Primary Culture and Characterization of Postnatal Retinal Precursor Cells and Their Potential for Repair of the Damaged Ganglion Layer.

Retinal precursor cells (RPCs) derived from embryonic, mesenchymal, induced pluripotent, and neonatal sources have demonstrated significant potential in cell replacement therapies for retinal degeneration. Early postnatal RPCs (PND0-PND8) can integrate into the adult diseased retina; however, their limited survival reduces long-term therapeutic efficacy. In contrast, RPCs from later neonatal stages (>PND15), being more lineage-committed, may yield improved regenerative outcomes, particularly for Müller glia and photoreceptors. In this study, we evaluated RPCs isolated from PND12-PND15 Wistar rat pups obtained from age-matched breeding pairs to assess their pluripotency and regenerative capacity in vivo. A modified protocol incorporating EDTA in nuclease-free buffers was standardized for retinal dissection, processing, and incubation, followed by culture in neurobasal medium supplemented with B27 and FGF2. Undifferentiated neurospheres expressing stem cell markers Nanog and Sox2 were dissociated at 96 hr and transplanted into an adult N-methyl-d-aspartate-induced retinal degeneration model. Compared with early postnatal RPCs (PND2-PND4), which formed smaller, slow-growing neurospheres (2.58 ± 0.15 µm) with rapid differentiation, PND12-PND15 RPCs generated larger, stable neurospheres (4.5 ± 0.18 µm) within 48 hr. Pilot data indicate enhanced potential of these cells in promoting retinal ganglion layer repair.