Human Fibroblast Sheet Promotes Human Pancreatic Islet Survival and Function In Vitro

In previous work, we engineered functional cell sheets using bone marrow-derived mesenchymal stem cells (BM-MSCs) to promote islet graft survival. In the present study, we hypothesized that a cell sheet using dermal fibroblasts could be an alternative to MSCs, and then we aimed to evaluate the effects of this cell sheet on the functional viability of human islets. Fibroblast sheets were fabricated using temperature-responsive culture dishes. Human islets were seeded onto fibroblast sheets. The efficacy of the fibroblast sheets was evaluated by dividing islets into three groups: the islets-alone group, the coculture with fibroblasts group, and the islet culture on fibroblast sheet group. The ultrastructure of the islets cultured on each fibroblast sheet was examined by electron microscopy. The fibroblast sheet expression of fibronectin (as a component of the extracellular matrix) was quantified by Western blotting. After 3 days of culture, islet viabilities were 70.2 ± 9.8%, 87.4 ± 5.8%, and 88.6 ± 4.5%, and survival rates were 60.3 ± 6.8%, 65.3 ± 3.0%, and 75.8 ± 5.6%, respectively. Insulin secretions in response to high-glucose stimulation were 5.1 ± 1.6, 9.4 ± 3.8, and 23.5 ± 12.4 μIU/islet, and interleukin-6 (IL-6) secretions were 3.0 ± 0.7, 5.1 ± 1.2, and 7.3 ± 1.0 ng/day, respectively. Islets were found to incorporate into the fibroblast sheets while maintaining a three-dimensional structure and well-preserved extracellular matrix. The fibroblast sheets exhibited a higher expression of fibronectin compared to fibroblasts alone. In conclusion, human dermal fibroblast sheets fabricated by tissue-engineering techniques could provide an optimal substrate for human islets, as a source of cytokines and extracellular matrix

Cooperation by fibroblasts and bone marrow-mesenchymal stem cells to improve pancreatic rat-to-mouse islet xenotransplantation.

Experimental and clinical experiences highlight the need to review some aspects of islet transplantation, especially with regard to site of grafting and control of the immune response. The subcutaneous space could be a good alternative to liver but its sparse vasculature is its main limitation. Induction of graft tolerance by using cells with immunoregulatory properties is a promising approach to avoid graft rejection. Both Fibroblasts and Mesenchymal Stem Cells (MSCs) have shown pro-angiogenic and immunomodulatory properties. Transplantation of islets into the subcutaneous space using plasma as scaffold and supplemented with fibroblasts and/or Bone Marrow-MSCs could be a promising strategy to achieve a functional extra-hepatic islet graft, without using immunosuppressive drugs. Xenogenic rat islets, autologous fibroblasts and/or allogenic BM-MSCs, were mixed with plasma, and coagulation was induced to constitute a Plasma-based Scaffold containing Islets (PSI), which was transplanted subcutaneously both in immunodeficient and immunocompetent diabetic mice. In immunodeficient diabetic mice, PSI itself allowed hyperglycemia reversion temporarily, but the presence of pro-angiogenic cells (fibroblasts or BM-MSCs) within PSI was necessary to improve graft re-vascularization and, thus, consistently maintain normoglycemia. In immunocompetent diabetic mice, only PSI containing BM-MSCs, but not those containing fibroblasts, normalized glycemia lasting up to one week after transplantation. Interestingly, when PSI contained both fibroblasts and BM-MSCs, the normoglycemia period showed an increase of 4-times with a physiological-like response in functional tests. Histology of immunocompetent mice showed an attenuation of the immune response in those grafts with BM-MSCs, which was improved by co-transplantation with fibroblasts, since they increased BM-MSC survival. In summary, fibroblasts and BM-MSCs showed similar pro-angiogenic properties in this model of islet xenotransplantation, whereas only BM-MSCs exerted an immunomodulatory effect, which was improved by the presence of fibroblasts. These results suggest that cooperation of different cell types with islets will be required to achieve a long-term functional graft

Evolution of bioluminescent cells embedded in PSI and subcutaneously transplanted in both immunocompetent and immunodeficient mice.

<p>A) <i>Immunocompetent mice</i>. Relative luminescence signal of 10⁶ allogenic Luc[+]-BM-MSCs transplanted alone into PSI (PSI-5M_luc) or co-transplanted with 10⁶ autologous Luc[-]-fibroblasts (PSI-5FM_luc), as well as representative IVIS® lumina images of previously described mice, 12 days after transplantation. B) <i>Immunodeficient mice</i>. Relative luminescence signal of 10⁶ allogenic Luc[+]-BM-MSCs transplanted alone into PSI (PSI-M_luc) or co-transplanted with 10⁶ autologous Luc[-]-fibroblasts (PSI-FM_luc), as well as luminescence signal of 10⁶ allogenic Luc[+]-fibroblasts transplanted alone into PSI (PSI-F_luc). Representative IVIS® lumina images of previously described mice, 12 days after transplantation. Values related to day 0 (100%) are represented as mean ± SEM. For all groups n=5. (*) p<0.05; (**) p<0.01; (***) p<0.001.</p

Histology of subcutaneous islet xenografts in immunocompetent diabetic mice.

<p>A) Representative images of subcutaneously transplanted islets alone (ISC), PSI without cells and PSI containing: 10⁶ autologous fibroblasts (PSI-5F), 10⁶ allogenic BM-MSCs (PSI-5M), or 10⁶ of both autologous fibroblasts and allogenic BM-MSCs (PSI-5FM). Islet grafts were retrieved seven days after transplantation in diabetic mice. Samples were stained with H and E, or labelled with anti-insulin or anti-MPO antibodies. Black arrows point to islets. B) Quantification of the number of MPO-positive cells per field present on the leukocyte infiltration of the islet graft. C) Quantification of the insulin-positive area per section present on the islet graft. (**) p<0.01; (***) p<0.001.</p