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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.
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.
摘要：
背景：建立稳定的猪胚胎干细胞（pESCs）有助于基础和生物医学研究，包括比较发育生物学，以及评估干细胞疗法的安全性。尽管有这些优势,从体外囊胚获得的大多数pESCs需要复杂的培养基和饲养层，常规使用，基因改造,和分化为特定的细胞类型困难。我们旨在建立具有单细胞传代能力的pESCs，高增殖潜能，并且使用简化的无血清培养基从体外来源的胚泡长期培养中稳定。
方法：我们使用各种基础培养基（DMEM/F10（1：1），DMEM/F12和a-MEM）和因子（FGF2，IWR-1，CHIR99021和WH-4-023）。在饲养或无饲养条件下分析建立的pESC的多能性和自我更新能力。最终,我们在无血清条件下开发了由FGF2，IWR-1和WH-4-023组成的简化培养基（FIW）。
结果：pESC-FIW细胞系能够以短细胞倍增时间进行单细胞传代，并表达多能性标记POU5F1，SOX2和NANOG，以及细胞表面标记SSEA1、SSEA4和TRA-1-60。pESC-FIW显示稳定的增殖速率和正常的核型，即使经过50个通道。转录组分析显示，pESC-FIW与报道的在复杂培养基中维持的pESC相似，并显示出胃泌素上胚细胞特征。使用mTeSR™在纤连蛋白包被的平板上在无饲养条件下维持pESC-FIW多次传代，用于无饲养培养的商业培养基，表现出与在饲养条件下观察到的特征相似的特征。
结论:这些结果表明,WNT和SRC的抑制足以建立能够在无血清条件下进行单细胞传代和无饲养细胞扩增的pESC。pESCs易于维护，有利于其在农业和生物医学基因编辑技术中的应用。以及血统承诺研究。
方法：我们使用各种基础培养基（DMEM/F10（1：1），DMEM/F12和a-MEM）和因子（FGF2，IWR-1，CHIR99021和WH-4-023）。在饲养或无饲养条件下分析建立的pESC的多能性和自我更新能力。最终,我们在无血清条件下开发了由FGF2，IWR-1和WH-4-023组成的简化培养基（FIW）。
结果：pESC-FIW细胞系能够以短细胞倍增时间进行单细胞传代，并表达多能性标记POU5F1，SOX2和NANOG，以及细胞表面标记SSEA1、SSEA4和TRA-1-60。pESC-FIW显示稳定的增殖速率和正常的核型，即使经过50个通道。转录组分析显示，pESC-FIW与报道的在复杂培养基中维持的pESC相似，并显示出胃泌素上胚细胞特征。使用mTeSR™在纤连蛋白包被的平板上在无饲养条件下维持pESC-FIW多次传代，用于无饲养培养的商业培养基，表现出与在饲养条件下观察到的特征相似的特征。
结论:这些结果表明,WNT和SRC的抑制足以建立能够在无血清条件下进行单细胞传代和无饲养细胞扩增的pESC。pESCs易于维护，有利于其在农业和生物医学基因编辑技术中的应用。以及血统承诺研究。
