Abstract
Abstract: :
Purpose: We have previously reported that bone marrow (BM) derived lineage minus hematopoietic stem cells (Lin– HSC) have both vasculo– and neurotrophic effects after intravitreal injection into mouse eyes. In this study we demonstrate that similar populations of cells exist in human (BM) and exhibit similar rescue properties when injected into the eyes of SCID mice with retinal degeneration. Methods: Human BM (hBM) mononuclear cells were obtained from BM of healthy volunteers. To isolate a Lin– cell population from hBM mononuclear cells, the following antibodies were used with MACS separation system; CD2, 3, 4, 11a, 11b, 14, 16, 19, 33, 38, 45RA, 64, 68, 86, 235a (Pharmingen). To visualize the injected human cells, cells were labeled with dye (cell tracker green CMFDA, Molecular Probes) before injection. Cells (approximately 5x105/0.5µl) were injected intravitreally into rd1/rd1 mice using a 33–gauge needled–syringe (Hamilton). To increase the accuracy of data, we injected Lin– cells into one eye and control cells into the contralateral eye in the same animal and compared the effect. Retinas were harvested at various time points, fixed and then stained with lectin or antibodies to CD31 followed by Alexa 488 or 594 conjugated secondary antibodies. Vascular images were obtained using a BioRad MP2100 confocal microscope equipped with a Spectra–Physics Tsunami Ti:sapphire laser and Lasersharp Software. Results: The injected hLin– HSC migrated to and targeted sites of retinal angiogenesis in a fashion identical to that observed when mouse Lin–HSC were injected. In addition to the vascular targeting, the human stem cells also provided a robust rescue effect on both the vascular and neuronal cell layers of the rd1/rd1 mice. Conclusions: These results confirm the presence of cells in hBM that target retinal vasculature and can prevent its degeneration in mice with inherited retinal degeneration. While several recent reports have described partial phenotypic rescue in mice and dogs with retinal degeneration after viral based gene therapy with the wild type gene, this is the first report of a cell based therapeutic effect achieved by vascular rescue with human cells.
Keywords: retinal degenerations: cell biology • retinal neovascularization • degenerations/dystrophies