LAUSR

Abstract Introduction: We recently showed improved engraftment and diabetes reversal when mature islets or stem-cell-derived beta cells (SCIPC) were cotransplanted with parathyroid gland (PTG). The CD34+ vascular endothelial progenitor cells, comprising 3-5% of the PTG, recapitulated this effect, whereas the CD34- cells were less effective. We conducted this study to test the hypothesis that PTG, particularly the CD34+ cells, protect islets by accelerating and enhancing neoangiogenesis. Materials and Methods: Human PTG was digested enzymatically and CD34+ and CD34- cells were purified using fluorescence-activated cell sorting. One quarter of a PTG, 1000IEQ human islets, 150,000 PTG CD34+ cells and 150,000 PTG CD34- cells were transplanted in the subcutaneous space (SQ) of immunodeficient NSG mice. Sham control mice received incisions without cell transplant. Skin flaps surrounding the transplant sites were created to reveal the vasculature at day 5 and 14 post-transplant and documented. Vessel area percentage and the number of vascular junctions in skin flaps was quantified using AngioTool (NIH/NCI). The mice were sacrificed and skin tissue was prepared for histology by fixing in 4% PFA and imbedding in OCT. Cryosections were stained with anti-human von Willebrand factor (vWF) antibody conjugated to Alexa 488, anti-mouse CD31 conjugated to Alexa 647 and DAPI and imaged with confocal microscopy. Results and Discussion: At 5 days post-transplant, skin flaps with PTG CD34+ transplant had 34.7% vessel area with 119.5 junctions. In comparison, PTG CD34- skin flaps had 28.7% vessel area and 83.5 junctions and sham skin flaps had 18.5% vessel area and 31.5 junctions. At 14 days post-transplant, CD34+ skin flaps had 33.4% vessel area and 101 junctions, the CD34- skin flaps had 19.5% vessel area and 38 junctions, and sham skin flaps had 21% vessel area and 10 junctions. Histologic assessment of skin that received human PTG and CD34+ cell transplants showed an abundance of human vWF staining, demonstrating human-derived vessels. These human vessels were surround by mouse vessels identified by mouse CD31-specific staining. Human vWF and mouse CD31 signals were observed in same vessels, suggesting chimeric vessel formation and anastomosis of human and mouse vessels. In contrast, the sham had no human blood vessels and the islet and 34- transplants were dominated by mouse vessels. Conclusion:  PTG, and in particular the CD34+ cells, induced early and sustained angiogenesis as evidenced by increased vessel area percentage and vascular junctions, compared with sham, islet and CD34- transplants. The vascular network is contributed by donor tissue and mouse vessels in PTG and PTG CD34+ transplants, but mostly by mouse vessels in PTG CD34- and islet transplants.