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OBJECTIVE: Human embryonic stem cells (hESCs) exist in two different states of pluripotency, na€ ıve and primed, which represent the earlier human blastocyst stage and the more advanced epiblast-like stage, respectively. Na€ ıve hESCs differ from primed cells in their typical colonies morphology and their tolerance to single-cell passaging. These and other phenotypic differences may facilitate culture of na€ ıve cells, conferring a practical advantage for their usage in cell transplantation. The aim of the current study was to compare the cellular phenotype of na€ ıve and primed hESCs.DESIGN: A laboratory study using hESCs. MATERIALS AND METHODS: Four hESC lines were included in this study: Lis 38_N, Lis 39_N, Lis 45_N and Lis 46_N, all of which were derived and cultured in na€ ıve conditions. After 20 passages, half of the na€ ıve cells were transferred to primed conditions, while the other half continued to grow under naive conditions. After 10 additional passages, each sub-culture was confirmed as na€ ıve or primed by the expression of na€ ıve markers using qRT-PCR and immunofluorescence. Then each sub-culture was phenotypically characterized for cell proliferation using doubling time assay, cell cycle distribution using EdU incorporation during the s-phase and FACS analysis, and clonogenicity by low-density plating followed by alkaline-phosphatase staining.RESULTS: All hESC lines were characterized as pluripotent by RT-PCR and immunofluorescence for pluripotency markers. Na€ ıve hESCs that were converted to primed expressed significantly decreased mRNA levels of the na€ ıve markers KIF17, TFCP2L1 and STELLA, compared to their naive counterparts (p<0.05). While na€ ıve cells exhibit nuclear staining for TFE3, converted primed cells displayed cytoplasmic staining. The doubling time of na€ ıve cells (18.1AE3.99) was significantly shorter than that of primed cells (33.7AE7.15) (p<0.01). Respectively, significantly higher fraction of cells in S-phase was detected in na€ ıve cells (45.3AE2.1) compared with primed cells (35.4AE2.9) (p< .005). Additionally, na€ ıve cells displayed a $ 1.7-fold higher cloning efficiency from a single cell (39.8AE11) compared to primed cells (23.1AE4.8) (p<0.05).CONCLUSIONS: Our results indicate that na€ ıve hESCs have higher proliferation rate and survived better under stress conditions compared to primed cells. These phenotypic characteristics suggest that na€ ıve hESC may be favorable for practical applications than primed hESC.

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