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UPREGULATION OF NEUROTROPHINS IN HUMAN RETINAL PROGENITORS AND RETINAL PIGMENT EPITHELIUM (RPE) CELLS CO-CULTURED IN THE NASA BIOREACTOR. K Dutt and R. Kumar. Department of Pathology, Morehouse School of Medicine in Atlanta, GA

     Age related Macular Degeneration (AMD) and retinitis pigmentosa (RP) remain the leading causes of blindness.  Tissue transplantation is a promising approach to treatment of these diseases. Scarcity of tissues for replacement, however, is a major obstacle. We have previously reported that formation of 3D structures is promoted when retinal progenitors and RPE are co-cultured in the bioreactor.  The purpose of the present work is an attempt to identify neurotrophins, which might be altered in the 3D environment of the bioreactor. Methods: Human retinal progenitors and RPE were cultured alone and as co-cultures in the bioreactor.  Tissue structures generated were evaluated by scanning and transmission electron microscopy, immunocytochemistry and Western blot analysis.   RT-PCR was performed using appropriate primers; amplified products generated for different cycles were evaluated. Results: Retinal-RPE co-cultured in the bioreactor on cytodex beads or as beadless co-cultures show a higher degree of differentiation and layering.  Several retinal cell types could be identified in multi-layered structures generated in the bioreactor by immunophenotyping, of which photoreceptors were most well defined and differentiated.  RT-PCR data revealed that neurotrophins bFGF, TGFa, CNTF and BDNF are all upregulated in cells co-cultured in the bioreactor as compared to monolayer cultures.  There was a significant correlation between higher level of BDNF, CNTF and the complexity of tissue-like structures generated in the bioreactor. Conclusion:  NASA’s bioreactor possibly promotes tissue formation by upregulation of neurotrophins.  Our results appear significant not only for tissue generation, but also in light of findings by others that the same neurotrophins can rescue degenerating photoreceptors in animal models of RP. 

     (Supported by NASA grants NAG9-964 (KD) and NCC9-112 (KD). )