Poster V13 - RNA Sequencing and transfection of R28 retinal progenitor cell line with DNA topoisomerase-2 beta (Top2b)

Introduction: Diabetic retinopathy (DR) is a progressive and irreversible loss of vision in individuals with diabetes mellitus due to the breakdown of the inner blood retinal barrier. Subsequent angiogenesis of leaky blood vessels into the retina produces permanent photoreceptor cell death. There is no current treatment for diabetic retinopathy and preventative care is limited due to the often-late diagnosis. Regenerative medicine to restore vision is a potential treatment for diabetic retinopathy, but the delivery of transplanted progenitor cells to the injury site remains a challenge. Guiding the migration of transplanted cells poses a novel solution for delivery to injured sites. This project examined the transfection of cultured R28 cells with DNA topoisomerase-2 beta (Top2b), which is implicated in cell migration, proliferation, and regulation of chemotactic receptors such as VEGFR1, FGFR1, and CXCR4. The R28 cell line was examined using RNA sequencing to identify markers of different progenitor populations and the effect on transfection.

Materials and

Methods: RNA sequencing was performed on R28 cells plated on laminin coated TC plates at a density of 50,000 cells per well. Lipofectamine 3000 (Thermofisher) was used to transfect and overexpress Top2B into R28 cells, and a lentiviral vector (Origene) was used to transduce Top2B silencing, with respective positive controls. Puromycin was used to select for modified cells. Cells were fixed and immunostained for chemotactic receptor expression of VEGFR1, FGFR1, and CXCR4.

Results, Conclusions, and Discussions:

Results: After quality control was performed, the RNA seq of 8,712 R28 cells revealed five separate cell types within the heterogeneous cell line, with 52% of the R28s expressing as Muller glia (MG) precursor cells, 21% in a differentiation state of photoreceptors, 22% in a proliferative state, and smaller sub groups. R28s plated on laminin transfected to overexpress Top2b showed a significant increase in CXCR4 receptor expression as compared with the plasmid control (p=0.0307) , lentiviral control (p < 0.0001), and lentiviral knockdown (p < 0.0001) conditions.

Conclusion: The incidence of vision loss from DR in the USA is rapidly increasing with limited preventative methods or treatments. The overexpression of Top2b resulting in an increase in chemotactic receptor CXCR4 expression in R28 cells could provide an avenue for stimulating greater cell migration to target damaged areas of the retina. The presence of multiple cell types within this progenitor cell line could also provide a useful scaffold using R28s with overexpressed chemotactic receptors by multiple cell types for comprehensive targeting of retinal neurons.

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