Maintenance of feeder free anchorage independent cultures of ES and iPS cells by retinol/vitamin A.

Pluripotent embryonic stem (ES) cells derived from mammalian blastocyst and the adult fibroblast derived induced pluripotent stem (iPS cells) exhibit complete potential to form cells representing all the primary germ layers such as mesoderm, endoderm and ectoderm. These cells are usually co-cultured with mouse embryonic fibroblast feeders to prevent spontaneous differentiation. Feeder free cultures can provide substantial advantage to improve the efficiency and consistency of the culture conditions. In these studies, we demonstrate that a small dietary compound retinol, the alcohol form of vitamin A has capacity to regulate the pluripotency of pluripotent stem cells and maintain highly enriched population of pluripotent ES and iPS cells in feeder free suspension cultures. Retinol maintains long-term cultures of undifferentiated cells via elevated expression of stem cell specific transcription factors Nanog and Oct4. The studies provide evidence that retinol regulates the self-renewal of pluripotent stem cells via the over expression of insulin like growth factor II (IGFII) that engages PI3 kinase signaling pathway via IGF1 receptor tyrosine kinase. The ES cells retain capacity to generate high degree germline competent chimeric animals after microinjection into blastocysts. The studies offer a convenient system for long term maintenance of pluripotent stem cells via the activation of intracellular machinery for self-renewal by a physiologically relevant compound for large-scale production of high quality pluripotent stem cells.