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Brian G. Ballios, Laura Clarke, Molly S. Shoichet, Derek van der Kooy; A Bioengineered Delivery System For The Transplantation Of Mouse Retinal Stem Cell-derived Rod Photoreceptors Encourages Cell Survival And Integration. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2694.
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© ARVO (1962-2015); The Authors (2016-present)
Adult retinal stem cell (RSCs) derived from the ciliary epithelium of mice can give rise to all retinal cell types. RSC-derived photoreceptors have demonstrated functional recovery in mouse models of disease. The potential of RSC-derived rods in adult mouse transplantation models has been limited by poor cell distribution, survival and integration into host tissue. An injectable and biodegradable hydrogel material, a blend of hyaluronan and methylcellulose (HAMC), has shown promise in overcoming the cell distribution barrier. Here we report a mechanism through which HAMC directly supports the survival and integration of post-mitotic RSC-derived rods in vitro and in vivo.
RSC-derived rods were pre-differentiated on laminin substrate in the presence of taurine/retinoic acid, which can increase the percentage of rods differentiating in clonal RSC colonies to over 95% of the population. At 28 days, induction factors were removed and cells were exposed to either HAMC, HA-only, MC-only or serum free media (SFM) for 7 days. Survival was assessed with ethidium homodimer and phenotype by immunocytochemical (ICC) staining for rhodopsin. RSC-derived rods were harvested at various pre-differentiation time points and injected subretinally into adult mice. Cell distribution was analyzed between neural retina, subretinal space, and retinal pigment epithelium (RPE) in the HAMC v. saline vehicles, with or without addition of DL-alpha-aminoadipic acid (AAA).
Post-mitotic RSC-derived rod photoreceptors showed significantly improved survival in HA-containing mixtures (HAMC, HA-only: 90%) compared to those without (MC-only, SFM: 60%), with no change in rhodopsin expression. Activated caspase-3 levels showed that the pro-survival effect was through inhibition of apoptosis. ICC and Q-PCR analysis revealed the expression of the hyaluronan receptor CD44 on RSC-derived rods. The binding of HA to CD44 is known to influence cellular migration and survival. CD44-/- RSC-derived rods did not exhibit the pro-survival effect seen with HA-containing mixtures, demonstrating the specificity of this interaction. Transplantation of wild-type RSC-derived rods in HAMC+AAA results in enhanced survival and integration into neural retina, which is lost with CD44-/- cells.
HAMC directly influences RSC-derived rod survival in vitro and in vivo through the CD44 receptor. While glial barriers present a ceiling to cell integration numbers, cell survival in vivo is a critical pre-requisite for transplant efficacy.
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