P-193

Incorporation of rat vascular progenitor cells into mosaic islet clusters to promote engraftment of transplanted pancreatic islets

D. Penko¹, C. Mee², D. Mohanasandarum², P.T.H. Coates², C.S. Bonder³, C.F. Jessup^1
1 The University of Adelaide, School of Medicine, Adelaide, Australia; ² The Royal Adelaide Hospital, Central Northern Adelaide Renal and Transplant Service, Adelaide, Australia; ³ Hanson Institute, SA Pathology, Vascular Biology Laboratory, Adelaide, Australia

Pancreatic islet transplantation is an emerging cure for Type 1 Diabetes. Success is limited by death of the insulin-producing ?-cells within the islet, from insufficient provision of oxygen and nutrients via the vasculature. Vasculogenic endothelial progenitor cells (EPCs) have the potential to improve islet engraftment.

Objective: To incorporate EPCs and islet cells into functional mosaic clusters in vitro.

Methods: Rat bone marrow-derived EPC were enriched by culture with endothelial growth factors on fibronectin. Phenotype was confirmed by flow cytometry and Matrigel^TM assay. An embryoid body-forming medium enhanced the aggregation of dispersed rat islet cells and DiI-Acetylated Low Density Lipoprotein (DiI)-labeled EPCs over 3 days (d3) following centrifugation (400g/2min). Composition of clusters was examined via the ?-cell marker Newport Green (NG) and analysed by flow cytometry and confocal microscopy. In vitro function was measured by static glucose-stimulated insulin release in response to high (25mM) and low (2.8mM) glucose.

Results: In culture, EPCs upregulated endothelial markers VEGFR2 and VCAM1 by d14 and formed tube-like structures in Matrigel^TM. Mosaic clusters (diameter=142µm±32; n=20) contained more NG+ cells by d3 (76%) compared to clusters with islet cells alone (107µm±13; n=20; 49% NG+). DiI+EPC were distributed throughout mosaic clusters by confocal microscopy and comprised 41% of cells by d3. Mosaic clusters produced more insulin in high glucose (11347±1134pg/L) compared to clusters without EPC (6719±246pg/L) (Fig 1).

Conclusion: Mosaic clusters were functional in vitro and will be tested for their ability to enhance islet engraftment in vivo.

Figure 1. Glucose-stimulated insulin release in day 3 whole islets, reformed islets and mosaic clusters. Insulin secretion (y-axis) was compared between whole intact islets (solid bars), reformed islets (white bars) and mosaic clusters (hatched bars) in low (2.8mM) or high (25mM) glucose. Bars represent mean+/-SEM. (n=3-4 replicates/group; 10 clusters of 10 IEQ/test; *p=0.01 by students t-test, **p<0.05 by one-way ANOVA).

P-193

Incorporation of rat vascular progenitor cells into mosaic islet clusters to promote engraftment of transplanted pancreatic islets

D. Penko¹, C. Mee², D. Mohanasandarum², P.T.H. Coates², C.S. Bonder³, C.F. Jessup^1
1 The University of Adelaide, School of Medicine, Adelaide, Australia; ² The Royal Adelaide Hospital, Central Northern Adelaide Renal and Transplant Service, Adelaide, Australia; ³ Hanson Institute, SA Pathology, Vascular Biology Laboratory, Adelaide, Australia

Pancreatic islet transplantation is an emerging cure for Type 1 Diabetes. Success is limited by death of the insulin-producing ?-cells within the islet, from insufficient provision of oxygen and nutrients via the vasculature. Vasculogenic endothelial progenitor cells (EPCs) have the potential to improve islet engraftment.

Objective: To incorporate EPCs and islet cells into functional mosaic clusters in vitro.

Methods: Rat bone marrow-derived EPC were enriched by culture with endothelial growth factors on fibronectin. Phenotype was confirmed by flow cytometry and Matrigel^TM assay. An embryoid body-forming medium enhanced the aggregation of dispersed rat islet cells and DiI-Acetylated Low Density Lipoprotein (DiI)-labeled EPCs over 3 days (d3) following centrifugation (400g/2min). Composition of clusters was examined via the ?-cell marker Newport Green (NG) and analysed by flow cytometry and confocal microscopy. In vitro function was measured by static glucose-stimulated insulin release in response to high (25mM) and low (2.8mM) glucose.

Results: In culture, EPCs upregulated endothelial markers VEGFR2 and VCAM1 by d14 and formed tube-like structures in Matrigel^TM. Mosaic clusters (diameter=142µm±32; n=20) contained more NG+ cells by d3 (76%) compared to clusters with islet cells alone (107µm±13; n=20; 49% NG+). DiI+EPC were distributed throughout mosaic clusters by confocal microscopy and comprised 41% of cells by d3. Mosaic clusters produced more insulin in high glucose (11347±1134pg/L) compared to clusters without EPC (6719±246pg/L) (Fig 1).

Conclusion: Mosaic clusters were functional in vitro and will be tested for their ability to enhance islet engraftment in vivo.

Figure 1. Glucose-stimulated insulin release in day 3 whole islets, reformed islets and mosaic clusters. Insulin secretion (y-axis) was compared between whole intact islets (solid bars), reformed islets (white bars) and mosaic clusters (hatched bars) in low (2.8mM) or high (25mM) glucose. Bars represent mean+/-SEM. (n=3-4 replicates/group; 10 clusters of 10 IEQ/test; *p=0.01 by students t-test, **p<0.05 by one-way ANOVA).