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4. In vitro cellular reprogramming to model gonad development and its disorders

Author

Gonen, Nitzan, Eozenou, Caroline, Mitter, Richard, Elzaiat, Maëva, Stévant, Isabelle, Aviram, Rona, Bernardo, Andreia Sofia, Chervova, Almira, Wankanit, Somboon, Frachon, Emmanuel, Commere, Pierre-Henri, Brailly-Tabard, Sylvie, Valon, Léo, Barrio Cano, Laura, Levayer, Romain, Mazen, Inas, Gobaa, Samy, Smith, James C., McElreavey, Kenneth, Lovell-Badge, Robin, Bashamboo, Anu, Bar-Ilan University [Israël], The Francis Crick Institute [London], Génétique du Développement humain - Human developmental genetics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Imperial College London, Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology, Institut Pasteur [Paris] (IP), Biomatériaux et Microfluidiques (plateforme) - Biomaterials and Microfluidics (platform), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Mort cellulaire et homéostasie des épithéliums / Cell death and epithelial homeostasis, National Research Center [Caire, Egypte], This work is funded in part by a research grant (40000767) from the European Society of Pediatric Endocrinology (to A.B.) and by the Agence Nationale de la Recherche (ANR, ANR-10-LABX-73 REVIVE, ANR-17-CE14-0038-01, and ANR 20CE14 0007 to K.M., and ANR-19-CE14-0022 and ANR-19-CE14-0012 to A.B.). N.G., A.S.B., R.M., J.C.S., and R.L.-B. were funded by the Francis Crick Institute. The Francis Crick Institute receives its core funding from Cancer Research UK (CC2116), the UK Medical Research Council (CC2116), and the Wellcome Trust (CC2116). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. . N.G., I.S., and R.A. are funded by the ERC Starting Grant EnhanceSex (101039928). A.S.B. was also funded by the British Heart Foundation (BHF-FS/12/37/29516) and the Wellcome Trust (210987/Z/18/Z)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-17-CE14-0038,MGonDev,Etude des mécanismes du développement des gonades chez l'homme(2017), ANR-20-CE14-0007,Goldilocks,Analyse intégrée du rôle du facteur de transcription SF-1 / NR5A1 et de ses gènes cibles dépendants du dosage dans la fonction gonadique et les troubles du développement sexuel (DSD)(2020), ANR-19-CE14-0022,SexDiff,Régulation de la détermination du sexe et de la différenciation ovarienne : implications dans les troubles du développement sexuel(2019), and ANR-19-CE14-0012,RNA-SEX,Fonction de l'ARN hélicase dans la détermination du sexe chez les vertébrés et les troubles du développement du sexe chez l'homme (DSD)(2019)

Subjects
Model organisms, Chemical Biology & High Throughput, Multidisciplinary, MESH: Humans, FOS: Clinical medicine, Stem Cells, [SDV]Life Sciences [q-bio], Genome Integrity & Repair, Neurosciences, Gene Expression, MESH: Gonadal Dysgenesis, 46,XY, Tumour Biology, MESH: Induced Pluripotent Stem Cells, MESH: Cellular Reprogramming, MESH: Male, Signalling & Oncogenes, MESH: Gonads, MESH: Animals, Genetics & Genomics, MESH: Mice, MESH: Female, Developmental Biology, Computational & Systems Biology
Abstract

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro–derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9–mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Published
2023
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5. PMON275 GONAD-on-CHIP to study early gonad development and DSD

Author

Bashamboo, Anu, primary, Gonen, Nitzan, additional, Eozenou, Caroline, additional, Mitter, Richard, additional, Bernardo, Andreia Sofia, additional, Chervova, Almira Chervova, additional, Frachon, Emmanuel, additional, Commère, Pierre-Henri, additional, Brailly-Tabard, Sylvie, additional, Mazen, Inas, additional, Gobaa, Samy, additional, Smith, James, additional, McElreavey, Kenneth, additional, and Lovell-Badge, Robin, additional

Published
2022
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7. In-vitro cellular reprogramming to model gonad development and its disorders

Author

Gonen, Nitzan, primary, Eozenou, Caroline, additional, Mitter, Richard, additional, Bernardo, Andreia, additional, Chervova, Almira, additional, Frachon, Emmanuel, additional, Commère, Pierre-Henri, additional, Mazen, Inas, additional, Gobaa, Samy, additional, McElreavey, Kenneth, additional, Lovell-Badge, Robin, additional, and Bashamboo, Anu, additional

Published
2021
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14. Microfabricated Device for High-Resolution Imaging of Preimplantation Embryos.

Author

Vandormael-Pournin S, Frachon E, Gobaa S, and Cohen-Tannoudji M

Subjects
Animals, Electroporation methods, Embryo Culture Techniques methods, Female, Male, Mice, Mice, Inbred C57BL, Microinjections methods, Blastocyst ultrastructure, Microscopy, Confocal methods
Abstract

The mouse preimplantation embryo is an excellent system for studying how mammalian cells organize dynamically into increasingly complex structures. Accessible to experimental and genetic manipulations, its normal or perturbed development can be scrutinized ex vivo by real-time imaging from fertilization to late blastocyst stage. High-resolution imaging of multiple embryos at the same time can be compromised by embryos displacement during imaging. We have developed an inexpensive and easy-to-produce imaging device that facilitates greatly the imaging of preimplantation embryo. In this chapter, we describe the different steps of production and storage of the imaging device as well as its use for live imaging of mouse preimplantation embryos expressing fluorescent reporters from genetically modified alleles or after in vitro transcribed mRNA transfer by microinjection or electroporation.

Published
2021
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