Avoiding immunological rejection in regenerative medicine.

One of the major goals of regenerative medicine is repair or replacement of diseased and damaged tissues by transfer of differentiated stem cells or stem cell-derived tissues. The possibility that these tissues will be destroyed by immunological rejection remains a challenge that can only be overcome through a better understanding of the nature and expression of potentially immunogenic molecules associated with cell replacement therapy and the mechanisms and pathways resulting in their immunologic rejection. This review draws on clinical experience of organ and tissue transplantation, and on transplantation immunology research to consider practical approaches for avoiding and overcoming the possibility of rejection of stem cell-derived tissues.

Functional characterization of late outgrowth endothelial progenitor cells in patients with end-stage renal failure.

Renal transplantation is potentially curative in renal failure, but long-term efficacy is limited by untreatable chronic rejection. Endothelial damage contributes to chronic rejection and is potentially repairable by circulating endothelial progenitor cells (EPC). The frequency and function of EPC are variably influenced by end-stage renal failure (ESRF). Here, we isolated and functionally characterized the late outgrowth EPC (LO-EPC) from ESRF patients to investigate their potential for endothelial repair. Patients with ESRF generated more LO-EPC colonies than healthy controls and had higher plasma levels of IL-1rα, IL-16, IL-6, MIF, VEGF, Prolactin, and PLGF. Patients' LO-EPC displayed normal endothelial cell morphology, increased secretion of PLGF, MCP-1, and IL-1ß, and normal network formation in vitro and in vivo. They demonstrated decreased adhesion to extracellular matrix. Integrin gene profiles and protein expression were comparable in patients and healthy volunteers. In some patients, mesenchymal stem cells (MSC) were co-isolated and could be differentiated into adipocytes and osteocytes in vitro. This is the first study to characterize LO-EPC from patients with ESRF. Their behavior in vitro reflects the presence of elevated trophic factors; their ability to proliferate in vitro and angiogenic function makes them candidates for prevention of chronic rejection. Their impaired adhesion and the presence of MSC are areas for potential therapeutic intervention.

Lentiviral-mediated overexpression of Bcl-xL protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis.

BACKGROUND: Endothelial cells (EC) respond to mild injurious stimuli by upregulating anti-apoptotic gene expression to maintain endothelial integrity. EC dysfunction and apoptosis resulting from ischemia/reperfusion injury may contribute to chronic allograft rejection. We optimized conditions for lentiviral vector (LVV) transduction of rat aortic endothelial cells (RAEC) and investigated whether LVV delivery of the anti-apoptotic gene, Bcl-xL, protects RAEC from apoptotic death using in vitro models of hypoxia and ischemia/reperfusion injury. METHODS: LVV containing Bcl-xL were generated from a human immunodeficiency virus (HIV)-1 construct. EC were prepared from rat aorta. Hypoxia/reperfusion (H/R) or ischemia/reperfusion (I/R) injury was induced in vitro and apoptosis was assessed using caspase-3 activity, Annexin V/PI and TUNEL staining. RESULTS: After in vitro induction of H/R or I/R injury, RAEC showed duration-dependent apoptosis. We confirmed the damaging effect of the reperfusion phase. Endogenous Bax expression increased with I/R injury, whereas endogenous Bcl-xL remained constant. RAEC transduced with LVV expressing Bcl-xL were protected from early apoptosis caused by I/R injury, correlating with reduced cytochrome c release into the cytosol. CONCLUSIONS: Overexpressing Bcl-xL protects RAEC from I/R injury. This protective effect may be attributed to altering the balance of pro- and anti-apoptotic proteins, resulting in sequestration of the harmful Bax protein, and may open up new strategies for controlling chronic allograft rejection.