Fibrin supports human fetal islet-epithelial cell differentiation via p70(s6k) and promotes vascular formation during transplantation.

The human fetal pancreas expresses a variety of extracellular matrix (ECM) binding receptors known as integrins. A provisional ECM protein found in blood clots that can bind to integrin receptors and promote ß cell function and survival is fibrin. However, its role in support of human fetal pancreatic cells is unknown. We investigated how fibrin promotes human fetal pancreatic cell differentiation in vitro and in vivo. Human fetal pancreata were collected from 15 to 21 weeks of gestation and collagenase digested. Cells were then plated on tissue-culture polystyrene, or with 2D or 3D fibrin gels up to 2 weeks, or subcutaneously transplanted in 3D fibrin gels. The human fetal pancreas contained rich ECM proteins and expressed integrin αVß3. Fibrin-cultured human fetal pancreatic cells had significantly increased expression of PDX-1, glucagon, insulin, and VEGF-A, along with increased integrin αVß3 and phosphorylated FAK and p70(s6k). Fibrin-cultured cells treated with rapamycin, the mTOR pathway inhibitor, had significantly decreased phospho-p70(s6k) and PDX-1 expression. Transplanting fibrin-mixed cells into nude mice improved vascularization compared with collagen controls. These results suggest that fibrin supports islet cell differentiation via p70(s6k) and promotes vascularization in human fetal islet-epithelial clusters in vivo.

Aldehyde dehydrogenase 1 activity in the developing human pancreas modulates retinoic acid signalling in mediating islet differentiation and survival.

AIMS/HYPOTHESIS: Aldehyde dehydrogenase 1 (ALDH1), a human stem-cell marker, is an enzyme responsible for converting retinaldehydes to retinoic acids (RAs) to modulate cell differentiation. However, data on expression levels and functional roles of ALDH1 during human fetal pancreatic development are limited. The focus of this study was to characterise ALDH1 expression patterns and to determine its functional role in islet cell differentiation. METHODS: The presence of ALDH1 in the human fetal pancreas (8-22 weeks) was characterised by microarray, quantitative RT-PCR, western blotting and immunohistological approaches. Isolated human fetal islet-epithelial cell clusters were treated with ALDH1 inhibitors, retinoic acid receptor (RAR) agonists and ALDH1A1 small interfering (si)RNA. RESULTS: In the developing human pancreatic cells, high ALDH1 activity frequently co-localised with key stem-cell markers as well as endocrine transcription factors. A high level of ALDH1 was expressed in newly differentiated insulin(+) cells and this decreased as development progressed. Pharmacological inhibition of ALDH1 activity in human fetal islet-epithelial cell clusters resulted in reduced endocrine cell differentiation and increased cell apoptosis, and was reversed with co-treatment of RAR/RXR agonists. Furthermore, siRNA knockdown of ALDH1A1 significantly decreased RAR expression and induced cell apoptosis via suppression of the phosphoinositide 3-kinase (PI3K) pathway and activation of caspase signals. CONCLUSIONS/INTERPRETATION: Our findings indicate that ALDH1(+) cells represent a pool of endocrine precursors in the developing human pancreas and that ALDH1 activity is required during endocrine cell differentiation. Inhibition of ALDH1-mediated retinoid signalling impairs human fetal islet cell differentiation and survival.

Activation of Pancreatic Stellate Cells Is Beneficial for Exocrine but Not Endocrine Cell Differentiation in the Developing Human Pancreas.

Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8-22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14-16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.

beta1 integrin/FAK/ERK signalling pathway is essential for human fetal islet cell differentiation and survival.

beta1 integrin and collagen matrix interactions regulate the survival of cells by associating with focal adhesion kinase (FAK) and initiating MAPK/ERK signalling, but little is known about these signalling pathways during human fetal islet ontogeny. The purpose of this study was to investigate whether beta1 integrin/FAK activation of the MAPK/ERK pathway regulates human fetal islet cell expression of endocrine cell markers and survival. Isolated human (18-21 weeks fetal age) islet-epithelial cell clusters, cultured on collagen I, were examined using beta1 integrin blocking antibody, beta1 integrin siRNA and FAK expression vector. Perturbing beta1 integrin function in the human fetal islet-epithelial cell clusters resulted in a marked decrease in cell adhesion, in parallel with a reduction in the number of cells expressing PDX-1, insulin and glucagon (p < 0.05). beta1 integrin blockade disorganized focal adhesion contacts in the PDX-1(+) cells and decreased activation of FAK and ERK1/2 signalling in parallel with an increase in expression of cleaved caspases 9 and 3 (p < 0.01). Similar results were obtained following an siRNA knock-down of beta1 integrin expression. In contrast, over-expression of FAK not only increased phospho-ERK and the expression of PDX-1, insulin and glucagon (p < 0.05) but also abrogated the decreases in phospho-ERK and PDX-1 by beta1 integrin blockade. This study demonstrates that activation of the FAK/ERK signalling cascade by beta1 integrin is involved in the differentiation and survival of human fetal pancreatic islet cells.

Characterization and Differentiation of Sorted Human Fetal Pancreatic ALDHhi and ALDHhi/CD133+ Cells Toward Insulin-Expressing Cells.

The enzyme aldehyde dehydrogenase (ALDH) is found in developing and multipotent cell populations, and is important for the production and regulation of retinoic acid, which controls ß-cell differentiation in the pancreas. The role of ALDH-expressing cells in the formation of endocrine-like cells and co-localization with the putative stem cell marker CD133 has not been examined during human pancreatic development. This study focuses on the co-expression of CD133 on ALDH+ cells from the human fetal pancreas (18-22 weeks of fetal age) with transcription factors (TFs) central to endocrine cell development. Fluorescence-activated cell sorting demonstrated that cells with high ALDH activity (ALDHhi) had increased co-expression of CD133 and endocrine-lineage TFs when compared with cells with low ALDH (ALDHlo) expression. Hormone-expressing (insulin, somatostatin) and ductal cells (CK19) were noted in the ALDHhi population, while mesenchymal (vimentin) and endothelial (CD31) markers were predominantly found in ALDHlo cells. Culture of sorted ALDHhi or ALDHhi/CD133+ cells resulted in loss of endocrine TF, insulin, and CK19 expression. The formation of cell clusters from cultured ALDHhi or ALDHhi/CD133+ cells led to restored CK19 expression and showed endocrine TFs and insulin expression. In summary, pancreatic ALDHhi cells contain a heterogeneous CD133-enriched population with a subset of ß-cell associated markers in the developing human pancreas.

Maintaining human fetal pancreatic stellate cell function and proliferation require ß1 integrin and collagen I matrix interactions.

Pancreatic stellate cells (PaSCs) are cells that are located around the acinar, ductal, and vasculature tissue of the rodent and human pancreas, and are responsible for regulating extracellular matrix (ECM) turnover and maintaining the architecture of pancreatic tissue. This study examines the contributions of integrin receptor signaling in human PaSC function and survival. Human PaSCs were isolated from pancreata collected during the 2nd trimester of pregnancy and identified by expression of stellate cell markers, ECM proteins and associated growth factors. Multiple integrins are found in isolated human PaSCs, with high levels of ß1, α3 and α5. Cell adhesion and migration assays demonstrated that human PaSCs favour collagen I matrix, which enhanced PaSC proliferation and increased TGFß1, CTGF and α3ß1 integrin. Significant activation of FAK/ERK and AKT signaling pathways, and up-regulation of cyclin D1 protein levels, were observed within PaSCs cultured on collagen I matrix. Blocking ß1 integrin significantly decreased PaSC adhesion, migration and proliferation, further complementing the aforementioned findings. This study demonstrates that interaction of ß1 integrin with collagen I is required for the proliferation and function of human fetal PaSCs, which may contribute to the biomedical engineering of the ECM microenvironment needed for the efficient regulation of pancreatic development.

Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas.

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8-20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8-15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14-20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/exocrine granules.

SOX9 regulates endocrine cell differentiation during human fetal pancreas development.

The transition of pancreatic progenitor cells to mature endocrine cells is regulated by the sequential activation and interaction of several transcription factors. In mice, the transcription factor Sox9 has been shown to support endocrine cell differentiation. However, the functional role of SOX9 during pancreas development in the human has yet to be determined. The present study was to characterize SOX9 expression during human fetal pancreas development and examine its functional role by transfection with SOX9 siRNA or SOX9 expression vectors. Here we report that SOX9 was most frequently expressed in PDX1(+) cells (60-83%) and least in mature endocrine cells (<1-14%). The proliferation of SOX9(+) cells was significantly higher at 8-10 weeks than at 14-21 weeks (p<0.05) or 20-21 weeks (p<0.01). SOX9 frequently co-localized with FOXA2, NGN3 and transcription factors linked to NGN3 (NKX2.2, NKX6.1, PAX6). siRNA knockdown of SOX9 significantly decreased islet-epithelial cell proliferation, NGN3, NKX6.1, PAX6 and INS mRNA levels and the number of NGN3(+) and insulin(+) cells (p<0.05) while increasing GCG mRNA and glucagon(+) cells (p<0.05). Examination of SOX9 associated signaling pathways revealed a decrease in phospho-Akt (p<0.01), phospho-GSK3ß (p<0.01) and cyclin D1 (p<0.01) with a decrease in nuclear ß-catenin(+) (p<0.05) cells following SOX9 siRNA knockdown. In contrast, over-expression of SOX9 significantly increased the number of islet cells proliferating, NGN3, NKX6.1, PAX6 and INS mRNA levels, the phospho-Akt/GSK3ß cascade and the number of insulin(+) cells. Our results demonstrated that SOX9 is important for the expression of NGN3 and molecular markers of endocrine cell differentiation in the human fetal pancreas.

Integrin {alpha}3, but not {beta}1, regulates islet cell survival and function via PI3K/Akt signaling pathways.

ß1-integrin is a well-established regulator of ß-cell activities; however, the role of its associated α-subunits is relatively unknown. Previously, we have shown that human fetal islet and INS-1 cells highly express α3ß1-integrin and that collagens I and IV significantly enhance their survival and function; in addition, blocking ß1 function in the fetal islet cells decreased adhesion on collagen I and increased apoptosis. The present study investigates the effect of blocking α3. Using α3 blocking antibody or small interfering RNA, the effects of α3-integrin blockade were examined in isolated human fetal or adult islet cells or INS-1 cells, cultured on collagens I or IV. In parallel, ß1 blockade was analyzed in INS-1 cells. Perturbing α3 function in human islet or INS-1 cells resulted in significant decreases in cell function (adhesion, spreading, proliferation and Pdx1 and insulin expression/secretion), primarily on collagen IV. A significant decrease in focal adhesion kinase and ERK1/2 phosphorylation and increased caspase3 cleavage were observed on both collagens. These effects were similar to changes after ß1 blockade. Interestingly, only α3 blockade reduced expression of phospho-Akt and members of its downstream signaling cascades (glycogen synthase kinase ß and X-linked inhibitor of apoptosis), demonstrating a specific effect of α3 on the phosphatidylinositol 3-kinase/Akt pathway. These results suggest that α3- as well as ß1-integrin-extracellular matrix interactions are critical for modulating ß-cell survival and function through specialized signaling cascades and enhance our understanding of how to improve islet microenvironments for cell-based treatments of diabetes.