Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naive Pluripotency

Naive and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naive counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naive pluripotent state using LIF and inhibitors of glycogen synthase kinase 3 beta and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naive pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIFFGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naive PSCs.