Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts

Abstract

Purpose 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. Methods 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. Results 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. Conclusion We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues

“…The successful generation of in vitro blastocyst-like structures starting from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or epigenetically erased cells has been recently demonstrated [5,7,10,14,[28][29][30]. In the present manuscript, we describe a new approach that combines miR-200-mediated reprogramming ability with mechanical stimuli to create in vitro 3D spherical structures that are phenotypically similar to natural embryos.…”

“…In this context, we have recently reported the possibility of generating 3D multicellular spherical structures that are remarkably similar to natural embryos. We start with easily obtainable terminally differentiated cells and combine the use of epigenetic cues to erase the original cell phenotype [12,13] and mechanical stimuli to ease cell aggregation and encourage pluripotency [14,15]. The epigenetic modifier used in that experiment was the well-known eraser agent 5-azacytidine (5-aza-CR), which is able to induce both direct and indirect demethylating events [12].…”

“…To this purpose, cells belonging to the two experimental groups were transfected with miR-200s to induce a high plasticity state. Subsequently, miR200-reprogrammed young and aged cells were either driven towards the TR lineage by using an ad hoc induction protocol [24] or encapsulated into polytetrafluoroethylene (PTFE) micro-bioreactors to support tridimensional cell aggregation and encourage pluripotency [14,15,25]. Lastly, the generated TR-like cells and ICM-like spheroids were co-cultured in the same micro-bioreactor and then transferred to microwells to encourage differentiation and favor blastoid generation [14].…”

“…Blastoids were generated by assembling TR-like cells and ICM-like spheroids, as previously described [14]. More in detail, the TR-like cells differentiated for 11 days were detached from culture dishes and resuspended in G1-PLUS medium (Vitrolife, Massa, Italy) at 3 × 10 4 cells/30 µL concentration.…”

3D culture applied to reproduction in females: possibilities and perspectives

“…The LM also can be used as a micro-bioreactors, to promote 3D cell rearrangement to extend and stably maintain high plasticity in vitro ( Pennarossa et al, 2019 , 2020a ). Arcuri et al (2024) and Pennarossa et al (2023) , generated inner cell mass (ICM)-like organoids using a LM micro-bioreactor to promote 3D cell rearrangement and boost pluripotency. The cells encapsulated into micro-bioreactors rearrange in 3D ICM-like structures, creating structures called epiBlastoids, which can be used as models for studying early embryo development ( Arcuri et al, 2024 ; Pennarossa et al, 2023 ).…”