Incorporation of Transdifferentiating RPE Cells into the Retinal Ganglion Cell Layer

Abstract

Abstract: : Purpose: Our previous studies indicate that cultured RPE cells can be guided to differentiate towards various types of retinal neurons. This study aims at evaluating the behavior and the developmental potential, after microinjection into the eye, of cultured RPE cells induced by bFGF and cath5 to transdifferentiate towards retinal ganglion cells. Methods: RPE cells were isolated from day 6 chick embryos and were cultured in the presence of bFGF and infected with RCAS-cath5. Cells in the culture were harvested and microinjected into embryonic chick eye or adult mouse eye damaged with NMDA. At various time points after cell injection, the eyes were fixed and the distributions of grafted cells within the retina were analyzed. Results: In the presence of bFGF and with ectopic expression of cath5, cultured RPE cells began to express retinal ganglion cell markers, such as RA4, 3A10, and 4H6. When microinjected into the developing chick eye, the majority of these transdifferentiating RPE cells, recognized either by the presence of a viral protein or by a tracer dye (DiI or Hoechst), was found in the ganglion cell layer within the retina. Morphologically these cells exhibited extensive differentiation with elaborate dendritic and axonal processes. Double labeling experiments showed that these cells, although residing in the ganglion cell layer, did not express detectable levels of Brn3A, Islet-1, or Pax6. When injected into adult mouse eyes, some of these transdifferentiating chick RPE cells were found in the ganglion cell layer, even though they lacked significant morphological differentiation. Conclusions: Chick RPE cells, undergoing transdifferentiation towards retinal ganglion cells under the induction of bFGF and cath5, could potentially migrate into the ganglion cell layer of adult mouse retina. When microinjected into the developing chick eye, the transdifferentiating RPE cells could be incorporated into the ganglion cell layer and developed elaborate cellular processes. It remains to be demonstrated whether these cells will function as bona fide retinal ganglion cells.

Keywords: ganglion cells • regeneration • transplantation