cBMSCs into IPCs.

Control of Hyperglycemia Using Differentiated and Undifferentiated Mesenchymal Stem Cells in Rats with Type 1 Diabetes

Due to their ability in self-renewing and differentiation into a wide variety of tissues, mesenchymal stem cells (MSCs) exhibit outstanding potential for regenerative medicine. This study was aimed at investigating different aspects of MSC therapy in controlling hyperglycemia in streptozotocin-induced diabetes rats. Using an islet cell differentiation protocol, bone marrow (BM) MSCs were differentiated into insulin-producing cells (IPCs). The differentiation process was evaluated by immunocytochemistry, reverse transcriptase PCR, and dithizone staining. Diabetic animals in 4 diabetic individual groups received normal saline, BM-MSCs, coadministration of BM-MSCs with supernatant, and IPCs. Blood glucose and insulin levels were monitored during the experiment. Immunohistochemical analysis of the pancreas was performed at the end of the experiment. Administration of BM-MSCs could not reverse glucose and insulin levels in experimental animals as efficiently as cotransplantation of BM-MSCs with supernatant. The effect of coadministration of BM-MSCs with supernatant and transplantation of IPCs on controlling hyperglycemia is comparable. Immunohistochemical analysis showed that number and size of islets per section were significantly increased in groups receiving IPCs and BM-MSC-supernatant compared to the MSC group of animals. In conclusion, coadministration of BM-MSCs with supernatant could be used as efficiently as IPC transplantation in controlling hyperglycemia in diabetic rats.