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

Genes differentially expressed in PSI group vs. PSI-F group.

<p>Heat map represents the relative expression levels of identified genes (logFC2 PSI vs. PSI-F) in each day as determined in a GeneChip Mouse Gene 2.0 Array analysis (being green the higher expression and red the lower). Right column indicates gene name. Additionally, in each day the heat map for any individual gene represents the variation of its expression during all the samples taken for our analysis (0, 1, 3, 7 and 10 days). Genes have been arranged from maximum to minimum fold expression differences in any given day after transplantation.</p

Fibroblasts accelerate islet revascularization and improve long-term graft survival in a mouse model of subcutaneous islet transplantation

<div><p>Pancreatic islet transplantation has been considered for many years a promising therapy for beta-cell replacement in patients with type-1 diabetes despite that long-term clinical results are not as satisfactory. This fact points to the necessity of designing strategies to improve and accelerate islets engraftment, paying special attention to events assuring their revascularization. Fibroblasts constitute a cell population that collaborates on tissue homeostasis, keeping the equilibrium between production and degradation of structural components as well as maintaining the required amount of survival factors. Our group has developed a model for subcutaneous islet transplantation using a plasma-based scaffold containing fibroblasts as accessory cells that allowed achieving glycemic control in diabetic mice. Transplanted tissue engraftment is critical during the first days after transplantation, thus we have gone in depth into the graft-supporting role of fibroblasts during the first ten days after islet transplantation. All mice transplanted with islets embedded in the plasma-based scaffold reversed hyperglycemia, although long-term glycemic control was maintained only in the group transplanted with the fibroblasts-containing scaffold. By gene expression analysis and histology examination during the first days we could conclude that these differences might be explained by overexpression of genes involved in vessel development as well as in β-cell regeneration that were detected when fibroblasts were present in the graft. Furthermore, fibroblasts presence correlated with a faster graft re-vascularization, a higher insulin-positive area and a lower cell death. Therefore, this work underlines the importance of fibroblasts as accessory cells in islet transplantation, and suggests its possible use in other graft-supporting strategies.</p></div

Subcutaneous transplantation of PSI in immunodeficient diabetic mice.

<p>(A) Follow up of NFBG in the following groups (n = 8 per group): (▲, Diabetic) non-transplanted diabetic mice; (♦, ISC) diabetic mice subcutaneously transplanted with free islets; (■, PSI) diabetic mice subcutaneously transplanted with PSI; (●, PSI-F) diabetic mice subcutaneously transplanted with PSI containing 2 x 10⁵ fibroblasts. ^†p<0.001 vs. PSI and PSI-F; ^#p<0.05 vs. PSI-F; ^‡p<0.01 vs. PSI-F; ^§p<0.001 vs. PSI-F. (B) Kaplan-Meier mice survival curve. Groups are the same as detailed in A (n = 8 per group). Mean survival time (MST) is indicated for each group. ^†p<0.01 vs. Diabetic; ^#p<0.001 vs. PSI and PSI-F; ^‡p<0.001 vs. Diabetic. (C) Blood glucose responses to IPGTTs, 90 days after transplantation. Groups are the same as detailed in A, except for (◊, Control) non-transplanted healthy mice (n = 8 per group, except for PSI where n = 5). (D) Area under the curve (AUC) data obtained from IPGTTs. *p<0.05; **p<0.01.</p

Pathways represented by specifically expressed genes in PSI-F (www.babelomics.org).

<p>Pathways represented by specifically expressed genes in PSI-F (<a href="http://www.babelomics.org" target="_blank">www.babelomics.org</a>).</p

Histology of subcutaneous islet grafts along the first 10 days after transplantation.

<p>(A) Histological images of CD31 (to detect vessels) and insulin (to detect beta cells) immunostaining of PSI and PSI-F grafts at days 1, 3, 7 and 10 after transplantation (Black arrows point to islets and red arrows point to vessels). (B) Quantification of vessels in the subcutaneous grafts after CD31 immunostaining at days 1, 3, 7 and 10 after transplantation. (C) Quantification of apoptotic cells per area in PSI and PSI-F grafts at days 1, 3, 7 and 10 after transplantation. *p<0.05; **p<0.01; ***p<0.001.</p

Genes expressed on the first ten days after transplantation on subcutaneous islet grafts.

<p>(A) Pattern of expression of Interleukin 6, Interleukin 1 beta, Chitinase 3, Regenerating islet-derived 1 and Regenerating islet-derived 3 gamma after transplantation. (B) Number of genes specifically expressed in PSI, PSI-F and commonly expressed in both groups when compared with Diabetic group.</p