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Jorn Lakowski, Yating Han, Rachael A. Pearson, Robin R. Ali, Jane C. Sowden; Effective Transplantation Of Photoreceptor Precursor Cells Selected Via Cell Surface Antigen Expression. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2225.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerative diseases resulting in the loss of photoreceptors are a major cause of untreatable blindness. Cell replacement therapy represents a feasible future treatment option. We previously demonstrated that post mitotic yet immature photoreceptor precursors expressing an Nrl.GFP transgene can integrate into the diseased murine retina and restore some visual function. As genetic modification of donor cells derived from stem cell cultures is not desirable for therapy, we have developed cell selection strategies using cell surface antigens and tested whether transplantation competency is maintained after fluorescence-activated cell (FAC) sorting.
Affymetrix microarrays were used to analyse the transcriptome of postnatal day 4 Nrl.GFP expressing precursors and genes encoding cell surface molecules were identified using gene ontology terms. Highly expressed cell surface antigens were verified by flow cytometry and RT-PCR. To test the feasibility of using cell surface markers for retinal therapy, FAC sorted cells were transplanted sub-retinally into the normal or diseased mouse eye. At 3 weeks post-transplantation, the number of newly integrated photoreceptor cells in the outer nuclear layer (ONL) was analyzed.
34 cell surface molecules expressed in P4 photoreceptor precursors were identified. Expression of Nt5e, which encodes a GPI-anchored protein was 23 fold higher in P4 Nrl.GFP+ve compared with P4 Nrl.GFP-ve cells. Cells from developing retinae were FAC sorted based on Nt5e expression using CD73 antibody and transplanted into adult retinae. Nt5e expressing cells migrated into the ONL, acquired the morphology of mature photoreceptors and expressed outer segment and synaptic markers. The median integration efficiency of the Nt5e-sorted cells was 4.35 fold higher than that of unsorted cells and is equivalent to sorting based on Nrl.GFP expression.
Transplantation competent rod precursor cells can be can be isolated from a complex mixture of cells using cell surface antigens without loss of viability or integration competence for the purpose of retinal therapy.
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