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[In vitro differentiation of embryonic stem cells mediated by the supernatant of retinal cells and retinoid acid].

PURPOSE
To analyse the differentiation features of embryonic stem cells which were induced by the supernatant of retinal cells and retinoid acid (RA).


METHODS
Embryonic bodies (EB) were gained when embryonic stem cells (ESC) were propagated 1 generation after anabiosis. 3.5d EB without digestion were induced at four conditions sorted as condition A, B, C and D. A contains only retinoid acid (RA) in medium; B contains RA and the supernatant from retinal neurons and glia of mouse; C contains RA and the supernatant from retinal pigment epithelium of human; D contains RA and the supernatant of fetal retinal glia. The changes of embryonic stem cells were observed and the immune cytochemistry was adopted to detect the nestin, glial fibrillary acid protein (GFAP), cytokeratin, microtubule-associated protein 2 (MAP-2) and rhodopsin in induced cells.


RESULTS
(1) The changes of differentiation features of EB: The characteristics for early stage were almost same at the four conditions. The cells with various shapes could be seen around EB. EB were dispersed 3 weeks later. Additionally, the cells which were no more abounded and not clear boundary appeared at condition A, the round cells with high transparency at condition B, EB and the cells around EB with apparent pigment at condition C, the large cells with simplex shape and glia-like appearance at condition D. (2) Immune cytochemistry showed that the most induced cells were MAP-2 (+) cells, no nestin (+) cells appeared at the four conditions. There were no GFAP, cytokeratin or rhodopsin positive cells in condition A; However, GFAP (+), cytokeratin (+) and rhodopsin(+) cells could be seen at condition B, cytokeratin positive cells at condition C, GFAP positive cells at condition D.


CONCLUSION
RA can induce embryonic stem cells into neurons in second induction. Several kinds of the supernatant from retinal cells have an effect on induction of ESC. ESC can be differentiated into cells associated with the origin of the supernatant.