OBJECTIVETo understand and improve the transdifferentiation of pancreatic stem cells into islet cells through isolation, cultivation and transdifferentiation of adult human pancreatic duct cells.

METHODSA portion of adult human pancreas was digested with collagenase, followed by incontinuous density gradient to separate islets from the acinar and ductal tissue. Duct epithelial cells were cultivated in CMRL1066 and then in serum-free DMEM/F12 medium with the addition of growth factors for 27 days. Samples were taken at different time points for light and electron microscopic examination and for immunocytochemical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19. Amylase and insulin contents in the medium were assayed.

RESULTSA large number of duct epithelial cells were harvested after the isolation of islets. Some duct epithelial cells were PDX-1 and CK-19 positive at day one and duct epithelial cells proliferated and expanded rapidly and then transdifferentiated into stem cells and finally 3D islets. 760 islets were harvested from each gram of pancreatic tissue on day 27.

CONCLUSIONSAdult pancreatic duct cells are potential stem cells and could be transdifferentiated into islet cells in vitro under appropriate conditions.

Adult ; Cell Differentiation ; physiology ; Humans ; Islets of Langerhans ; cytology ; Pancreas ; cytology ; Stem Cells ; cytology

BACKGROUNDGlobally, 180 million people suffer from diabetes mellitus. Islet transplantation is believed to be an almost ideal therapy for insulin-dependent patients. How to maintain the viability and the function of isolated human islets is a challenge in clinical practice. Sertoli cells are considered 'nurse cells' in the seminiferous tubules and have been used in cell graft protocols for neurodegenerative diseases and diabetes in many studies. Many researchers have used immature murine testes as the primarily source of Sertoli cells in islet transplantation because they are easily purified. Mature human Sertoli cells have been seldom investigated. In the present study, we developed a method for the isolation and culture of Sertoli cells derived from adult human testes, and investigated their effects on the function of allogeneic islets when they were cultured together in vitro.

METHODSAdult Sertoli cells were prepared successfully by two-step enzyme digestion with trypsin, collagenase and hyaluronidase. They were identified by morphological characteristics and their activity was determined by MTT colorimetry over a 28-day culture time in vitro. A glucose-stimulated insulin secretion test was performed to detect the effects of Sertoli cells on allogeneic islets' function when they were co-cultured for 21 days in vitro.

RESULTSIn cultured cells, mature human Sertoli cells accounted for more than 90% of total cells. The activity of Sertoli cells reached 95% and they remained highly cytoactive for a long time in vitro (P > 0.05). Compared with the islets cultured alone, the co-cultured islets with allogeneic Sertoli cells maintained higher sensitivity to glucose stimulation for the duration of the experiment (P < 0.01).

CONCLUSIONSA method of isolation and culture of Sertoli cells from adult testes has been established. Sertoli cells could enhance allogeneic islets' function when they were co-cultured in vitro. They could be a helper cell in islet transplantation.

Adult ; Cell Separation ; methods ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Humans ; Islets of Langerhans ; physiology ; Islets of Langerhans Transplantation ; Male ; Sertoli Cells ; cytology ; physiology

OBJECTIVETo isolate human amniotic mesenchymal cells (hAMCs) and investigate their transdifferentiation ability into islet-like cells in vitro.

METHODSHuman amnion was treated with the trypsin/EDTA to remove the amniotic epithelial cells and then incubated with collagenase I and dispase at 37 degrees celsius; overnight. The cells were collected by centrifugation and identified for the expressions of vimentin and SSEA-4 using immunofluorescence assay and for CD29, CD90, CD34, and CD45 using flow cytometry. RT-PCR was performed to detect the expressions of ACTG2, ACTA2, MMP2, Cripto, Sox2, LEFTYA, nanog, and Oct-4 in the cells. The differentiation potential of the isolated cells into inslet-like cells was assessed after a 14-day induction with the inducing factors by RT-PCR and immunofluorescence assay.

RESULTSThe hAMCs were capable of in vitro proliferation and passaging for 10 passages while retaining the normal karyotype. The isolated cells were positive for staining of vimentin and SSEA-4 and negative for CD34 and CD45; the CD29 and CD90 cells accounted for (91.5∓9.93)% and (48.7∓9.47)% of the cells, respectively. The hAMCs expressed several pluripotency-related genes, including Cripto, Sox2, LEFTYA, nanog, and Oct-4. After induction, endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon.

CONCLUSIONWe have successfully established the method for isolating hAMCs, which possess the potential of differentiation into islet-like cells in vitro.

Amnion ; cytology ; Cell Culture Techniques ; methods ; Cell Transdifferentiation ; physiology ; Cells, Cultured ; Female ; Humans ; Islets of Langerhans ; cytology ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo obtain massive human pancreatic islets with modified techniques and evaluation of the islets for the clinical allo-transplantation to treat type I and II diabetes.

METHODS28 consecutive adult human pancreata were isolated with modified automated techniques. Islets were purified using continuous density gradient. The islet yield was counted with international standard known as islet equivalent (IEQ). The function of the isolated islets was evaluated by measuring DNA/insulin ratio, static glucose stimulating test in vitro and transplanting the islets into diabetic nude mice in vivo followed by abdominal glucose tolerance test and C peptide measurement.

RESULTSThe yield of 28 consecutive human pancreata isolations ranged from 5 000 to 1 030 000 IEQs/pancreas with the average of 291 635 IEQs/pancreas. The first 13 isolations yielded 49 123 IEQs/pancreas, 846 IEQs/g and, purity 87% in average. The remained 15 isolations after the modifications yielded 501 813 IEQs/pancreas, 7 003 IEQs/g and purity 89% in average. The results of in vitro SGS showed good response to the different glucose concentration. 34 diabetic nude mice were transplanted under the renal capsule with the freshly isolated islets. 29 out of 34 diabetic mice obtained normoglycemia within 12 hours and the glucose tolerance tests were near normal. Serum C peptide level of transplanted mice is close to that of the control group.

CONCLUSIONSMassive human islets can be isolated with the modified techniques. Quality assessment of these islets both in vitro and in vivo has indicated that these high quality human islets could be used for the clinical allogeneic islet transplantation.

Adult ; Animals ; Cell Separation ; methods ; Diabetes Mellitus, Experimental ; surgery ; Glucose ; Humans ; In Vitro Techniques ; Islets of Langerhans ; cytology ; drug effects ; physiology ; Islets of Langerhans Transplantation ; Mice ; Mice, Nude ; Transplantation, Heterologous

OBJECTIVETo investigate the feasibility and benefits of co-culture of cryopreserved islets with small intestinal submucosa (SIS).

METHODSPurified rat islets cryopreserved for one month were divided into SIS group and control group, and after culture in standard islet culture media RPMI1640 for 1 week, the morphology and function of the islets were assessed.

RESULTSThe SIS protects the fragile islets from damage by cryopreservation, and increased the recovery from (60.6-/+3.3)% to (91.7-/+1.8) % (P<0.05). Compared with the control group, incubation of the islets of the SIS group in high-glucose (16.7 mmol/L) solution resulted in significantly enhanced insulin secretion (23.7-/+1.6 vs 12.5-/+1.1 mU/L, P<0.05). When the islets were incubated in high-glucose solution containing theophylline, the calculated stimulation index of SIS group was about 3-fold higher than that of the control group.

CONCLUSIONCo-culture of cryopreserved rat islets with SIS can increase the recovery of islet cells and improve their function.

Animals ; Coculture Techniques ; Cryopreservation ; methods ; Glucose ; pharmacology ; Insulin ; secretion ; Intestinal Mucosa ; cytology ; drug effects ; physiology ; Intestine, Small ; cytology ; drug effects ; physiology ; Islets of Langerhans ; cytology ; drug effects ; physiology ; Male ; Rats ; Rats, Wistar ; Theophylline ; pharmacology

OBJECTIVETo explore the mechanisms of differentiation and development of pancreatic endocrine cells as well as pancreatic regeneration.

METHODSHuman embryonic pancreatic tissue at 7-14 weeks of gestation was collected. Diabetes mellitus rat model was induced with 65 mg/kg of streptozotocin. Insulin, glucagon, somatostatin, nestin, and cytokeratin 19 (CK19) of pancreatic tissues were observed by immunohistochemistry.

RESULTSAt 9 weeks of gestation, pancreatic epithelial cells began to co-express insulin, glucagon, somatostatin, and CK19 before migration. Islet cells gradually congregated along with the increase of aging, and at 14 weeks of gestation histological examination showed islet formation. At 12 weeks of gestation, nestin-positive cells could be seen in the pancreatic mesenchyme. During early embryogenesis, islet cells of pancreatic ducts co-expressed insulin, glucagon, and somatostatin. During pancreatic regeneration after damage, nestin expression of islet cells increased.

CONCLUSIONIn the early stage of embryogenesis, islet cells of primary pancreatic ducts can be differentiated to multipotential endocrine cells before migration. During tissue regeneration, pancreatic stem cells may differentiate and proliferate to form pancreatic islet.

Animals ; Cell Differentiation ; Diabetes Mellitus, Experimental ; chemically induced ; metabolism ; pathology ; Embryonic Development ; physiology ; Epithelial Cells ; cytology ; physiology ; Humans ; Insulin-Secreting Cells ; cytology ; physiology ; Islets of Langerhans ; cytology ; physiology ; Male ; Pancreas ; cytology ; embryology ; physiology ; Pancreatic Ducts ; cytology ; embryology ; physiology ; Rats ; Rats, Sprague-Dawley ; Regeneration ; physiology ; Stem Cells ; cytology ; metabolism ; physiology

OBJECTIVEInterferon-γ (IFN-γ) plays an important role in apoptosis and was shown to increase the risk of diabetes. Visfatin, an adipokine, has anti-diabetic, anti-tumor, and regulating inflammatory properties. In this study we investigated the effect of visfatin on IFN-γ-induced apoptosis in rat pancreatic β-cells.

METHODSThe RINm5F (rat insulinoma cell line) cells exposed to IFN-γ were treated with or without visfatin. The viability and apoptosis of the cells were assessed by using MTT and flow cytometry. The expressions of mRNA and protein were detected by using real-time PCR and western blot analysis.

RESULTSThe exposure of RINm5F cells to IFN-γ for 48 h led to increased apoptosis percentage of the cells. Visfatin pretreatment significantly increased the cell viability and reduced the cell apoptosis induced by IFN-γ. IFN-γ-induced increase in expression of p53 mRNA and cytochrome c protein, decrease in mRNA and protein levels of anti-apoptotic protein Bcl-2 were attenuated by visfatin pretreatment. Visfatin also increased AMPK and ERK1/2 phosphorylation and the anti-apoptotic action of visfatin was attenuated by the AMPK and ERK1/2 inhibitor.

CONCLUSIONThese results suggested that visfatin protected pancreatic islet cells against IFN-γ-induced apoptosis via mitochondria-dependent apoptotic pathway. The anti-apoptotic action of visfatin is mediated by activation of AMPK and ERK1/2 signaling molecules.

Adenylate Kinase ; metabolism ; Animals ; Apoptosis ; physiology ; Cell Line ; Cytokines ; physiology ; Flow Cytometry ; Interferon-gamma ; physiology ; Islets of Langerhans ; cytology ; MAP Kinase Signaling System ; Nicotinamide Phosphoribosyltransferase ; physiology ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction

Dysfunction of the pancreatic beta-cell is an important defect in the pathophysiological changes of type 2 diabetes, and type 2 diabetes is evidently associated with obesity. But the role of the adipocyte in the dysfunction of the pancreatic beta-cell remains unknown. In the present study, we examined the direct effects of 3T3-L1 adipocytes on the expression of ATP-sensitive potassium channels (K(ATP) channels) in MIN6 insulin-secreting cells. MIN6 cells were divided into two groups as control group, where MIN6 cells were cultured in normal culture medium, and coculture group, where MIN6 cells were cocultured with differentiated 3T3-L1 adipocytes for 1 week. Semi-quantitative RT-PCR was employed to measure the expression of K(ATP) channel subunit Kir6.2 in MIN6 cells. Fura-2 was used to reflect changes in intracellular calcium concentration ([Ca(2+)](i)) in MIN6 cells. The secretary function of MIN6 cells from both groups was estimated by radioimmunoassay method. The results showed that the Kir6.2 cDNA levels corrected by GAPDH cDNA levels after densitometric analysis were 0.989+/-0.035 in control group and 0.726+/-0.087 in coculture group. The expression of Kir6.2 was significantly decreased in MIN6 cells in the coculture group as compared with that in control. MIN6 cells cocultured with 3T3-L1 adipocytes lost the ability to increase [Ca(2+)](i) when stimulated by tolbutamide (0.1 mmol/L), a highly selective KATP channel closer. In contrast, MIN6 cells in control group had typical responses to tolbutamide with a significant increase in [Ca(2+)](i). The magnitudes to basal levels of [Ca(2+)](i) after tolbutamide stimulation were 1.520+/-0.203 in control and 1.114+/-0.097 in coculture group (P<0.05, n=6). MIN6 cells in control showed a significant increase in insulin secretion from 0.38+/-0.099 mU/min to 2.87+/-0.248 mU/min after being stimulated by tolbutamide, whereas MIN6 cells in coculture group did not increase insulin secretion when stimulated by tolbutamide (0.21+/-0.055 mU/min to 0.22+/-0.082 mU/min). It is demonstrated that 3T3-L1 adipocytes decrease the expression of K(ATP) channels in MIN6 cells through secreting certain factors, which impair the secretary function of MIN6 cells. The present results indicate that adipocytes are directly involved in pancreatic beta-cell dysfunction, which may facilitate the development of type 2 diabetes.
3T3 Cells ; Adipocytes ; cytology ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Coculture Techniques ; Gene Expression ; Hypoglycemic Agents ; pharmacology ; Insulin ; biosynthesis ; Insulin Resistance ; Islets of Langerhans ; cytology ; metabolism ; Mice ; Potassium Channels, Inwardly Rectifying ; biosynthesis ; genetics ; physiology ; Tolbutamide ; pharmacology ; Transcription, Genetic ; drug effects

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Diabetes Mellitus ; therapy ; Immunohistochemistry ; Islets of Langerhans ; cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Polymerase Chain Reaction ; Radioimmunoassay ; Rats ; Rats, Sprague-Dawley

The regional distribution and relative frequency of some endocrine cells in the principal pancreatic islets of two teleosts, Silurus asotus Linne (Siluridae) and Siniperca scherzeri Steindachner (Centropomidae), which have similar feeding habits, were observed using specific antisera against insulin, glucagon, somatostatin and bovine pancreatic polypeptide (bovine PP) using the peroxidase antiperoxidase (PAP) method. Spherical to spindle shaped cells were demonstrated in the principal pancreatic islets in both species of teleost fishes. However, they were not detected in the exocrine portions nor the pancreatic ducts. Insulin-immunoreactive cells were located in the central regions of the principal pancreatic islets at high frequency in both species. Glucagonimmunoreactive cells were restricted to the peripheral regions of the principal pancreatic islets in both species. They formed a mantle zone in the peripheral regions of Silurus asotus with moderate frequency, and occupied a narrower mantle zone in Siniperca scherzeri with moderate frequency. In addition, glucagonimmunoreactive cell cores were also found in the peripheral zone of some principal pancreatic islets of Siniperca scherzeri. Somatostatin-immunoreactive cells were dispersed in the central zone of the principal pancreatic islets of Silurus asotus with moderate frequency, but were located in the peripheral regions with low frequency in Siniperca scherzeri. Bovine PPimmunoreactive cells were found in the peripheral region and the mantle zone of the principal pancreatic islets with low and rare frequency, respectively in both species. In conclusion, the regional distribution and relative frequency of endocrine cells in the principal pancreatic islets of Silurus asotus showed general patterns similar to those of other teleostean fishes. But, some speciesdependent distributional patterns and/or relative frequencies, particularly in glucagon-, somatostatin- and bovine PP-immunoreactive cells, were detected in the principal pancreatic islets of Siniperca scherzeri
Animals ; Catfishes/*anatomy & histology/physiology ; Cattle ; Glucagon/analysis/immunology ; Immune Sera/diagnostic use/immunology ; Immunohistochemistry/veterinary ; Insulin/analysis/immunology ; Islets of Langerhans/*cytology/physiology ; Pancreatic Polypeptide/analysis/immunology ; Perciformes/*anatomy & histology/physiology ; Somatostatin/analysis/immunology