Retinol, the alcohol form of vitamin A, maintains pluripotency of mouse embryonic stem cells (ESCs) by the overexpression of Nanog, which is a key transcription factor for their self-renewal. ESCs represent the most promising source of all types of cells for regenerative medicine and drug discovery. These cells maintain pluripotency through a complex interplay of different signaling pathways and transcription factors including leukemia inhibitory factor (LIF), homeodomain protein Nanog, and Oct3/4. Nanog, however, plays a key role in maintaining the pluripotency of mouse and human ESCs. Overexpression of nanog by heterologous promoters can maintain pluripotency of ESCs in the absence of LIF. Also, Nanog alone is sufficient for the self-renewal of ESCs while maintaining the Oct4 levels. Normally, mouse and human ESCs are cultured over mouse embryonic fibroblasts as feeders to maintain pluripotency. Although feeder cells provide important growth-promoting factors, their use involves several cumbersome and time-consuming steps. Here we demonstrate that retinol can support feeder-independent self-renewal of ESCs in long-term cultures without affecting their pluripotency. The effect of retinol is independent of the strain background, and the cells maintain complete potential to differentiate into all the primary germ layers in embryoid bodies and in chimeric animals. Self-renewal of ESCs by retinol is not mediated by retinoic acid. The studies demonstrate for the first time that a physiologically relevant small molecule has growth-promoting effect on the self-renewal of ESCs by activating the endogenous machinery to overexpress a critical gene for pluripotency.
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