Use of miR-200-Mediated Reprogramming and Mechanical Cues to Generate Human Blastocyst Models.

Several early developmental events remain unclear due to ethical and technical limitations related to the use of natural embryos. To overcome this problem, over the last decade, different approaches aimed at the generation of in vitro blastocyst-like models have been developed.Here, we describe a protocol that combines miR-200-mediated cell reprogramming and mechanical stimuli to create 3D spheroids phenotypically similar to natural embryos. Specifically, dermal fibroblasts are reprogrammed exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either differentiated into trophoblast (TR)-like cells, using an ad hoc induction protocol, or encapsulated into polytetrafluoroethylene (PTFE) micro-bioreactors to generate inner cell mass (ICM)-like spheroids. Subsequently, TR-like cells and ICM-like spheroids are encapsulated and co-cultured in the same micro-bioreactor and then transferred to microwells to allow further differentiation and boost blastoid development.The generated 3D artificial blastoids, phenotypically similar to natural embryos, could find a useful application in the field of assisted reproduction technologies to further characterize the events taking place during early embryogenesis as well as to study embryo disorders.