Table1_Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines.DOCX

Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly (p < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.

卵裂球聚集技术是提升囊胚发育潜能的极具前景的手段，其可通过增加内细胞团数量，为嵌合动物制备以及胚胎干细胞系的建立提供有力支持。本研究明确了利用植物血凝素-L（PHA-L）开展卵裂球聚集的最优条件，并通过与通用卵裂球聚集方法“井中井”（Well of the Well, WOW）对比，验证了PHA-L的聚集效率。结果显示，采用15 μg/ml的PHA-L处理144 h，可有效实现三枚去透明带2细胞期胚胎（TZ2Es）的卵裂球聚集，并提升其经孤雌激活（PA）后的胚胎发育能力。与WOW法相比，经PHA-L培养的TZ2Es卵裂球聚集率显著更高，且差异具有统计学意义（93.5 ± 1.9% vs. 78.0 ± 8.5%，p < 0.05）。此外，本研究结果证实，通过PHA-L介导的TZ2Es聚集，可改善孤雌激活来源囊胚的质量，同时提升猪胚胎干细胞样细胞（pESLCs）的接种效率与集落质量。研究还观察到，PHA-L诱导获得的pESLC可维持未分化状态，并表达典型的胚胎干细胞多能性标志物，具备拟胚体（EB）形成能力，且可分化为三胚层的细胞谱系。猪卵裂球聚集技术有望提升胚胎质量、胚胎干细胞建系效率以及拟胚体形成效率，同时可应用于囊胚互补系统以及嵌合动物的制备。