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U. Vossmerbaeumer, S. Kuehl, H. Kluter, H. Eichler, J.B. Jonas, K. Bieback; Cytoengineering of Retinal Pigment Epithelium Cells From Ocular Progenitor Reservoirs . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3062.
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Purpose: Degenerative processes in the RPE play a pivotal role in the development of age related maculopathy. The resulting nutritional deficit to the photoreceptors compromises their function. Repair of the lesions by implantation of RPE cells generated from progenitor cells might preserve function. We investigate methods for isolation, cultivation and differentiation of cells from ocular tissues for eventual use in subretinal reconstructive therapy. Methods: The ciliary marginal zone (CMZ) was resected from enucleated rat and human eyes and digested by Collagenase, Hyaluronidase, Trypsin and DNAse. Suspension culture of isolated cells was established to allow formation of spherical cell conglomerates using optimized medium for primary neuronal cells containing 20ng/ml bFGF and 10 ng/ml EGF. Mitotic activity was ascertained by BrdU. Removal of cytokines and addition of FCS served to induce adherence and differentiation of the obtained spheres on a suitable surface. Immunostainings were performed to assess differentiation status. Phagocytosis of FITC–labelled latex beads was performed to proof functionality of differentiated cells. Results: CMZ derived cells were cultured and expanded for over six months. Formation of spherical cellular conglomerates in early suspension culture was taken as primary morphological sign of the presence of progenitor cells. Further evidence of their being initially undifferentiated was supplied by expression of Nestin. After exposure to FCS and an adherent surface we observed expression of markers that are accepted as indicative for differentiation: MAP–2 for neuronal, GFAP for glial and Bestrophin as well as Cytokeratine 8/18 for RPE properties, respectively. Intracellular uptake of latex beads was found denoting phagocytotic activity as functional characteristic of RPE cells. Conclusions: We succeeded in the isolation and cultivation of cells from human and rodent ciliary marginal zone which showed characteristics of progenitor cells. Subsequently, these cells were positive for neuronal, glial and RPE markers. Still, however, differentiation under culture conditions remains a predominantly stochastic process. For therapeutic purposes, methods of unidirectional induction of differentiation seem desirable. Thus the CMZ appears a candidate region as a reservoir for regenerative ocular tissue in view of retina reconditioning therapy.
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