Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment.     

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.     

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.     

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.     

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 from human pancreatic islet-derived progenitor cells following transplantation in mice

Stem/progenitor cells hold promise for alleviating/curing type 1 diabetes due to the capacity to differentiate into functional insulin-producing cells. The current study aims to assess the differentiation potential of human pancreatic IPCs (islet-derived progenitor cells). IPCs were derived from four human donors and subjected to more than 2000-fold expansion before turning into ICCs (islet-like cell clusters). The ICCs expressed ISL-1 Glut2, PDX-1, ngn3, insulin, glucagon and somatostatin at the mRNA level and stained positive for insulin and glucagon by immunofluorescence. Following glucose challenge in vitro, C-peptide was detected in the sonicated ICCs, instead of in the conditioned medium. To examine the function of the cells in vivo, IPCs or ICCs were transplanted under the renal capsule of immunodeficient mice. One month later, 19 of 28 mice transplanted with ICCs and 4 of 14 mice with IPCs produced human C-peptide detectable in blood, indicating that the in vivo environment further facilitated the maturation of ICCs. However, among the hormone-positive mice, only 9 of 19 mice with ICCs and two of four mice with IPCs were able to secrete C-peptide in response to glucose.     

Fibrin scaffold enhances function of insulin producing cells differentiated from human umbilical cord matrix-derived stem cells

Tissue engineering is a new strategy which proposed to treat numerous human diseases nowadays. Three dimensional (3D) scaffolds fill the gap between two dimensional cell culture (2D) and animal tissues through mimicking the environmental behaviors surrounding the cells. In this study, hUCMs into insulin producing cells in fibrin scaffold were differentiated compare to conventional culture condition. Differentiation rate was estimated by real time PCR, immunocytochemistry (ICC) and the chemiluminesence (CLIA) and enzyme immunoassay (EIA). Real time PCR's results showed an increasing expression in NKX2.2, PDX1 and INS (producing the hormone insulin) genes in fibrin scaffold. Furthermore ICC analysis exhibited that insulin and pro-insulin proteins were more in fibrin scaffolds. CLIA and EIA on insulin and C peptide secretion indicated that both of groups were sensitive to the glucose challenge test but significant higher response was observed in fibrin scaffold (6.5 fold in 3D, 1.8 fold in 2D culture). It could be concluded that differentiation of hUCM cells into insulin producing cells in fibrin scaffold 3D culture system is much more efficient than 2D conventional culture system.     

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.     

Differentiation of microfluidic-encapsulated trabecular meshwork mesenchymal stem cells into insulin producing cells and their impact on diabetic rats

Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.     

Bottom-up signaling from HGF-containing surfaces promotes hepatic differentiation of mesenchymal stem cells

The capacity of stem cells to differentiate into specific cell types makes them very promising in tissue regeneration and repair. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, hepatocyte growth factor (HGF), an important morphogen in liver development, was co-printed with collagen I (Col) to create arrays of protein spots on glass. Human adipose stem cells (ASCs) were cultured on top of the HGF/Col spots for 2 weeks. The effects of surface-immobilized HGF on hepatic differentiation of ASCs were analyzed using RT-PCR, ELISA and immunocytochemistry. Stimulation of stem cells with HGF from the bottom-up caused an upregulation in synthesis of α-fetoprotein and albumin, as determined by immunocytochemistry and ELISA. RT-PCR results showed that the mRNA levels for albumin, α-fetoprotein and α1-antitrypsin were 10- to 20-fold higher in stem cells cultured on the HGF/Col arrays compared to stem cells on Col only spots. Our results show that surfaces containing HGF co-printed with ECM proteins may be used to differentiate mesenchymal stem cells such as ASCs into hepatocyte-like cells. These results underscore the utility of growth factor-containing culture surfaces for stem cell differentiation.     

<Emphasis Type="Italic">In vitro</Emphasis> preconditioning of insulin-producing cells with growth factors improves their survival and ability to release insulin

Glucose-induced oxidative stress in the diabetic pancreas directly affects viability and the consequent therapeutic outcome of transplanted stem cells. Pretreatment of stem cells with growth factors induces tolerance in them against various stresses (hypoxia, thermal or hyperglycaemic). This study investigated the effect of pretreatment on insulin-producing cells (IPCs) differentiated from adipose-derived mesenchymal stem cells (ADMSCs), with a combination of stromal cell-derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) against hyperglycaemic stress (17 or 33 mM glucose). The results showed that IPCs pretreated with a combination of SDF1α and bFGF exhibited maximally alleviated apoptosis, senescence and cell damage with a concomitantly increased release of insulin, enhanced cell proliferation and greater up-regulation of Insulin 1, Insulin 2, Ngn3, Pdx1 and Nkx6.2 when stressed with 33 mM glucose. These findings may offer an improved therapeutic outcome for the treatment of diabetes.     

Pancreatic islet-like clusters from periosteum-derived progenitor cells

Recent studies comparing the insulin-producing cell (IPC) differentiation capacity of mesenchymal stem cells (MSCs) derived from four different sources (bone marrow, Wharton’s jelly, adipose tissue, and the periosteum) demonstrated that IPC differentiation of periosteum-derived progenitor cells (PDPCs) progressed faster than any other MSCs within 7 days, indicating that PDPCsare most suited to IPC differentiation. Here, two different cell culture methods, adhesion and cluster culture, were assessed for their ability to support in vitro IPC differentiation. The induction of IPC differentiation was confirmed by RTqPCR analysis of insulin gene expression levels and immunofluorescence analysis of insulin protein. An enzyme-linked immunosorbent assay was used to quantify secreted insulin. PDPC-derived IPCs from cluster cultures demonstrated a significantly increased expression of insulin and an enhanced secretion of insulin of insulin protein in response to glucose compared to IPCs derived from adhesion cultures. Thus, pancreatic islet-like cluster cultures appear to provide the optimal conditions such as cluster culture for IPC differentiation of PDPCs.     

Isolation,characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.     

In vitro differentiation of human multilineage differentiating stress-enduring (Muse) cells into insulin producing cells

Mesenchymal stem cells (MSCs) is a heterogeneous population. Muse cells is a rare pluripotent subpopulation within MSCs. This study aims to evaluate the pulirpotency and the ability of Muse cells to generate insulin producing cells (IPCs) after in vitro differentiation protocol compared to the non-Muse cells. Muse cells were isolated by FACSAria III cell sorter from adipose-derived MSCs and were evaluated for its pluripotency. Following in vitro differentiation, IPCs derived from Muse and non-Muse cells were evaluated for insulin production. Muse cells comprised 3.2?±?0.7% of MSCs, approximately 82% of Muse cells were positive for anti stage-specific embryonic antigen-3 (SSEA-3). Pluripotent markers were highly expressed in Muse versus non-Muse cells. The percentage of generated IPCs by flow cytometric analysis was higher in Muse cells. Under confocal microscopy, Muse cells expressed insulin and c-peptide while it was undetected in non-Muse cells. Our results introduced Muse cells as a new adult pluripotent subpopulation, which is capable to produce higher number of functional IPCs.     

13C metabolic flux analysis clarifies distinct metabolic phenotypes of cancer cell spheroid mimicking tumor hypoxia

Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by ¹³C-metabolic flux analysis (¹³C-MFA). Principal component analysis of ¹³C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. ¹³C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

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。     

Electrospun poly-l-lactic acid/polyvinyl alcohol nanofibers improved insulin-producing cell differentiation potential of human adipose-derived mesenchymal stem cells

Combination of adipose-derived mesenchymal stem cells (ADSCs) and synthetic materials in terms of pancreatic tissue engineering can be considered as a treatment of diabetes. This study aimed to evaluate the differentiation of human ADSCs to pancreatic cells on poly-l -lactic acid/polyvinyl alcohol (PLLA/PVA) nanofibers as a three-dimensional (3D) scaffold. Mesenchymal stem cells (MSCs) were characterized for mesenchymal surface markers by flow cytometry. Then ADSCs were seeded on 3D scaffolds and treated with pancreatic differentiation medium. Immunostaining assay showed that ADSCs were very efficiently differentiated into a relatively homogeneous population of insulin-producing cells. Moreover, real-time RT-PCR results revealed that pancreas-specific markers were highly expressed in 3D scaffolds compared with their expression in tissue culture plates and this difference in expression level was significant. In addition, insulin and C-peptide secreted in response to varying concentrations of glucose in the 3D scaffold group was significantly higher than that in 2D culture. The results of the present study confirmed that PLLA/PVA scaffold seeded with ADSCs could be a suitable option in pancreatic tissue engineering.     

SHB and angiogenic factors promote ES cell differentiation to insulin-producing cells

The potential use of embryonic stem (ES) cells for cell therapy of diabetes requires improved methods for differentiation and isolation of insulin-producing beta-cells. The signal transduction protein SHB may be involved in both angiogenesis and beta-cell development. Here we show that cells expressing the pancreatic endodermal marker PDX-1 appear in the vicinity of vascular structures in ES cell-derived embryoid bodies (EBs) cultured in vitro. Moreover, overexpression of SHB as well as culture of EBs in presence of the angiogenic growth factors PDGF or VEGF enhanced the expression of PDX-1 and/or insulin mRNA. Finally, expression of GFP under control of the PDX-1 promoter in EBs allowed for the enrichment by FACS of cells expressing PDX-1, C-peptide, and insulin as determined by immunofluorescence. It is concluded that SHB and angiogenic factors promote the development of cells expressing PDX-1 and insulin in EBs and that such cells can be separated by FACS.