In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer
culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10-9 M and higher. Moreover, not only neural (Mash-1) and
neuroectodermal (Pax-6), but also endodermal (GATA-4, α-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer
only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.
Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferation, survival and differentiation in a wide range of cell types. Here we show that LIF potentiates retinoic acid-mediated neural induction in pluripotent P19 embryonal carcinoma cells. This activity of LIF was demonstrated by a profounded neural morphology followed by increased expression of neural-specific proteins (N-CAM, III β-tubulin, and GAP-43), up-regulation of early neural lineage-specific gene Mash-1, and down-regulation of early endoderm-specific genes α-fetoprotein and
GATA-4. Moreover, LIF also slows growth and increases the level of apoptosis in differentiating cells.