Pacherník J, Bryja V, Esner M, Kubala L, Dvorák P, Hampl A
Center of Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic.
Physiol Res. 2005;54(1):115-122. doi: 10.33549/physiolres.930526.
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, alpha-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.
在胚胎癌（EC）细胞和胚胎干细胞（ES）中，用视黄酸（RA）处理后进入的分化途径会因培养条件不同而有所变化。本研究采用小鼠EC细胞P19来研究血清对在简化单层培养中RA诱导的神经分化的影响。细胞形态和谱系特异性分子标志物的表达表明，在含血清条件下用RA处理后会出现非神经细胞类型，而在化学成分明确的无血清培养基中，10^(-9) M及更高浓度的RA会诱导大量神经分化。此外，在无血清条件下由RA驱动的分化早期，不仅神经（Mash-1）和神经外胚层（Pax-6）基因，而且内胚层（GATA-4、甲胎蛋白）基因也会表达。此外，通过荧光素酶报告基因检测确定，血清的存在与否不影响视黄酸反应元件（RARE）的活性。因此，小鼠EC细胞即使不在三维胚状体（EBs）中培养，暴露于RA时也能够产生神经细胞。然而，与标准的涉及EBs的方案不同，单层中的神经分化仅在避免血清因子复杂信号传导时才会发生。提出这种简单有效的策略作为体外神经分化研究的基础。
