Investigation on the pathways of embryonic stem cells differentiation into insulin-producing cells is of importance to pancreatic tissue-engineering. Instead of passing through the classic multi-step-inducing method, the expanded embryonic stem cells that were cultured and expanded in the presence of mouse embryonic fibroblast feed-layer and leukemia inhibitory factor (LIF) were induced into insulin-producing cells directly. The results showed a similar consequence from two different inducing cultures. Without passing through a so-called indispensable differentiation phase, the neural-precursor-cell-stage, the expanded embryonic stem cells could be induced into insulin-producing cells. The insulin-producing cells population resulting from our modified method were similar to that resulting from the classic multi-step method (passing through the neural-precursor-cells-stage), thus suggesting that neural-precursor-cell-phase is not the indispensable checkpoint of embryonic stem cell differentiation into insulin-producing cells. Embryonic stem cells can be induced into insulin-producing cell by classic multi-step inducing method or by direct inducing method.
Animals ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Insulin-Secreting Cells ; cytology ; Mice ; Neural Stem Cells ; cytology

Diabetes mellitus is a metabolic diseases, mainly including type 1 and type 2 diabetes. Treatment for type 1 and part of type 2 often involves regular insulin injection. However, this treatment neither precisely controls the blood sugar levels, nor prevents the diabetes complications. Transplantation of islets of Langerhans offers an attractive strategy for diabetes therapies, but its wide application has been limited by donor shortage and immunological rejection after transplantation. Stem cells with strong proliferation capacity and multipotential may be potential cell sources in diabetes therapies. For this, adult stem cells are interesting because of absence of teratoma formation and ethnical problems. Adult pancreatic stem cells (PSCs) really exist and could produce insulin-secreting cells both under the condition of pancreatic injury and in vitro culture, but lack of effective markers to enrich PSCs hampers the studies of exploring the expanding and differentiating conditions in vitro. Some other adult stem cells, such as hepatic stem cells, marrow stem cells or intestine stem cells, were also suggested to transdifferentiate into insulin-producing cells under special culture conditions in vitro or by genetic modifications. Moreover, transplanting these adult stem cells-derived insulin-secreting cells into the diabetic mouse could cure diabetes. Thus, adult stem cells would supply the abundant beta-cell sources for cell replacement therapy of diabetes.
Adult Stem Cells ; cytology ; transplantation ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Diabetes Mellitus ; therapy ; Insulin-Secreting Cells ; cytology

OBJECTIVETo investigate the effect of high glucose on mitochondrial respiratory chain function in INS-1 cells.

METHODSThe pancreatic beta cell line INS-1 was divided into the normal control (NC), high glucose (HG), and N-acetyl-L-cysteine (NAC) pretreatment groups, which were cultured for 72 h in the presence of 5.5 mmol/L glucose, 16.7 mmol/L glucose, and 16.7 mmol/L glucose with 1.0 mmol/L NAC, respectively. The activities of the enzyme complexes I and III of the respiratory chain in the cells were assessed with spectrophotometry, the ATP levels were examined using a luciferinluciferase kit, and insulin levels detected by radioimmunoassay.

RESULTSThe activities of the respiratory chain enzyme complexes I and III were 1.53-/+0.24 and 1.08-/+0.22 micromol.mg(-1).min(-1) in high glucose group, respectively, significantly lower than those in the normal control group (2.31-/+0.33 and 1.92-/+0.39 micromol.mg(-1).min(-1), P<0.01). ATP and insulin levels also decreased significantly in high glucose group as compared with those in the normal control group (P<0.01). The addition of NAC partially inhibited high glucose-induced decreases in the enzyme complex activities, ATP levels and insulin secretion (P<0.05).

CONCLUSIONThe respiratory chain function is positively correlated to insulin secretion in INS-1 cells, and exposure to high glucose causes impairment of the two enzyme complexes activities through oxidative stress, resulting in the mitochondrial respiratory chain dysfunction. High glucose-induced damages of the mitochondrial respiratory chain function can be partially inhibited by NAC.

Cell Respiration ; drug effects ; Cells, Cultured ; Glucose ; pharmacology ; Humans ; Insulin-Secreting Cells ; cytology ; physiology ; Mitochondria ; physiology ; Oxidative Stress ; drug effects

In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 microg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro.
Amnion ; cytology ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Humans ; Insulin-Secreting Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Pancreas ; physiology ; surgery ; Pancreatic Extracts ; pharmacology ; Rats ; Regeneration

In the conventional treatments of type I diabetes, there are various problems. As a new adequate treatment of diabetes, cell replacement therapy of diabetes has been applied and given research priority. We have investigated the applications of cell transplantation in the treatment of diabetes and have retrieved the relevant articles on cells transplantation for the treatment of diabetes. In this paper, we review the history, development, merits and demerits of cell transplantation and the recent advances in pancreatic islet transplantation research. The latest progress in the induction of stem cell to differentiate into the insulin-producing cells was also introduced.
Animals ; Diabetes Mellitus, Type 1 ; surgery ; therapy ; Humans ; Insulin-Secreting Cells ; cytology ; Islets of Langerhans Transplantation ; methods ; Stem Cell Transplantation

Diabetes mellitus is a complex metabolic disorder characterized by chronic hyperglycemia due to absolute or relative lack of insulin. Though great efforts have been made to investigate the pathogenesis of diabetes, the underlying mechanism behind the development of diabetes and its complications remains unexplored. Cumulative evidence has linked mitochondrial modification to the pathogenesis of diabetes and its complications and they are also observed in various tissues affected by diabetes. Proteomics is an attractive tool for the study of diabetes since it allows researchers to compare normal and diabetic samples by identifying and quantifying the differentially expressed proteins in tissues, cells or organelles. Great progress has already been made in mitochondrial proteomics to elucidate the role of mitochondria in the pathogenesis of diabetes and its complications. Further studies on the changes of mitochondrial protein specifically post-translational modifications during the diabetic state using proteomic tools, would provide more information to better understand diabetes.
Adipose Tissue ; metabolism ; Diabetes Complications ; Diabetes Mellitus ; metabolism ; pathology ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; metabolism ; Liver ; metabolism ; Mitochondria ; metabolism ; Muscle, Skeletal ; metabolism ; Proteome ; metabolism ; Proteomics

OBJECTIVETo investigate the differential potential of mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) into insulin-secreting cells and its inducing condition.

METHODSUCB nucleated cells (NCs) were isolated and cultured in Mesencult media. The obtained UCB MSC were purified by adherence method and expanded. Then they were induced with epidermal growth factor (EGF), B-mercaptoethanol and high concentration of glucose. The induced cells were identified by RT-PCR. Intracellular insulin was examined by immunocytochemistry. The quantity of insulin secretion and glucose-simulated insulin release were examined by chemiluminescence immunoassay. The induced cells were also transplanted into renal subcapsular space of STZ-induced hyperglycemic mice to observe the in vivo lowering effect on hyperglycemia.

RESULTSThe induced cells morphologically became round and were gathering into a mass. The expression of some genes related to pancreatic islet was found by RT-PCR. Chemiluminescence immunoassay showed insulin positivity and the cells secreted a low concentration of insulin [(0.37 +/- 0.06) mU/L]. The induced cells responded to high glucose challenge with a stimulation index of 1.76. After those cells grafted into renal sub-capsule there was an in vivo lowering effect on blood glucose level on STZ hyperglycemic mice.

CONCLUSIONMSCs from UCB can differentiated into insulin secreting cells.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Diabetes Mellitus, Experimental ; surgery ; Fetal Blood ; cytology ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; metabolism ; transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Mice ; Mice, Nude

BACKGROUNDPancreatic islet cell transplantation is an effective approach to treat type 1 diabetes. However, this therapy is not widely used because of the severe shortage of transplantable donor islets. This study investigated whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB) could be transdifferentiated into insulin producing cells in vitro and the role of extracellular matrix (ECM) gel in this procedure.

METHODSHuman UCB samples were collected and MSCs were isolated. MSCs specific marker proteins were analyzed by a flow cytometer. The capacities of osteoblast and adipocyte to differentiate were tested. Differentiation into islet like cell was induced by a 15-day protocol with or without ECM gel. Pancreatic characteristics were evaluated with immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Insulin content and release in response to glucose stimulation were detected with chemiluminescent immunoassay system.

RESULTSSixteen MSCs were isolated from 42 term human UCB units (38%). Human UCB-MSCs expressed MSCs specific markers and could be induced in vitro into osteoblast and adipocyte. Islet like cell clusters appeared about 9 days after pancreatic differentiation in the inducing system with ECM gel. The insulin positive cells accounted for (25.2 +/- 3.4)% of the induced cells. The induced cells expressed islet related genes and hormones, but were not very responsive to glucose challenge. When MSCs were induced without ECM gel, clusters formation and secretion of functional islet proteins could not be observed.

CONCLUSIONSHuman UCB-MSCs can differentiate into islet like cells in vitro and ECM gel plays an important role in pancreatic endocrine cell maturation and formation of three dimensional structures.

C-Peptide ; analysis ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Extracellular Matrix ; physiology ; Fetal Blood ; cytology ; Flow Cytometry ; Fluorescent Antibody Technique ; Glucagon ; analysis ; Humans ; Insulin ; analysis ; secretion ; Insulin-Secreting Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro.

METHODSThe hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS(1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups.

CONCLUSIONhAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.

Amnion ; cytology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; Flow Cytometry ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; RNA, Messenger ; genetics ; Trans-Activators ; metabolism ; beta 2-Microglobulin ; metabolism

BACKGROUNDIslet transplantation is an effective way of reversing type I diabetes. However, islet transplantation is hampered by issues such as immune rejection and shortage of donor islets. Mesenchymal stem cells can differentiate into insulin-producing cells. However, the potential of human umbilical cord mesenchymal stem cells (huMSCs) to become insulin-producing cells remains undetermined.

METHODSWe isolated and induced cultured huMSCs under islet cell culture conditions. The response of huMSCs were monitored under an inverted phase contrast microscope. Immunocytochemical and immunofluorescence staining methods were used to measure insulin and glucagon protein levels. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect gene expression of human insulin and PDX-1. Dithizone-staining was employed to determine the zinc contents in huMSCs. Insulin secretion was also evaluated through radioimmunoassay.

RESULTSHuMSCs induced by nicotinamide and β-mercaptoethanol or by neurogenic differentiation 1 gene (NeuroD1) transfection gradually changed morphology from typically elongated fibroblast-shaped cells to round cells. They had a tendency to form clusters. Immunocytochemical studies showed positive expression of human insulin and glucagon in these cells in response to induction. RT-PCR experiments found that huMSCs expressed insulin and PDX-1 genes following induction and dithizone stained the cytoplasm of huMSCs a brownish red color after induction. Insulin secretion in induced huMSCs was significantly elevated compared with the control group (t = 6.183, P < 0.05).

CONCLUSIONSHuMSCs are able to differentiate into insulin-producing cells in vitro. The potential use of huMSCs in β cell replacement therapy of diabetes needs to be studied further.

Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Cellular Reprogramming ; genetics ; physiology ; Female ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Cord ; cytology ; Wharton Jelly ; cytology