2. Academic Validation
  3. Establishment of porcine embryonic stem cells in simplified serum free media and feeder free expansion

Establishment of porcine embryonic stem cells in simplified serum free media and feeder free expansion

  • Stem Cell Res Ther. 2024 Aug 7;15(1):245. doi: 10.1186/s13287-024-03858-2.
Hyerin Choi 1 2,  Dongjin Oh 1 2,  Mirae Kim 1 2,  Ali Jawad 1 2,  Haomiao Zheng 1 2,  Lian Cai 1,  Joohyeong Lee 3,  Eunhye Kim 4,  Gabsang Lee 5,  Hyewon Jang 6,  Changjong Moon 6,  Sang-Hwan Hyun 7 8 9 10
Affiliations

Affiliations

  • 1 Veterinary Medical Center, College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Republic of Korea.
  • 2 Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea.
  • 3 Department of Companion Animal Industry, Semyung University, Jecheon, 27136, Republic of Korea.
  • 4 Laboratory of Molecular Diagnostics and Cell Biology, College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea.
  • 5 Department of Neurology, Institute for Cell Engineering, School of Medicine, Johns Hopkins Medicine, Baltimore, ML, USA.
  • 6 Department of Veterinary Anatomy and Animal Behavior, College of Veterinary Medicine, BK21 FOUR Program, Chonnam National University, Gwangju, Republic of Korea.
  • 7 Veterinary Medical Center, College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Republic of Korea. [email protected].
  • 8 Institute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of Korea. [email protected].
  • 9 Vet-ICT Convergence Education and Research Center (VICERC), Chungbuk National University, Cheongju, Republic of Korea. [email protected].
  • 10 Chungbuk National University Hospital, Cheongju, Republic of Korea. [email protected].
PMID: 39113095 DOI: 10.1186/s13287-024-03858-2
Abstract

Background: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium.

Methods: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions.

Results: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions.

Conclusions: These results indicated that inhibition of Wnt and Src was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

Keywords

Embryonic stem cells; Pig; Pluripotency; Self-renewal; Serum-free media.

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