Embryonic stem (ES) cells derived from the inner cell mass of a mammalian blastocyst represent unlimited source of all types
of cells for regenerative medicine and for drug discovery. Mouse and human ES cells require mouse embryonic fibroblast feeder
cells to maintain their undifferentiated state which involve additional time-consuming and labor-intensive steps. Recently
we reported a novel function of retinol, the alcohol form of vitamin A, in preventing the differentiation of mouse ES cells.
Retinol/vitamin A induces the overexpression of Nanog, a key transcription factor that is important for maintaining the pluripotency
of mouse and human ES cells. Further, retinol/vitamin A also supports feeder-independent culture of ES cells in long-term
cultures. The cells continue to maintain the expression of pluripotent cell-specific markers such as Nanog, Oct4, and Sox2
and form chimeric animals after injection into blastocysts. In this chapter, we describe feeder-independent cultures of mouse
ES cells in the medium supplemented with retinol. The ES cells are cultured over plates coated with gelatin in ES medium with
leukemia inhibitory factor (LIF) which is supplemented with 0.5 μM retinol/vitamin A. The cells are passaged every 3–5 days
by trypsinization. The pluripotency of the cells is tested by different undifferentiated ES cell-specific markers.