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

Abstract

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.

Vitamin A/Retinol and Maintenance of Pluripotency of Stem Cells

“…Retinol treatment of mouse ESCs and induced pluripotent stem cells (iPS) cells resulted in the elimination of differentiated cells leaving only pure population of undifferentiated cells [54] suggesting that the differentiated cells may have been eliminated via terminal differentiation due to their ability to metabolize retinol into retinoic acid. The undifferentiated characteristics of these cells were confirmed by generating chimeric animals via injection of cells into mouse blastocysts.…”

“…The undifferentiated characteristics of these cells were confirmed by generating chimeric animals via injection of cells into mouse blastocysts. The microinjection of cells resulted in the chimeric animals that exhibited high percentage of ESC contribution accompanied by germ line transmission of coat color of the ESCs [54]. …”

A Simple and Robust Method for Culturing Human‐Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin

“…Similarly, it is necessary to develop culture tools that are indispensable for maintaining the undifferentiated state of hiPSC cultures. As a good writing tool provides “paper and pencil to write a beautiful letter” and “an eraser to be able to correct mistakes,” hiPSC culture requires “good substrate and nutrient medium” to control the occurrence of deviating cells, with many studies having previously investigated hiPSC‐culture media and substrates . However, something equivalent to an eraser in this context does not yet exist, and removal technology involving not only the hiPSC‐nutrient medium and substrate, but also the unprecedented concept of removing deviating cells will be indispensable in the future.…”

“…[6][7][8] Recent studies showed that it is possible to maintain hiPSC pluripotency and self-renewal through artificial manipulation, and the exogenous expression of molecules associated with epigenetic regulation revealed that this can be achieved through addition or withdrawal of certain cytokines and/or growth factors or by changing the transcriptional activity of genes. [23][24][25][26][27][28][29][30][31] Methods based on culturing undifferentiated hiPSCs in monolayers have also been reported, although they present significant limitations. Specifically, hiPSC cultures are very difficult to handle, as they have a tendency to change state easily upon each passage or operation, which can lead to unintentional spontaneous differentiation.…”

A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency

“…Human embryonic stem (hES) cells can be maintained in an undifferentiated state by highly skilled researchers and engineers [1, 2]. In addition to skill development, reagents and automated instruments (e.g., kinase inhibitors [3, 4] and time‐lapse analyses [2, 5]) have been developed to maintain the undifferentiated potencies of stem cells.…”