Comparing the Cellular Phenotype of Naïve and Primed Human Embryonic Stem Cells

Introduction: Human embryonic stem cells (hESCs) exist in two different states of pluripotency, naïve and primed, which represent earlier human blastocyst stage and more advanced epiblast-like stage, respectively. Naïve hESCs seem to possess a broader developmental potential compared to primed hESCs, suggesting their use as a superior source of material for cell transplantation.

Aim: To compare the cellular phenotype of naïve and primed hESCs.

Methods: Four hESC lines were derived and cultured in naïve conditions. After 20 passages, half of them were transferred to primed conditions, and half continued to grow under naive conditions. After 10 additional passages, each sub-culture was confirmed as naïve or primed by expression of naïve markers using qRT-PCR and immunofluorescence. Then each sub-culture was phenotypically characterized for cell proliferation using doubling time assay, cell cycle distribution using labeled nucleotide incorporation and FACS analysis, and clonogenicity by low density plating followed by alkaline-phosphatase staining.

Results: Naïve hESCs that were converted to primed expressed significantly decreased mRNA levels of naïve markers KIF17, TFCP2L1 and STELLA, compared to their naive counterparts. While naïve cells exhibit nuclear staining for TFE3, converted primed cells displayed cytoplasmic staining. Naïve hESC showed higher proliferation rate, higher fraction of cells in S-phase and displayed a ∼ 1.5-fold higher cloning efficiency compared to converted primed cells.

Conclusions: Our results indicate that naïve hESCs have higher proliferation rate and survived better as single cells compared to primed cells. These phenotypic characteristics suggest that naïve hESC may be more favorable for practical applications than primed hESC.