Retinal ganglion cells differentiated in vitro from retinal stem/progenitor cells of the ciliary margin possess positional information and are competent to respond to axon guidance cues.

DI NAPOLI, JENNIFER; CAVALLERO, GUSTAVO; FIORE, LUCIANO; MEDORI, MARA; LUJILDE, NESTOR JAVIER; DIAZ CARRIZO, JOAQUIN; DEL RIO-TSONIS, KATIA; SANCHEZ, VIVIANA; CARRI, NÉSTOR GABRIEL; SCICOLONE, GABRIEL

San Diego

Congreso; Neuroscience 2013; 2013

Society for Neuroscience

Retinal ganglion cells differentiated in vitro from retinal stem/progenitor cells of the ciliary margin possess positional information and are competent to respond to axon guidance cues.   J. I. DI NAPOLI1, G. CAVALLERO1; L. FIORE1, M. S. MEDORI1, N. J. LUJILDE1, J. DIAZ CARRIZO1, K DEL RIO-TSONIS3,  V. SANCHEZ1, N. G. CARRI2, G. E. SCICOLONE1 1Institute of Cell Biology and Neuroscience (IBCN-UBA-CONICET), School of Medicine, UBA. Buenos Aires, Argentina. 2 Institute of Multidisciplinary Cell Biology (IMBICE-CIC-CONICET). La Plata, Argentina. 3. Department of Zoology, Miami University, Oxford, OH, USA.     Directing stem cells in vitro to differentiate to specific neurons with the ability to establish topographic ordered connections is a major objective in any strategy for regeneration. The Ciliary Margin (CM) of the embryonic chick eye possesses stem cells which progressively lose the capability to differentiate into retinal ganglion cells (RGCs). Specific combinations of insulin and Fibroblast Growth Factor 2 (FGF2) can stimulate the differentiation of the stem/progenitor cells of the chick CM to RGCs in vitro. The objectives of this work were: to obtain RGCs from neurospheres generated from the CM and to study whether these cells possess positional information that will allow them to respond to specific axon guidance cues. Since the Eph/ephrin system is the main molecular complex involved in axon guidance and the formation of the retinotectal topographic ordered connections, we centered our study in this system. Neurospheres were produced from CM cells obtained from nasal and temporal sides of eyes from 7 day embryos. After expansion, neurospheres were cultured over polylisine-laminin substrates and a curve of different concentrations of insulin/FGF2 combinations was assayed in order to determine the in vitro conditions that produce the highest percentage of RGCs within the cell population. Immunocytochemistry for EphAs and ephrin-As was performed. It was shown that these RGCs express the components of the Eph/ephrin system according to the topographic origin of the stem cells. Thus, nasal RGCs express higher levels of ephrin-As, whereas temporal RGCs express higher levels of EphAs. In order to determine whether these RGCs present topographic specific responses to axon guidance cues, we stimulated nasal and temporal differentiated RGCs with EphA3 ectodomain (EphA3-Fc) or with ephrin-A2 (ephrin-A2-Fc). According to their origin, temporal RGCs grew longer axons than nasal ones in control conditions, nasal RGCs grew longer axons with EphA3-Fc and temporal ones grew shorter axons with ephrin-A2. This axonal behavior is similar to the response that nasal and temporal RGCs have in vitro and in vivo during retinotectal mapping. These results suggest that the RGCs differentiated from the stem/progenitor cells of the CM possess positional information, and are competent to respond to axon guidance cues. This is important because this opens the possibility that grafts of stem cells could differentiate into competent RGCs which could restitute neural circuits.