本研究旨在开发一种新的协议，将表观遗传提示和机械刺激相结合，组装3D球形结构，任意定义为“epiBlastoids”，其表型与自然胚胎非常相似。使用三步方法生成epiBlastoids。在第一步中，成人皮肤成纤维细胞转化为滋养层（TR）样细胞，结合5-azacytidine的使用，抹去原有表型，与特定的诱导协议相结合，推动细胞向TR谱系发展。在第二步中，再次应用表观遗传抹去与机械感应相关提示相结合，生成内细胞团（ICM）样器官。具体而言，抹去的细胞被封装到微生物反应器中，以促进3D细胞重新排列和提高多能性。在第三步中，TR样细胞与ICM样的球体在同一微小反应器中共同培养。随后，新生成的胚囊体被转移到微孔中，以促进epiBlastoid的形成。成人皮肤成纤维细胞成功转移向TR谱系。经过表观遗传抹去和封装到微生物反应器中重新排列成3D ICM样结构的细胞。在同一微小生物反应器和微孔中共培养TR样细胞和ICM样球体会导致形成单一的结构，其形状均匀，类似于体内胚胎。CDX2 +细胞位于球体的外层，而OCT4 +细胞位于结构的内部。TROP2 +细胞显示YAP核积累，并积极转录成熟的TR标记物，而TROP2-细胞显示YAP细胞质分区，并表达多能性相关基因。我们描述了生成epiBlastoids的过程，它可能在辅助生殖领域找到有用的应用。 ©2023.作者(们)。

This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos.A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation.Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2- cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes.We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.© 2023. The Author(s).