Background: Human embryonic stem cells (hESCs), routinely derived from the inner cell mass (ICM) of blastocysts, are in a developmentally restricted primed state, unlike mouse ICM-derived ESCs that are considered to be in a naïve pluripotent state. The limitations of the primed hESCs include low derivation efficiency, laborious culture conditions, and extensive heterogeneity in differentiation potential, thus restricting their use for basic and applied research.
Aim: To derive naïve hESCs different from the conventional primed hESCs derived so far, using chemically modified medium.
Materials and Methods: Spare blastocysts were donated by couples undergoing PGD. ICMs were placed on feeder cells supplemented with naïve chemically defined medium in the absence of genetic manipulation. The medium contains on top of other integrids also the small molecule inhibitors developed by the Weizmann lab (Gafni, Ben-Yosef, Kalma, Hanna.Nature2013).
Results: Almost all ICMs plated for derivation attached and the cells proliferated and created large colonies. These colonies were further trypsinyzed into single cells and we successfully derived 15 naïve hESC lines, carrying mutations of human genetic disorders. The naïve cells retain molecular characteristics and functional properties that are highly similar to naïve mouse ESCs, i.e. tolerance to passaging as single cells, retaining a pre-inactivation X chromosome and global reduction in DNA methylation.
Conclusion: The naïve conditions supported for the first time the derivation of naïve human ESC lines, the largest collection of naïve PGD-hESCs ever reported. This unique collection provides an improved human in vitro model for studying genetic diseases, and developmental processes.
