Proliferation and differentiation of direct co-culture of bone marrow mesenchymal stem cells and pigmented cells from the ciliary margin

Damage of retinal ganglion cells (RGCs) is the major consequence of glaucoma and regeneration of RGCs is extremely difficult once the damage has occurred. Retinal stem cells (RSCs) are considered an ideal choice for RGC regeneration. Pigmented cells from the ciliary margin (PCMs) have great retinal differentiation potential and may be an ideal RSC candidate. However, the ciliary margin is too small, so the number of cells that can be obtained is limited. Bone marrow-derived mesenchymal stem cells (BMMSCs) are another type of stem cell that have been previously investigated for RGC regeneration. BMMSCs expand sufficiently, whereas the retinal differentiation of BMMSCs is insufficient. The aim of the present study was to investigate whether the co-culture of PCMs and BMMSCs may combine the advantages of both cell types to establish a novel and effective stem cell source for RGC regeneration. Primary rat PCMs and BMMSCs were isolated and co-cultured. Cell growth was observed by an inverted microscope and proliferation was monitored by an MTT assay. Cell cycle analysis was performed by using a flow cytometer, while the expression of the photoreceptor-specific homeobox gene (cone-rod homeobox, Crx) was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, retinal differentiation was confirmed by immunofluorescence staining of major markers of retinal differentiation, including rhodopsin, visual system homeobox 2 and heparin sulfate. The co-cultured cells expanded successfully, in a similar way to BMMSCs. In addition, the expression of Crx and retinal markers were significantly upregulated following BMMSC and PCM co-culture. The results of the present study demonstrated that the co-culture of BMMSCs and PCMs may be used as a source of RSCs.