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Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts

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Embryo Models In Vitro

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2767))

Abstract

Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.

Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined “epiBlastoids,” whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.

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Acknowledgments

This work was supported by Carraresi Foundation, PSR2021, and HORIZON-WIDERA-2021 project n#101079349 (OH-Boost). Authors are members of the Trans-COST Actions Taskforce on Covid-19.

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Authors and Affiliations

  1. Laboratory of Biomedical Embryology and Tissue Engineering, Department of Veterinary Medicine and Animal Sciences, Centre for Stem Cell Research, Università degli Studi di Milano, Lodi, Italy

    Sharon Arcuri, Georgia Pennarossa & Tiziana A. L. Brevini

  2. Department of Veterinary Medicine, University of Sassari, Sassari, Italy

    Sergio Ledda

  3. Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy

    Fulvio Gandolfi

Authors
  1. Sharon Arcuri
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  2. Georgia Pennarossa
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  3. Sergio Ledda
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  4. Fulvio Gandolfi
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  5. Tiziana A. L. Brevini
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Editor information

Editors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Magdalena Zernicka-Goetz

  2. Ottawa, ON, Canada

    Kursad Turksen

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Arcuri, S., Pennarossa, G., Ledda, S., Gandolfi, F., Brevini, T.A.L. (2023). Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts. In: Zernicka-Goetz, M., Turksen, K. (eds) Embryo Models In Vitro. Methods in Molecular Biology, vol 2767. Humana, New York, NY. https://doi.org/10.1007/7651_2023_486

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  • DOI: https://doi.org/10.1007/7651_2023_486

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3685-5

  • Online ISBN: 978-1-0716-3686-2

  • eBook Packages: Springer Protocols

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