Abstract
Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.
Highlights
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells, have the capacity to infinitely proliferate and the ability to differentiate into a large number of cell types
To develop a novel single-cell and feeder-free culture system for primate PSCs, we formulated the original MT-fCFA medium, a chemically defined medium supplemented with FGF and activin
We first attempted the adaptation of monkey ESCs previously established on conventional culture to the MT-fCFA medium
Summary
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the capacity to infinitely proliferate and the ability to differentiate into a large number of cell types. Human PSCs hold great potential for applications in drug discovery, disease modeling, and regenerative medicine. Monkey PSCs have valuable applications because monkeys share many physiological similarities with humans and are well-developed primate models of neurodegenerative disorders, autoimmune diseases, reproductive biology, infectious diseases, and behavior. Primate PSCs are thought to be identical to rodent epiblast stem cells (EpiSCs) [1]. EpiSC-like features make it difficult to culture primate PSCs in the undifferentiated state and to regulate differentiation into desired functional cells. A reliable and scalable culture system for supporting primate PSC maintenance is required in addition to efficient and reproducible differentiation techniques for preparing desired cells. There are two major obstacles in conventional culture systems, which impede the industrial and clinical application of primate PSCs
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