Int J Stem Cells.  2020 Mar;13(1):116-126. 10.15283/ijsc19094.

Protective Effect of Human Mesenchymal Stem Cells on the Survival of Pancreatic Islets

• Fumagalli
• Monfrini M
• Donzelli E
• Rodriguez-Menendez V
• Bonandrini B
• Figliuzzi
• Remuzzi A
• D’Amico G
• Cavaletti
• Scuteri A

• KMID: 2500571
• DOI: http://doi.org/10.15283/ijsc19094

Abstract

Background and Objectives
Transplantation of pancreatic islets is an intriguing new therapeutic option to face the worldwide spread problem of Type-I diabetes. Currently, its clinical use is limited by several problems, mainly based on the high number of islets required to restore normoglycaemia and by the low survival of the transplanted tissue. A promising attempt to overcome the limits to such an approach was represented by the use of Mesenchymal Stem Cells (MSC). Despite the encouraging results obtained with murine-derived MSC, little is still known about their protective mechanisms. The aim of the present study was to verify the effectiveness, (besides murine MSC), of clinically relevant human-derived MSC (hMSC) on protecting pancreatic islets, thus also shedding light on the putative differences between MSC of different origin.
Methods and Results
Threefold kinds of co-cultures were therefore in vitro set up (direct, indirect and mixed), to analyze the hMSC effect on pancreatic islet survival and function and to study the putative mechanisms involved. Although in a different way with respect to murine MSC, also human derived cells demonstrated to be effective on protecting pancreatic islet survival. This effect could be due to the release of some trophic factors, such as VEGF and Il-6, and by the reduction of inflammatory cytokine TNF-α.
Conclusions
Therefore, hMSC confirmed their great clinical potential to improve the feasibility of pancreatic islet transplantation therapy against diabetes.

Keyword

Type-I diabetes; Pancreatic islets; Mesenchymal stem cells; Soluble factors

Figure

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  Fig. 1 Islet morphology in co-cultures. (a) 500,000 hMSC were stained in red with the vital fluorescent dye DiI and direct cultured with 500 pancreatic islets stained in green with Calcein dye. hMSC were able to coat pancreatic islets. In green: pancreatic islets. In red: hMSC. (b) Pancreatic islets in directed co-culture with hMSC and (c) pancreatic islets co-cultured indirectly with hMSC at the optical microscope. Bar 150 μm.

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  Fig. 2 Survival of pancreatic islets. Pancreatic islet viability was investigated both by observing the presence and distribution of the vital dye Calcein, and by a pancreatic islet count. (a) Representative image of pancreatic islets cultured alone acquired by an inverted microscope: the number of viable cells able to convert the dye in the green fluorescent form is very limited. (b) Representative image of the healthier condition of pancreatic islets cultured in presence of hMSC, irrespective of the co-culture paradigm, showing a uniform spread of the vital dye Calcein up to 3 weeks. Bar 150 μm. (c) The graph showed the values obtained with the weekly count of viable islets alone or co-cultured. The values are shown as survival percentages and are expressed as the mean±SD of three independent experiments. *p<0.01 islets vs direct co-cultures, °p<0.05 islets vs indirect co-cultures and islets vs mixed co-cultures.

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  Fig. 3 Apoptosis detection. Pancreatic islets (Islets), direct co-cultures (Direct), indirect co-cultures (Indirect) and mixed co-cultures (Mixed) were analyzed with a specific antibody recognizing the active form of caspase 3 (a) and the active form of caspase 7 (b). In green the pancreatic islets stained with Calcein. In red the hMSC stained with the DiI dye. Bar 50 μm.

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  Fig. 4 Insulin release following glucose stimulation after 3 weeks of culture (T3). Islets in culture alone and in co-culture were exposed to different concentrations of glucose in the culture medium (20 mM and 1.67 mM). After one hour of exposure to each concentration, medium was collected and analyzed by ELISA. The results are expressed as mean±SD of three independent experiments. *p<0.01 vs islets, #p<0.05 vs islets.

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  Fig. 5 Evaluation of insulin expression in pancreatic islets alone or in direct co-cultures. Sections from pancreatic islets (a), direct co-cultures (b), hMSC cultured alone (c), mixed co-cultures (d), and indirect co-cultures with islets into the Transwell (e) and hMSC under the Transwell (f) were analyzed at a confocal microscope with a specific antibody for insulin. In green the pancreatic islets stained with Calcein. In red the hMSC stained with the DiI dye. Positive cells for insulin in blue. Bar 50 μm and 30 μm.

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  Fig. 6 Release of trophic factor analysis. Release of VEGF (a), TNF-α (b) and IL-6 (c) in medium after 3 weeks of culture (T3). The concentrations were determined by ELISA assay. The results are expressed as mean±SD of three independent experiments. ** p<0.001 vs islets.

Reference

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