Differentiation of Mesenchymal Stem Cells towards an insulin-releasing phenotype after co-culture with Pancreatic Islets

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001

Positive effect of Mesenchymal Stem Cells therapeutic administration on chronic Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001

A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets

The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs) with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an "insulin-releasing" phenotype. Two distinct mechanisms mediated these effects: i) the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii) MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility

Insulin release after glucose stimulation.

<p>Each week (up to 4 weeks of culture) pancreatic islets cultured alone or directly co-cultured with MSCs, and MSCs cultured alone were exposed to different glucose concentrations in the culture medium (20 mM and 1,67 mM Glucose), and the insulin release after each change was measured by an ELISA assay specific for this hormone. Results are expressed as mean ± SD of three independent experiments. * <i>P</i><0.05 Islets vs Islets+MSCs, ^○○<i>P</i><0.01 Islets+MSCs vs MSCs.</p

Insulin and Pdx1 detection.

<p>Insulin positive cells in pancreatic islets (a), in MSCs (b), and in direct co-cultures of pancreatic islets and MSCs (c and d). In green, calcein positive pancreatic islets. In red, MSCs stained with DiI. In blue, insulin positive cells. Bar 30 µm. Pdx1 positive cells in pancreatic islets (e), in MSCs (f), and in direct co-cultures of pancreatic islets and MSCs (g). In green, calcein positive pancreatic islets. In red, MSCs stained with DiI/Phalloidin. In blue, Pdx1 positive cells. Bar 30 µm.</p

Viability assessment of MSC-coated pancreatic islets.

<p>The viability of pancreatic islets coated with MSCs was assessed by using the vital fluorescent dye calcein, which evidenced in green only viable cells. Upper panel: pancreatic islets cultured alone stained with calcein after 2, 3 and 4 weeks of culture. Lower panel: pancreatic islets directly co-cultured with DiI red-stained MSCs which appear as yellow spots after 2, 3 and 4 weeks of culture. Arrows indicate pancreatic islets in which calcein did not spread uniformly. Bar 150 µm. b) Percentage of pancreatic islets' survival, expressed as mean ± SD. Calcein-positive pancreatic islets were counted and the survival percentage was calculated up to 4 weeks of culture.</p

MSC adhesion to pancreatic islets.

<p>MSCs previously stained with the red vital fluorescent dye DiI were directly added to green calcein-stained floating pancreatic islets. Attached MSCs are clearly visible as yellow spots (arrows) on green pancreatic islets after 1 week of culture (a) as well as after 4 weeks (b). In green: calcein stained pancreatic islets. In red: DiI stained MSCs. Bar 150 µm.</p