Human multipotent adult progenitor cells enhance islet function and revascularisation when co-transplanted as a composite pellet in a mouse model of diabetes

Abstract

Aims/hypothesis: Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival.

Methods: Islets (150) were co-transplanted, with or without human MAPCs (2.5 × 10⁵) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome.

Results: Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet-human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet-human MAPC composites.

Conclusions/interpretation: The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function.

Keywords: Islet; Revascularisation; Stem cells; Type 1 diabetes.

Fig. 1

Characterisation of human MAPCs. (a) Cell surface marker expression of human bone marrow-derived MAPCs. Flow cytometry histograms show the expression levels (peaks shaded dark grey) of selected markers associated with the characterisation of human MAPCs (CD44 and CD105) compared with negative isotype controls (peaks shaded light grey). (b) Culture medium of human MAPCs was analysed with human biomarker 40-Plex kit containing a pro-inflammatory panel, cytokine panel, chemokine panel, angiogenesis panel and vascular inflammation panel. Data are means ± SEM. SAA, serum amyloid A; sVCAM-1, soluble vascular cell adhesion molecule-1; CRP, C-reactive protein. (c) Pro-angiogenic properties of 2.5 × 10⁵ human MAPCs (hMAPC) with or without 150 C57BL/6 mouse islets in a CAM assay; 150 C57BL/6 mouse islets alone and BSA were used as negative controls and VEGF-A as positive control (mean ± SEM, n = 3–9 per group). **p < 0.01 and ***p < 0.001 for indicated comparisons

Fig. 2

In vivo function of a marginal islet mass co-transplanted with human MAPCs. (a) Blood glucose measurements of alloxan-induced diabetic C57BL/6 mice transplanted with 150 islets alone (control [n = 47]; white bars) or with 150 islets co-transplanted as separate (n = 52; dark-grey bars) or composite pellets (n = 40; light-grey bars) with 2.5 × 10⁵ human MAPCs. Floating bars extend from the minimum to the maximum, the line indicates the mean.*p < 0.05, **p < 0.01 and ***p < 0.001 vs islets alone (control). (b) Percentage of cured (black bars) and non-cured (grey bars) mice after islet transplantation. (c–f) AUCs and blood glucose measurements after IPGTTs in mice transplanted with a marginal islet mass alone (control, white circles) or combined with human MAPCs as a separate (crossed circles) or composite pellet (grey circles) 2 (c, d) and 5 (e, f) weeks after transplantation. **p < 0.01 for indicated comparisons. The box extends from the 25th to 75th percentiles, the line indicates the median, and the Tukey method was used to plot the whiskers and the outliers (solid black dots); (d, f) mean ± SEM

Fig. 3

Morphology and composition of islets co-transplanted with human MAPCs examined at 2 and 5 weeks post-transplantation. (a–c) Box and whiskers plots of mRNA levels of mouse insulin, glucagon and somatostatin in isolated islet grafts derived from mice transplanted with a marginal islet mass alone (control) or combined with human MAPCs as a separate or composite pellet. Values were normalised to the geometric mean of housekeeping genes. Data are expressed as $2^{-\mathrm{\Delta }\mathrm{\Delta }{\mathrm{C}}_{\mathrm{t}}}$. The box extends from the 25th to 75th percentiles, the line indicates the median, and the Tukey method was used to plot the whiskers and the outliers. Statistical significance was calculated using Mann–Whitney t tests. *p < 0.05 for indicated comparisons. (d–f) Box and whiskers plots of volumes of beta, alpha and delta cells of grafts derived from mice transplanted with a marginal islet mass alone or combined with human MAPCs as a separate or composite pellet. The box extends from the 25th to 75th percentiles, the line indicates the median, and the Tukey method was used to plot the whiskers and the outliers. Statistical significance was calculated using Mann–Whitney t tests. *p < 0.05 and **p < 0.01 for indicated comparisons. (g) Distribution of mouse insulin- (white), glucagon- (red), and somatostatin (green)-positive cells in islet grafts composed of islets and human MAPCs as separate (SEP) or composite (MIX) pellets or of islets alone (control) at 2 weeks post-transplantation. Stitched composite images are representative of sections from 4–8 different mice. Scale bar, 100 μm. Higher magnification of the boxed area in control demonstrates a compact graft with normal distribution of insulin, glucagon and somatostatin positivity. Higher magnification of the boxed area in the SEP and MIX groups shows spread-out grafts with high insulin and glucagon positivity

Fig. 4

Co-transplantation of islets with human MAPCs as composites promotes graft revascularisation in a marginal islet mass diabetic mouse model. (a) Representative sections of 5 week grafts consisting of mouse islets transplanted alone (control) or with human MAPCs as separate or composite pellets. Images are representative of insulin and endomucin (vessel) staining for 3 or 4 mice in each transplant group. Scale bar, 100 μm. (b–d) Assessment of vessel morphological variables was carried out as described in the Methods. Data are means ± SEM. Statistical analysis was performed using Mann–Whitney t tests. *p < 0.05 and **p < 0.01 for indicated comparisons