Multiple Human Progenitor Lineages Mediate the Endogenous Regeneration of Beta Cells Via Pro-Angiogenic and Putative Neogenic Function.

Abstract 2426

Poster Board II-403

In the context of regenerative therapies for diabetes, the contributions of transplanted stem cells are not limited to the direct replacement of damaged beta cells. We previously established that transplanted murine bone marrow (BM) stem cells stimulate endogenous beta cell proliferation and insulin production resulting in improved glycemic control. To enrich for analogous human progenitor subtypes that promote beta cell regeneration, we FACS-purified human BM and umbilical cord blood (UCB) cells based on aldehyde dehydrogenase (ALDH) activity, a conserved enzymatic function in multiple progenitor lineages. Compared to ALDH^lo cells, ALDH^hi cells from both human sources were enriched for progenitor phenotype, possessed multipotent hematopoietic colony forming cell function in vitro, and reconstitute hematopoiesis in NOD/SCID recipients. Human BM-derived ALDH^hi cells also contained multipotent mesenchymal colony forming cells (MCFC) (1 in 1.4×10³, n=5), whereas ALDH^lo cells established infrequent but expandable MCFC clones restricted to the adipose lineage. In contrast, both human UCB-derived ALDH-purified populations were devoid of MCFC capacity but the ALDH^hi population was enriched for endothelial colony forming cells (ECFC) (1 in 5.8×10⁴, n=4). To address beta cell regeneration in vivo, hyperglycemic (multiple low dose STZ-treated) NOD/SCID mice were tail vein injected with purified human ALDH^lo or ALDH^hi cells after sublethal (300cGy) preparative irradiation and blood glucose was monitored for >30 days. Compared to PBS injected controls (n=12), transplantation of BM-derived ALDH^lo (n=8) or ALDH^hi (n=10) cells induced a 2-fold decrease in systemic blood glucose concentrations within 7 days post transplantation, and significantly (p<0.01) reduced glycemia was maintained for >30 days. In contrast, the UCB-derived ALDH^hi population induced only a transient reduction of systemic blood glucose 7-18 days post-transplant, with the return of hyperglycemia and hypoinsulinemia by 30 days post-transplantation. Taken together, these data implicate the human BM mesenchmal lineage in hyperglycemia reduction. Although UCB-derived ALDH^hi cells did not augment the total islet number or beta cell mass at 30 days post-transplantation, ALDH^hi cell transplanted mouse pancreata contained significantly larger (p<0.05) insulin⁺ islets with increased vWF⁺ vessel density (p<0.05) compared to PBS controls, indicating that transiently reduced blood glucose may be due to beneficial pro-angiogenic effects of these cells on islet vascularization. To further investigate the direct role of transplanted MSC subtypes in glycemic recovery, ex vivo expanded ALDH^lo-MSC or ALDH^hi-MSC from 6 paired human BM samples were transplanted into hyperglycemic recipients and murine pancreata were analyzed for the endogenous regeneration of insulin⁺ islets. For 2 independent BM samples, transplantation of both ALDH^lo-MSC or ALDH^hi-MSC did not reduce established hyperglycemia, increase serum insulin, or show regeneration of insulin⁺ islets compared to PBS controls. For the remaining 4 BM samples, mice injected with cultured human BM ALDH^lo-MSC (n=16) or BM ALDH^hi-MSC (n=19) demonstrated permanently reduced systemic hyperglycemia and significantly increased serum insulin at 30 days post-transplantation (p<0.05). In contrast to UCB-transplanted mice, transplantation of cultured BM ALDH purified-MSC induced a significant increase (p<0.05) in islet number and beta cell mass, without an increase in islet size, suggesting that BM-derived MSC may initiate islet neogenesis. Thus, independent human BM samples showed variable capacity to regenerate islets, suggesting that cell type independent or extrinsic factors may also impact the regenerative capacity of human MSC. Finally, prolonged expansion of BM-derived MSC diminished the ability of transplanted cells to improve hyperglycemia. In summary, ALDH^hi mixed progenitor cells from human UCB contain pro-angiogenic progenitor subtypes that augment islet size and vascularization after transplantation, whereas expanded human BM-derived MSC increase regenerating islet number and total beta cell mass via a putative neogenic mechanism. Further characterization of the divergent pro-angiogenic and neogenic mechanisms by which specific cell types promote beta cell regeneration may provide new approaches for cellular therapy in diabetes.