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Establishment of Pig Embryonic Stem Cells using Simplified Serum-free media and Adaptation of Feeder-free conditions

ABSTRACT: Most porcine embryonic stem cells (pESC) derived from in vitro blastocysts Still require complex media compositions and feeder layers which make difficulty to adapt their broad and routine use. We derived pESC using simplified culture media (FIW) consisting of small molecules FGF2, IWR-1, and WH-4-023 in serum-free medium. We established several pESC-FIW lines capable of single cell passaging with short cell doubling time (about 12 hours). These cells expressed pluripotency markers, OCT4, SOX2, and NANOG, as well as cell surface markers, SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed stable proliferation rate and normal karyotype even after long-term culture over 50 passages. Established pESC showed formative characteristics based on the negative expression for the naive marker KLF4 and primed marker T, whereas strong expression for the formative marker OTX2 with high levels of related genes. Transcriptome analysis showed that pESC-FIW had similar cellular identities to reported pESC maintained in complex media formulations and had characteristics of gastrulating epiblast cells. In addition, pESC-FIW could be maintained for multiple passages in a feeder-free condition on fibronectin coated plate using mTeSR™, a commercial medium for feeder-free culture. These results indicate that the canonical WNT (IWR-1) inhibition and the SRC inhibition (WH-4-023) are sufficient to establish pESC capable of single cell passaging and feeder-free expansion under the serum-free condition. Easy to maintain-pESC can be a tool for gene editing application useful for agriculture and biomedicine, and lineage commitment studies.

ORGANISM(S): Sus scrofa

PROVIDER: GSE240454 | GEO | 2024/08/07

REPOSITORIES: GEO

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