232 Combined effect of miR-200b/c and mechanical stimuli to generate blastoids in vitro
G. Pennarossa A , R. Pasquariello B , S. Arcuri A , S. Ledda C , F. Gandolfi B and T. Brevini AA Laboratory of Biomedical Embryology, Department of Veterinary Medicine and Animal Sciences and Center for Stem Cell Research, Università degli Studi di Milano, Milan, Italy
B Laboratory of Biomedical Embryology, Department of Agricultural and Environmental Sciences: Production, Landscape, Agroenergy, Università degli Studi di Milano, Milan, Italy
C Department of Veterinary Medicine, Università degli Studi di Sassari, Sassari, Italy
Reproduction, Fertility and Development 35(2) 245-245 https://doi.org/10.1071/RDv35n2Ab232
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Mammalian embryogenesis is driven by complex interactions that coordinate morphogenesis, coupling biomechanical and biochemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryonic development. Here, we describe a three-step method that combines the use of miR-200b/c and mechanical stimuli to generate 3D spherical structures, arbitrarily defined “blastoids,” whose phenotype is remarkably similar to natural embryos. In the first step, we reprogram porcine adult dermal fibroblasts into trophoblast (TR)-like cells, using miR-200b/c to induce a transient high plasticity state, followed by an ad hoc induction protocol (Arcuri 2021 Methods Mol. Biol; Arcuri 2021 Front. Vet. Sci). In the second step, we combine mechanosensing-related stimuli by cells encapsulation in PTFE micro-bioreactors with miR-200b/c treatment for the generation of ICM-like spheroids (Pennarossa 2020 JoVE). In the third step, we co-culture reprogrammed TR-like cells with ICM-like spheroids in the same micro-bioreactors for two days and then transfer them into AggreWell™ plates for four days, to encourage further differentiation and allow blastoid formation. At the end of step one, cells display a mature TR morphology, with epithelioid shape, round nuclei, and well-defined borders. They transcribe the TR mature markers GCM1, PPAG3, PAG6, HSD17B1, CYP11A1, and IFNG, which were originally absent in untreated fibroblasts. In the second step, fibroblasts exposed to miR-200b/c and encapsulated in PTFE micro-bioreactors aggregate in 3D spherical structures with uniform size geometry. This is paralleled by the onset of the pluripotency-related markers OCT4, NANOG, REX1, and SOX2, completely absent in untreated cells, and by downregulation of the fibroblast-specific markers VIM and THY1. At the end of the third step (six days), reprogrammed TR-like cells and ICM-like spheroids rearrange in single 3D spheroids with size raging between 100 and 200 μm and maintain stable and high transcription levels for the pluripotency-related genes OCT4, NANOG, REX1, and SOX2 as well as for the mature TR cell markers GCM1, PPAG3, PAG6, HSD17B1, CYP11A1, and IFNG. Overall, the procedure here described is a novel strategy for the in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. This method represents a notable advancement in organoid technology, does not require retroviral gene transfection, and leads to the generation of blastoids to study early embryogenesis as well as embryo disorders.
This research was funded by Carraresi Foundation, PSR2020, PSR2021.
