Differentiation Of Pancreatic Stem Cells Research Articles

Pancreatic stem cells differentiate into insulin-secreting cells on fibroblast-modified PLGA membranes

Diabetes mellitus is an epidemic worldwide. Pancreatic stem cells can be induced to differentiate into insulin-secreting cells, this method is an effective way to solve the shortage of islet donor. Poly (lactic acid-co-glycolic acid (PLGA) copolymer is an excellent scaffold for tissue engineering as it presents good biocompatibility and film forming properties. In this study, we adopted biological methods, using fibroblast-coated PLGA diaphragm to form a biological membrane, and then pancreatic stem cells were cultured on the fibroblast-modified PLGA membrane and the two-step induction method was utilized to induce the differentiation of pancreatic stem cells into insulin-secreting cells. The proliferation and differentiation of pancreatic stem cells on the fibroblast-modified PLGA membrane as well as the expression of genes related to the differentiation of pancreatic stem cells were examined in both normal and induced cultures to explore the potential of fibroblast-modified PLGA membrane for the transplantation to treat diabetes mellitus. The results indicated that fibroblasts can effectively improve the cell compatibility and histocompatibility of the PLGA membrane and promote the proliferation and differentiation of pancreatic stem cells. After induction, real-time fluorescence quantitative PCR (FQRT-PCR) results showed there were more Notch receptors and its ligands expressed in the membranes of pancreatic stem cells than non-induced pancreatic stem cells or fibroblast. Semiconductor quantum dot coupled-anti-complex probe experiments revealed that induced pancreatic stem cells had higher expression levels of Notch 2 and Delta-like 1 than non-induced ones, which may regulate the expression of Neurogenin-3 (Ngn3) and Hairy/Enhancer of split-1 gene (Hes1) through Notch signaling interaction between fibroblasts and pancreatic stem cells as well as enhance the proliferation of pancreatic stem cells and their differentiation into insulin-secreting cells. Further, our study suggests that the fibroblast-modified PLGA membrane can be used as matrix material composed of pancreatic stem cells or other stem cells to construct artificial islet tissue for the treatment of diabetes mellitus.

Construction of functional pancreatic artificial islet tissue composed of fibroblast-modified polylactic- co-glycolic acid membrane and pancreatic stem cells.

Objective To improve the biocompatibility between polylactic- co-glycolic acid membrane and pancreatic stem cells, rat fibroblasts were used to modify the polylactic- co-glycolic acid membrane. Meanwhile, we constructed artificial islet tissue by compound culturing the pancreatic stem cells and the fibroblast-modified polylactic- co-glycolic acid membrane and explored the function of artificial islets in diabetic nude mice. Methods Pancreatic stem cells were cultured on the fibroblast-modified polylactic- co-glycolic acid membrane in dulbecco's modified eagle medium containing activin-A, β-catenin, and exendin-4. The differentiated pancreatic stem cells combined with modified polylactic- co-glycolic acid membrane were implanted subcutaneously in diabetic nude mice. The function of artificial islet tissue was explored by detecting blood levels of glucose and insulin in diabetic nude mice. Moreover, the proliferation and differentiation of pancreatic stem cells on modified polylactic- co-glycolic acid membrane as well as the changes on the tissue structure of artificial islets were investigated by immunofluorescence and haematoxylin and eosin staining. Results The pancreatic stem cells differentiated into islet-like cells and secreted insulin when cultured on fibroblast-modified polylactic- co-glycolic acid membrane. Furthermore, when the artificial islet tissues were implanted into diabetic nude mice, the pancreatic stem cells combined with polylactic- co-glycolic acid membrane modified by fibroblasts proliferated, differentiated, and secreted insulin to reduce blood glucose levels in diabetic nude mice. Conclusion Pancreatic stem cells can be induced to differentiate into islet-like cells invitro. Invivo, the artificial islet tissue can effectively regulate the blood glucose level in nude mice within a short period. However, as time increased, the structure of the artificial islets was destroyed due to the erosion of blood cells that resulted in the gradual loss of artificial islet function.

The Role of Epigenetic Regulation and Pluripotency-Related MicroRNAs in Differentiation of Pancreatic Stem Cells to Beta Cells.

In this study, we aimed to research the effects of class-I HDACs and glucose on differentiation of pancreatic islet derived mesenchymal stem cells (PI-MSCs) to beta cells. Beta cell differentiation determined by flow cytometric analysis and gene expression levels of PDX1, PAX4, PAX6, NKX6.1, NGN3, INS2, and GLUT2. As a result the valproic acid, is an inhibitor of class-I HDACs, caused the highest beta cell differentiation in PI-MSCs. However, the cells in this group were at early stages of differentiation. Glucose co-administration to this group carried the differentiation to higher levels, but these newly formed beta cells were not functional. Moreover, reduction in the levels of pluripotency factors that Oct3/4, c-Myc, and Nanog were parallel to beta cell differentiation. Also, the levels of HDAC1 and acetylated H3/H4 were increased and methylated H3 was decreased by VPA treatment. In addition, we have detected over expression in genes of miR-18a-5p, miR-19b-5p, miR-30d-3p, miR-124, miR-146a-5p, miR-184, miR-335, and miR-433-5p in parallel to beta cell differentiation. As the conclusion, this study is important for understanding the epigenetic mechanism that controls the beta cell differentation and it suggests new molecules that can be used for diagnosis, and treatment of diabetes. J. Cell. Biochem. 119: 455-467, 2018. © 2017 Wiley Periodicals, Inc.

Human Fetal Liver Stromal Cell Co-Culture Enhances the Differentiation of Pancreatic Progenitor Cells into Islet-Like Cell Clusters

Recent advance in directed differentiation of pancreatic stem cells offers potential to the development of replacement therapy for diabetic patients. However, the existing differentiation protocols are complex, time-consuming, and costly; thus there is a need for alternative protocols. Given the common developmental origins of liver and pancreas, we sought to develop a novel protocol, devoid of growth factors, by using liver stromal cells (LSCs) derived from human fetal liver. We examined the effects of the LSCs on the differentiation of pancreatic progenitor cells (PPCs) into islet-like cell clusters (ICCs). PPCs and LSCs isolated from 1st to 2nd trimester human fetal tissues underwent co-cultures; differentiation and functionality of ICCs were determined by examining expression of critical markers and secretion of insulin. Co-culture with 2nd but not 1st trimester LSCs enhanced ICC differentiation and functionality without the use of exogenous differentiation 'cocktails'. Differential expression profiles of growth factors from 1st versus 2nd trimester fetal liver were compared. Many morphogenic factors were expressed by LSCs, while insulin-like growth factor 1 (IGF1) was identified as one of the key molecules responsible for the ICC differentiation. This is the first report showing that an LSC-induced microenvironment can enhance ICC differentiation and functionality. Further modifications of the stroma microenvironment may offer an alternative, efficient and cost-effective approach to providing islets for transplantation.

Better Induction and Differentiation Strategy for Rat Pancreatic Stem Cells: Transplant in Liver Niche

Current in vitro induction protocols cannot generate mature islet cells from stem cells. Transplantation into the liver niche may greatly contribute to the maturity of pancreatic stem cells (PSCs) due to its similarity to the pancreas. To determine the effect of the liver niche on the differentiation of PSCs, we used neonatal Wistar rat pancreata for cultivation of PSCs, which were transplanted into diabetic Wistar rats via the portal vein or beneath the renal capsule. After transplantation, we measured random blood glucose, weight, and serum insulin and performed an intraperitoneal glucose tolerance test. Specimens were examined by immunofluorescence. As a result, highly proliferating harvested cells showed the characteristics of stem cells. The PSCs could be induced to form large islet-like structures (150–200 μm diameter) in the liver, which resulted in better therapeutic efficacy. In contrast, there were smaller islet-like structures (about 50 μm diameter) when PSCs were transplanted beneath the renal capsule. These findings demonstrated that the liver niche benefits the in vivo differentiation of PSCs into endocrine and exocrine cells that may contribute to the generation of insulin producing cells.

Directed differentiation of pancreatic stem cells by soluble and immobilised signalling factors

Recent findings demonstrate that adult stem cells exhibit a much higher differentiation potential than previously expected and that the sources within the adult organism seem to be surprisingly manifold. In this study, we investigated adult stem cells isolated from mouse and rat pancreas and showed for the first time their potential to differentiate towards osteoblasts, chondrocytes and adipocytes. These pancreatic stem cells (PSCs) have previously been described to differentiate spontaneously into cell types of all three germ layers. We could now demonstrate that a directed differentiation is also possible and compared a variety of cytokines as inducers. In addition to today's practice of applying only soluble differentiation factors we investigated the use of immobilised cytokines. For protein immobilization different strategies were evaluated such as covalent linkage of insulin-like growth factor-1 (IGF-1) to amino- or epoxy-silanised glass or affinity binding of the biotinylated cytokine to neutravidin-coated surfaces. Our results show that cytokines could be immobilised functionally, retaining their biological activity to effectively induce stem cell differentiation in long-term cell culture experiments. Our results emphasised the high differentiation potential of mouse and rat PSCs, showing that a directed cytokine-induced differentiation of both rodent PSC types is possible and demonstrating that differentiation factors, immobilised to different surfaces, can successfully trigger a stem cell's fate.

Transplantation of bone marrow derived cells promotes pancreatic islet repair in diabetic mice

The transplantation of bone marrow (BM) derived cells to initiate pancreatic regeneration is an attractive but as-yet unrealized strategy. Presently, BM derived cells from green fluorescent protein transgenic mice were transplanted into diabetic mice. Repair of diabetic islets was evidenced by reduction of hyperglycemia, increase in number of islets, and altered pancreatic histology. Cells in the pancreata of recipient mice co-expressed BrdU and insulin. Double staining revealed β cells were in the process of proliferation. BrdU + insulin − PDX-1 + cells, Ngn3 + cells and insulin + glucagon + cells, which showed stem cells, were also found during β-cell regeneration. The majority of transplanted cells were mobilized to the islet and ductal regions. In recipient pancreas, transplanted cells simultaneously expressed CD34 but did not express insulin, PDX-1, Ngn3, Nkx2.2, Nkx6.1, Pax4, Pax6, and CD45. It is concluded that BM derived cells especially CD34 + cells can promote repair of pancreatic islets. Moreover, both proliferation of β cells and differentiation of pancreatic stem cells contribute to the regeneration of β cells.

Proliferation and differentiation of duct epithelial cells after partial pancreatectomy in rats

The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. There was significant difference between the experimental group and the control group (P<0.001). Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.

Induction of PDX-1-positive cells in the main duct during regeneration after acute necrotizing pancreatitis in rats.

Pancreatic regeneration involves two pathways; proliferation and differentiation of pancreatic progenitor cells, which probably exist in pancreatic ductal epithelium, and replication of pre-existing differentiated acinar, islet, and ductal epithelial cells. During pancreatic development, differentiated cells arise from the ductal progenitor cells expressing the pancreatic/duodenal homeobox-1 (PDX-1) homeodomain transcription factor. The aims of this study were to characterize cell proliferation and differentiation during regeneration after acute necrotizing pancreatitis and to evaluate the role of PDX-1-positive stem cells. Necrotizing pancreatitis was induced in rats by retrograde intraductal infusion of sodium taurocholate. Cell types were classified into five categories: main, large, and small ductal epithelial cells, tubular complexes and acinar cells. Each category was scored using a 5-bromo-2'-deoxyuridine (BrdU) labelling index (LI) at various time points after induction of pancreatitis. Tissue sections were also immunostained for PDX-1 to determine the source of pancreatic stem cells. Acinar necrosis was observed at 24 h after induction of pancreatitis and most lobules were filled with tubular complexes on day 5. Subsequently, newly formed acinar cells were observed on day 7, but the lobular architecture returned to normal appearance on day 28. Proliferation started in the main and large ducts at 24 h; marked mitotic activity was evident in small ductal epithelial cells and tubular complexes on day 3, and in acinar cells on day 7. At 24 h after induction of pancreatitis, epithelial cells of the main duct with PDX-1-positive nuclei were greatly increased, simultaneously with the peak LI of BrdU. These results suggest that regeneration after necrotizing pancreatitis involves proliferation and differentiation of pancreatic progenitor cells, and that ductal epithelial cells with PDX-1-positive nuclei may contribute to the differentiation of pancreatic stem cells in the main duct.

Human Pancreatic Ductal Cells: Large-Scale Isolation and Expansion

The in vitro differentiation of pancreatic stem cells has recently been shown to represent a new source of beta cells for cell therapy in diabetes. Human ductal cell differentiation, in vitro, has been documented in three-dimensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex vivo are mandatory for clinical relevance. We compared three sources of human ductal cells (hDC) (method A1-2, B, and C). The classical main duct isolation of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fraction in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal component in the form of ductal cysts, as we described previously; monolayers derived from digested cysts were 80% ductal (CK19). Method C: initially adherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7) ductal cells. One-week exocrine cultures were amylase negative and 46% (CK19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81+/-12% (B), 91+/-2% (C). Extrapolating total yields we obtained (+/-SEM): 10.5+/-4.6 x 10(3) (A1), 36+/-18 x 10(3) (A2), 292+/-50 x 10(6) (B), 1696+/-526 x 10(6) (C) viable hDC per pancreas. A secondary monolayer expansion of cyst-derived hDC (method B) was achieved with NuSerum (4.2-fold on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plastic). First passage exocrine-derived ductal cells also responded to matrix and to growth factors, albeit not significantly. In conclusion, this study demonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. If their differentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic and autologous diabetes cell therapy.