The combination of bFGF and CHIR99021 maintains stable self-renewal of mouse adult retinal progenitor cells

Millions of people are affected with retinal diseases that eventually cause blindness, and retinal progenitor cell (RPC) transplantation is a promising therapeutic avenue. However, RPC expansion and the underlying regulation mechanisms remain elusive.

Present study developed an approach to maintain long-term self-renewal of RPCs from adult retinal tissues and revealed that activation of the non-canonical Wnt5A/Ca2+ pathway may participate in regulating RPC self-renewal in vitro. This study presents a very promising platform to expand RPCs for future therapeutic application.

Adult mouse neural RPCs (mNRPCs) were isolated and amplified with the combination of basic fibroblast growth factor (bFGF) and glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021. The progenitor characteristics were evaluated with RT-PCR, immunocytochemistry (ICC), western blot, flow cytometry, and transcriptome analysis prior to transplantation. By treating cells with or without bFGF and CHIR99021 at different time points, the mechanism for mNRPCs' self-renewal was investigated by transcriptome analysis and western blot assay.

mNRPCs were self-renewing in the presence of bFGF and CHIR99021 and showed prominent RPC characteristics. bFGF was essential in promoting cell cycle by facilitating G1/S and G2/M transitions. bFGF combined with CHIR99021 activated the non-canonical Wnt5A/Ca2+ pathway and form a calcium homeostasis. In addition, the self-renewing mNRPCs could differentiate into rod photoreceptor-like cells and retinal pigment epithelium (RPE)-like cells by in vitro induction. When green fluorescent protein (GFP)-labeled cells were transplanted into the subretinal space (SRS) of Pde6b (rd1) mice (also known as RD1 mice, or rodless mice), the cells survived for more than 12 weeks and migrated into the retina. Parts of the recipient retina showed positive expression of photoreceptor marker rhodopsin. Transplanted cells can migrate into the retina, mainly into the inner cell layer (INL) and ganglion cell layer (GCL). Some cells can differentiate into astrocytes and amacrine cells. Cultured mNRPCs did not form tumors after transplanted into NOD/SCID mice for 6 months. Conclusions: Present study developed an approach to maintain long-term self-renewal of RPCs from adult retinal tissues and revealed that activation of the non-canonical Wnt5A/Ca2+ pathway may participate in regulating RPC self-renewal in vitro. This study presents a very promising platform to expand RPCs for future therapeutic application.