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DENSITY DEPENDENT MODULATION OF DIFFERENTIATION IN HUMAN RETINAL PROGENITORS.  Kamla Dutt, T. Lindsay, R. Kumar, I. Ezeonu, Department of Pathology, Morehouse School of Medicine, Atlanta, GA

   Purpose: To examine the role of cell density in cell commitment and differentiation in retinal progenitors. Specifically to investigate theresponse to exogenously added growth factors and RPE secreted growth factors in progenitors.  Methods: Non-transformed human retinal progenitors (Ret 1-4, 208) plated at the density of 1x10⁴ cell/cm² (low), 3x10⁴ cells/cm² (medium) and 1 x 10⁵/cells/cm² (high) in serum free medium were either exposed to bFGF, TGFa, BDNF, CNTF (100 ng/ml) or retinal pigment epithelium (RPE) conditioned medium (100%, 50%, 25% 12.5%).  Emerging phenotypes were scored by morphological analysis, immunophenotyping (for opsin, D₄, D₂ D₃ receptors, tyrosine hydroxylase and Protein Kinase C). RT-PCR and western analyses were performed for identification of secreted products.  Results: At low density 90% of precursors differentiated into photoreceptors in response to bFGF, TGFa in serum free conditions. At medium and high cell densities 30% and 25%  of the cells, respectively, acquired photoreceptor phenotype. In contrast, BDNF and CNTF enhanced both multipolar (neuronal) and photoreceptor cell differentiation in low-density cultures.  Conditioned medium from RPE at 50% concentration induced photoreceptor phenotype in low-density cultures on day 2 and 3 in 67% and 87% of cells, respectively, whereas 100% conditioned medium induced multipolar neuronal differentiation in 84% and 87% of the cells on day 2 and 3, respectively.  Brdu labeling confirms that cell commitment occurs in the latter part of the cell cycle.  Conclusions: Cell density serves as a mechanism by which cells sense their external environment and control their response to growth factors.  bFGF and TGFa predominantly induce photoreceptor phenotype in low-density culture.  On the other hand, BDNF and CNTF promote both multipolar ganglionic and photoreceptor phenotype.  RPE cells secrete multiple factors, with varying effects on cell commitment at different concentrations and densities.

(Supported by NASA growth NCC-9-112 (KD) NAG 9-96-4-(KD) and NAG 9-1169 (KD).

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