Stem cells obtained from retinal ciliary margin possess positional values.

MAHECHA CASTAÑEDA JG; SPELZINI G; SANCHEZ V; MEDORI M; CARRI N; OLMOS CARREÑO C; DI NAPOLI J; SCICOLONE G

Medellín

Congreso; LATIN AMERICAN SOCIETY FOR DEVELOPMENTAL BIOLOGY MEETING 2017; 2017

Latin American Society for Developmental Biology

The chicken embryonic retina presents multipotent stem cells located in the ciliary margin (CM). Large-scale expansion of neural stem and progenitor cells will be essential for the medical treatment of the large number of patients suffering from neurodegenerative disorders. The neurosphere assay constitutes an in vitro procedure to expand stem/progenitor cells and a suitable model to study their behavior.Previously we cultured dissociated cells obtained from chicken embryonic nasal and temporal CM, favoring neurospheres formation. After expansion, neurospheres were cultured on polylysine-laminin substrate with different concentrations of fibroblast growth factor 2 (FGF2) and insulin and we demonstrated that these conditions are sufficient to induce retinal ganglion cells (RGCs) differentiation at different stages of development.The purpose of this work was to determine the spatial arrangement of the stem cells located in expanding neurospheres and the behavior of RGCs obtained from neurospheres cultured in differentiation conditions, analyzing whether they have positional values.We employed neurospheres obtained from chicken embryos CMs of 7 days of development (E7). They were cultured in expanding-conditions (floating neurospheres) to evaluate the expression pattern of molecules which may maintain positional values in stem cells along the neurospheres radial axis. Furthermore, neurospheres were cultured in differentiation conditions (on laminin substrate) to evaluate the behavior of RGCs axons.We showed that: 1) Laminin-1 was expressed in the center of expanding neurospheres and its expression was significantly associated with the neurosphere size. 2) Mitoses were located in the peripheral area where the Eph/Ephrin system and N-cadherin expression were predominant. 3) RGCs presented differential pattern of expression of the Eph/ephrin system according to their site of origin (nasal vs temporal CM) and presented differential axonal response to axon guidance molecules like EphA3 and ephrin-A2.These results show that: Stem cells obtained from CM at E7 express molecules which maintain positional values along the radial axis of the neuroespheres (laminin-1 in the central area / the Eph/Ephrin system and N-cadherin in the peripheral area). RGCs obtained from neurospheres cultured in differentiation conditions have the same positional values such as the RGCs in the tangential axis of the developing retina (EphA3 is predominant in nasal neurospheres whereas ephrin-A2 is predominant in temporal ones). These results support the possibility to employ stem cells to replace depleted RGCs. Supported by grants of UBA and CONICET