Naïve hESCs have an in-vitro advantage over primed cells in early but not in late culture

Introduction: Human embryonic stem cells (hESCs) exist in two different states of pluripotency, naïve and primed, which represent preimplantation blastocyst and postimplantation epiblast, respectively.

Aim: To compare the cellular phenotype and genetic stability of naïve and primed hESCs in early and late passages.

Methods: Four hESC lines were derived in naïve conditions and matched primed counterparts were obtained by transferring half of the cells to primed conditions. Phenotypic characterization included cell proliferation using doubling time, cell-cycle distribution using FACS analysis and clonogenicity by Alkaline-phosphatase staining. Genetic stability was evaluated by chromosomal microarray analysis (CMA).

Results: In early passage (p30), naïve hESCs showed significantly higher proliferation rate and respectively more cells in S-phase, as well as higher cloning efficiency compared to primed cells. In later passage (p50), one naïve line preserved higher proliferation rate, while all other lines showed higher proliferation rate of the primed cells, and corresponding fractions of cells in S-phase. In all 4 lines, naïve cells displayed higher clonogenicity compared to primed cells. No genetic aberrations were observed in p30, whereas several genetic changes were detected in p50 both in naïve and primed hESCs.

Conclusions: Genetic stability of hESCs was not affected by their pluripotency state (naïve or primed), but rather by the prolonged culture. Naïve cells display selective advantage in early passage, which subside over time in culture, probably due to yet unknown genetic or epigenetic changes. These genetic and phenotypic characteristics should be taken into consideration when designating hESCs for clinical applications.