Investigation of the effect of pancreatic decellularized matrix on encapsulated Islets of Langerhans with mesenchymal stem cells.

OBJECTIVE: In this study, it was aimed to provide a therapeutic approach for T1DM by encapsulating the pancreatic islets with mesenchymal stem cells and decellularized pancreatic extracellular matrix to support the survival of islets while maintaining their cellular activity. METHOD: Pancreatic extracellular matrix was decellularized using different concentrations of detergent series. After the preparation of the protein-based tissue extracellular matrix was shown to be free of cells or any genetic material by molecular, immunofluorescence and histochemical techniques. Following the homogenization of the decellularized pancreatic extracellular matrix and the analysis of its protein composition by LC-MS, the matrix proteins were incorporated with pancreatic islets and rat adipose tissue-derived MSCs (rAT-MSCs) in alginate microcapsules. Glucose-stimulated insulin secretion property of the islet cells in the microbeads was evaluated by insulin ELISA. The gene expression profile of the encapsulated cells was analyzed by Real-Time PCR. RESULTS: Unlike the protein composition of whole pancreatic tissue, the decellularized pancreas matrix was free of histone proteins or proteins originated from mitochondria. The protein matrix derived from pancreatic tissue was shown to support the growth and maintenance of the islet cells. When compared to the non-encapsulated pancreatic islet, the encapsulated cells demonstrate to be more efficient in terms of insulin expression. CONCLUSION: The extracellular pancreatic matrix obtained in this study was directly used as supplementary in the alginate-based microcapsule enhancing the cell survival. The tissue matrix protein and alginate had a synergistic effect on total insulin secretion, which might have the potential to overcome the insulin deficiency. Despite the improvement in the cell viability and the number, the efficiency of the insulin secretion in response to glucose stimulation from the alginate microcapsules did not meet the expectation when compared with the non-encapsulated pancreatic islets.

Mesenchymal stem cells and ligand incorporation in biomimetic poly(ethylene glycol) hydrogels significantly improve insulin secretion from pancreatic islets.

The main goal of this study was to investigate pancreatic islet function with mesenchymal stem cells (MSCs) in a ligand-functionalized poly(ethylene glycol) (PEG) hydrogel for the treatment of type 1 diabetes (T1D). Rat bone marrow-derived MSCs (rBM-MSCs) were encapsulated within synthetic PEG hydrogel, and cell viability and apoptosis within this 3D environment was examined in detail. ATP content and caspase-3 activity of encapsulated MSCs showed that fibronectin-derived RGDS, laminin-derived IKVAV and/or insulinotropic glucagon-like peptide (GLP-1) were required to maintain MSC survival. Incorporation of these peptides into the hydrogel environment also improved pancreatic islet viability, where combinations of peptides had altered effects on islet survival. GLP-1 alone was the leading stimulator for insulin secretion. Cell adhesion peptides RGDS and IKVAV improved insulin secretion only when they were used in combination, but could not surpass the effect of GLP-1. Further, when pancreatic islets were co-encapsulated with MSCs within synthetic PEG hydrogel, a two-fold increase in the stimulation index was measured. Synergistic effects of MSCs and peptides were observed, with a seven-fold increase in the stimulation index. The results are promising and suggest that simultaneous incorporation of MSCs and ECM-derived peptides and/or GLP-1 can improve pancreatic islet function in response to altered glucose levels in the physiological environment. Copyright © 2014 John Wiley & Sons, Ltd.

Role of Mesenchymal Stem Cells Transfected With Vascular Endothelial Growth Factor in Maintaining Renal Structure and Function in Rats with Unilateral Ureteral Obstruction.

OBJECTIVES: Mesenchymal stem cells hold promise for renal disease treatment. Vascular endothelial growth factor may heal tubule-interstitial fibrosis in unilateral ureteral obstruction by inhibiting epithelial-mesenchymal transition. We investigated the protective effect of vascular endothelial growth factor in transfected mesenchymal stem cells in unilateral ureteral obstruction-induced renal injury in rats. MATERIALS AND METHODS: Male Wistar Albino rats (32 rats; weight, 250-300 g) were divided into 4 equal groups: group 1, control; group 2, unilateral ureteral obstruction; group 3, unilateral ureteral obstruction and mesenchymal stem cells; and group 4, unilateral ureteral obstruction and vascular endothelial growth factor-transfected mesenchymal stem cells. Vascular endothelial growth factor-transfected mesenchymal stem cells were administered intravenously before onset of unilateral ureteral obstruction. On day 14, the rats were killed and kidneys were retrieved. Tubular necrosis, mononuclear cell infiltration, and interstitial fibrosis were evaluated in paraffin blocks. We evaluated green fluorescent protein-positive and vascular endothelial growth factor-positive cells; anti-inflammatory (Prostaglandin E2 receptor) and interleukin 1 receptor antagonist), proinflammatory/anti-inflammatory (interleukin 6), and proinflammatory (MPO) cytokine expression levels; and levels of nitric oxide; transforming growth factor ß1, E-cadherin, and hydroxyproline. RESULTS: Green fluorescent protein-positive cells were negative in the renal parenchyma in groups 1 and 2 and positive in groups 3 and 4. Vascular endothelial growth factor levels were significantly higher in group 4. Transforming growth factor ß1, nitric oxide, and E-cadherin levels were significantly higher in the unilateral ureteral obstruction than control group; however, in the study groups, these values were not significantly different from the unilateral ureteral obstruction group. In stem cell-transplanted tissue samples, EP3, interleukin 1 receptor antagonist, and interleukin 6 levels were elevated, but MPO expression levels were low. Although there were significant differences for tubular necrosis and fibrosis in group 2, there were significant reductions in tubular injury and fibrosis in groups 3 and 4. CONCLUSIONS: Systemic stem cells transplanted into the kidney protected against unilateral ureteral obstruction-induced renal epithelial-mesenchymal transition and renal fibrosis.

Adipose tissue-derived mesenchymal stromal cells efficiently differentiate into insulin-producing cells in pancreatic islet microenvironment both in vitro and in vivo.

BACKGROUND AIMS: Differentiation or reprogramming of stem cells could be achieved by remodulating the microenvironment, which regulates the fate of cells by soluble factors and contacts. By providing an in vivo-like microenvironment, directional and functional differentiation of stem cells could be achieved in vitro. In this study, the differentiation of mesenchymal stromal cells (MSCs) derived from rat tissues (adipose, rAT; bone marrow, rBM) were analyzed by in vitro and in vivo co-culture experiments. The insulin-producing capacities of islets transplanted under the renal kidney capsule with rAT- and rBM-MSCs were compared and the reduction of hyperglycemia symptoms in rat models was examined. METHODS: MSCs prelabeled with green fluorescence protein were co-cultured with islets directly. The insulin production of cells was determined by immunostaining and ELISA. Streptozotocin-induced diabetic rat models were created and MSCs were co-transplanted with the islets under the kidney capsule to confirm the in vitro results. RESULTS: MSCs were differentiated into insulin-producing cells after 38 days of co-culture, confirmed by insulin and C-peptide stainings. In vivo functional studies revealed that the co-culture of islets with MSCs provided higher differentiation efficiency. The weight gain measurement and glucose tolerance test in the rat group co-transplanted of rAT-MSCs and islets indicate a better recovery than islet-alone transplants and co-transplants of islets and rBM-MSCs. CONCLUSIONS: rAT-MSCs could be considered as the cell of choice for cell-based treatment of type 1 diabetes. Because the co-transplantation of islets with MSCs increases the number of insulin-producing cells, this method was suggested for clinical applications.

Reduction of lesion in injured rat spinal cord and partial functional recovery of motility after bone marrow derived mesenchymal stem cell transplantation.

AIM: This study aimed to analyze the effect of rat bone marrow-mesenchymal stem cells (rBM-MSCs) delivery on lesion site after spinal cord injury, and to observe the functional recovery after transplantation. MATERIAL AND METHODS: MSCs were isolated from rat femurs and tibias. The experimental rat population was divided into four groups: only laminectomy (1); laminectomy+trauma (2); laminectomy+trauma+PBS (3); laminectomy+trauma+MSCs (4). Their motility were scored regularly. After 4-weeks, rats were sacrificed, and their spinal cords were examined for GFP labeled rBM-MSCs by immunostainings. RESULTS: In the early posttraumatic period, the ultrastructures of spinal cord tissue were preserved in Group 4. The majority of cells forming the ependymal region around the central canal were found to be MSCs. The gray-and-white-matter around the ependymal region were composed of Nestin+/GFAP+ cells, with astrocytic-like appearance. The scores showed significant motor recovery in Group 4, especially in hind limb functions. However, no obvious change was observed in other groups. CONCLUSION: The increase Nestin+/GFAP+ cells in the gray-and-white-matter around the ependymal region could indicate the potential to self-renew and plasticity. Thus, transplantation of rBM-MSCs might be an effective strategy to improve functional recovery following spinal cord trauma. In conclusion, molecular factors in cell fate decisions could be manipulated to enhance reparative potential of cell-based therapy.

Bone marrow-derived mesenchymal stem cells co-cultured with pancreatic islets display ß cell plasticity.

The direct co-culturing effect of rat bone-marrow-derived mesenchymal stem cells (rBM-MSCs) on the pancreatic-islets (PIs) was studied to obtain functional islet cells. MSCs were isolated from rat bone marrow and cultivated under standard conditions. Following their characterization, the rBM-MSCs were directly (with cell-islet contact) co-cultured with recovered PIs together with the single cell cultures of those cell cultures as a control. The effect of direct co-cultures of rBM-MSCs with the PIs of normal rats was investigated using immunophenotypical and functional methods. The change in the amount of insulin secretion was evaluated as an indicator for differentiation of rBM-MSCs. One approache for in vitro differentiation to achieve reprogramming for differentiation into suitable cell types by changing the microenvironment of the cells to provide signals that might activate metabolic pathways is to use co-cultures with the microenvironment of the specific cells of the desired cell type, tissue/organ extracts, extracellular matrix compounds or biologically absorbable materials. Differentiated rBM-MSCs were found to be immunopositive for the specific insulin-producing cell marker, insulin, but not in undifferentiated rBM-MSCs. The functionality tests by ELISA confirmed that insulin secretion of co-cultured MSCs with islets was higher than that of islets. These evidences indicated that PIs could be regarded as critical components of the stem cell niche, such that MSCs can be differentiated into insulin-producing cells (IPCs). Moreover, direct cell-to-cell contact might provide additional and independent support. This approach would circumvent the need for PI-stem cell co-culture and could potentially facilitate the production of functional IPCs for future clinical applications.

A comprehensive characterization study of human bone marrow mscs with an emphasis on molecular and ultrastructural properties.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) continue to draw attention of researchers in the fields of basic science and medicine due to their indispensible regenerative, reparative, angiogenic, anti-apoptotic, and immunosuppressive properties, all of which collectively point out their enormous therapeutic potential. There is still, however, a need for further investigation of their characteristics to broaden their field of use and learn much more about how to control their fate and improve their therapeutic effectiveness. hBM-MSCs were extensively characterized in terms of their growth characteristics, genetic stability, and differentiation capability to the mesodermal and ectodermal cell lineages; a special emphasis was given to their phenotypic and ultrastructural properties. Expression of embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog was shown with real-time PCR. Transmission electron microscopy revealed the ultrastructural characteristics of hBM-MSCs; they had pale, irregularly shaped and large euchromatic nuclei, and two distinct areas in their cytoplasm: an intensely stained inner zone rich in mitochondria and rough endoplasmic reticulum (rER) with dilated cisternae and a relatively peripheral zone poor in organelles. hBM-MSCs expressed adipogenic (adipophilin and PPARγ), myogenic (desmin, myogenin, α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP, ß3-tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, type I collagen), and chondrogenic (type II collagen, SOX9) markers either at RNA or protein level even under basal conditions, without any stimulation towards differentiation. The differentiation potential of hBM-MSCs to adipogenic, osteogenic, and neurogenic lineages was shown by using the relevant differentiation factors.

Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics.

BACKGROUND AIMS: Stem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat. METHODS: Immunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied. RESULTS: We found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells. CONCLUSIONS: The immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.