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064

RETINOÏC ACID-INDUCED NEURONAL DIFFERENTIATION OF P19 CELLS CAN INVOLVE REDUCTION OF OXYTOCIN RECEPTOR EXPRESSION

V Arsenault, J Paquin, M Jankowski, J Gutkowska

Montreal, Quebec

INTRODUCTION: We showed that retinoic acid (RA)-induced (10–7-10–9 M) differentiation of P19 embryonal stem cells into cardiomyocytes is OT receptor (OTR)-dependent since it is blocked by an OT antagonist. However RA-mediated differentiation of P19 cells is dose-dependent and at high concentration (10–6 M) RA induces neuronal phenotype. The OT/OTR system is expressed in the central and cardiac nervous system and influences behavior, cognition and cardiac contractility.

OBJECTIVE: We investigated the possibility that OT/OTR is involved in RA-induced neuronal differentiation of P19 cells.

METHODS: To induce differentiation, P19 cells were cultured in aggregates for 4 days in the presence of the following agents: RA (10–6 M), OT (10–7 M), OTA (10–7 M), and the combinations OT+RA and OTA+RA. Then, aggregated cells were trypsinized and grown in monolayers for few more days in serum-free culture medium and in absence of the agents. Similarly treated cells, but not exposed to the agents, served as a control. Cell cultures were collected at different time-points to analyze morphology and OTR expression.

RESULTS: At day 4 aggregates treated with RA had lowered OTR transcript level comparing to aggregates treated with OT or OTA. The OTR values in OT+RA and OTA+RA treatments were in between those of RA and OT/OTA treatments but the differences were not statistically significant. The RA, OT+RA and OTA+RA treatments generated cell cultures containing mostly (³90%) neurons at day 9. Non-induced cells remained in the undifferentiated state, as assessed by immunoreactivity of embryonal stem cell marker, SSEA-1. In OT and OTA-treated cell cultures we found undifferentiated cells and cells of undefined phenotype exhibiting thin neurite-like extensions. Phenotypical characterization of these cells is currently underway.

CONCLUSION: In the P19 cell model, OT and OTA do not exhibit significant neurogenic or antineurogenic properties. The neurogenic action of RA could depend on its reducing effect on OTR expression during aggregation.

DNC

Canadian Institutes of Health Research

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