Abstract
Abstract: :
Purpose: The amphibian eye, which grows throughout life, contains stem cells in the ciliary margin zone. Although the adult mammalian eye doesn't grow during adult life, we have demonstrated that the adult human eye also contains retinal stem cells (RSCs) in the same region (Coles et al, 2004). Now, we are interested in better characterizing these RSCs and their progeny Methods: We isolated retinal stem cells able to proliferate extensively and analyzed their gene expression pattern using microarray. Clonogenic expanded hRSCs gave rise to more than 100 billion cells in 120 days. To further characterize their stemness potential, we performed an expression analysis using the Affymetrix® U133 Plus 2.0 GeneChip. Results: During expansion, RSCs expressed genes common to stem cells, such as nucleostemin, nestin, Bmi1, Notch2, ABCG2, c–kit and its ligand, as well as cyclin D3 which acts downstream of c–kit. After differentiation, the cells derived from the hRSCs expressed genes with different functions in neurons such as genes coding for the skeleton (Map2, Tau), for the synaptic vesicles proteins (SNAPAP, SYPL, SNARE), for neuronal receptors (Gly–R, GABAR1, GABARP), and for a transcription factor necessary for neurogenesis (Neurogenin1). Moreover specific markers of retinal cells are also expressed as shown by the presence of RPE65, EFEMP1 (both proteins are expressed in retinal pigmented cells) and opsin, rom1, peripherin (specific to photoreceptors). In one of the cell line, we also detected proteins specific to retinal neurons: calbindin, syntaxin, and recoverin. We confirmed the expression of several genes by RT–PCR and immunostaining. We are currently pursuing the characterization. Conclusions: We showed that the hRS/PCs that expand easily share common characteristics with other stem cell populations. Furthermore, these cells differentiated along the neural lineage and expressed markers of several retinal cells types.
Keywords: retinal culture • gene microarray • ciliary body