206 COMPARATIVE EVALUATION OF VARIOUS PROTOCOLS FOR NEURAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

Abstract

Mouse embryonic stem (ES) cells derived from the inner cell mass of blastocysts can differentiate into neuronal cells by treatment with retinoic acid (RA). ES cells cultured as aggregates and as single cell suspensions were then exposed to RA which induced multiple phenotypes of neuronal cells. Differentiation was dependent on the concentration of RA and the time of exposure. In this study, we cultured ES cells as a suspension in which they formed embryoid bodies (EBs). The EBs were treated with varying concentrations of RA for differing times. We used increasing concentrations of RA (50 nM, 100 nM, 1 �M, and 3 �M) prepared from a stock of 10 mM RA in DMSO. Immunocytochemistry staining was carried out on 2, 5, 7, and 9 days of culture. We formed EBs for 4 days with standard ES cell medium (without LIF) plus an additional 4 days of treatment with 1 �M RA. ES cells were treated with 1 �M RA for 2 days in suspension culture. Two-day-old EBs plated on culture dishes were treated with 1 �M RA for 3 days. To test for the effect of RA concentration on embryonic differentiation, 2-day-old EBs were treated with 50 nM, 100 nM, 1 �M, and 3 �M RA for 3 days. The time-dependent effects of RA on the 4-/4+ RA group were investigated. Results showed that neuronal precursor cells appeared on the second day of culture; they were stained with nestin antibody. On the 5th day of culture, neurons were detected with NCAM antibody. On the 7th day of culture, glial cells were observed with GFAP, and on the 9th day of culture GFAP, expression increased. In EBs that were plated and then treated with RA, the same results were obtained. RA induced neuronal differentiation in a concentration-dependent manner. Low concentrations (50 nM and 100 nM) of RA induced neuronal differentiation besides mesenchymal differentiation; however, higher concentrations (1 �M and 3 �M) of RA did not induce mesenchymal differentiation. The most efficient neuronal differentiation was obtained at 3 mM RA concentration. This study was performed in TUBITAK Research Institute for Genetic Engineering and biotechnology.