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T. V. Johnson, N. D. Bull, K. R. Martin; Müller Glial Reactivity Prevents Retinal Integration of Intravitreally Transplanted Stem Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6232.
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© ARVO (1962-2015); The Authors (2016-present)
Intraocular stem cell transplantation may be therapeutic for retinal neurodegenerative disease via neuronal replacement and/or neuroprotection. However, efficacy is presently hindered by the extremely low level of graft migration into the neural retina. Our aim was to identify the major barrier to retinal integration of intravitreally transplanted stem cells, which we hypothesized to include the cellular and/or extracellular matrix (ECM) components of the inner limiting membrane (ILM). We used a novel in vitro retinal explant system, in vivo stem cell transplantation, and selective disruption of different ILM components to characterize the key inhibitory elements of the ILM.
Bone marrow-derived mesenchymal stromal cells (MSCs) were co-cultured on the vitreal surface of retinal explants. Retinal MSC migration was compared between control explants and explants in which: a) portions of the ILM (including basal lamina and Müller cell endfeet) were removed by mechanical peeling, N=8 per group; b) the inner basal lamina was digested with collagenase (0.2U or 0.05U, N=4 per group); c) Müller reactivity was selectively modulated with alpha aminoadipic acid (AAA, 200µg, N=8 per group). In vivo, MSCs were injected intravitreally two days after intravitreal AAA (N=4) or saline (N=3) injection into rats with laser-induced ocular hypertension. Cell integration, ILM integrity, and glial reactivity were assessed by immunohistochemistry (IHC).
The amount of peeled ILM was positively correlated with the number of MSCs migrating into the retina (R2=0.83). Enzymatic digestion of the basal lamina was robust as demonstrated by anti-collagen IV and anti-laminin IHC, but did not enhance migration of MSCs. In contrast, AAA treatment downregulated glial fibrillary acid protein, vimentin, and nestin expression in Müller glia and dramatically enhanced the percentage of MSCs that had migrated into the retina after 7d (39.3±11.4% vs 0.8±0.4%, mean±SEM, p<0.01). The positive effect of AAA treatment on retinal graft migration was preserved in vivo (31.4±6.0 vs. 7.6±1.5 integrated MSCs per cryosection, p=0.02).
Our results demonstrate that while the ECM of the inner basal lamina is neither necessary nor sufficient to prevent migration of grafted cells into the neural retina, Müller glial reactivity is associated with poor graft migration and targeted disruption of Müller reactivity dramatically improves the structural integration of intravitreally transplanted cells.
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