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

Nitzan Gonen1,2, Caroline Eozenou3, Richard Mitter4

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Summary
This summary is machine-generated.

Scientists developed a new in vitro system using pluripotent stem cells to study gonadal development and disorders of sex development (DSDs). This model successfully differentiates cells into gonadal progenitors, aiding research into DSD mechanisms.

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Area of Science:

  • Developmental Biology
  • Stem Cell Biology
  • Genetics

Background:

  • Gonadal fate determination during embryonic development involves complex signaling pathways.
  • Disorders of Sex Development (DSDs) arise from errors in these pathways, leading to developmental inconsistencies.
  • A lack of accessible in vitro systems hinders the study of sex determination and DSDs.

Purpose of the Study:

  • To establish robust protocols for differentiating mouse and human pluripotent stem cells into gonadal progenitors.
  • To create a functional in vitro model for studying mechanisms of sex determination and DSDs.
  • To investigate the role of specific genetic variants in DSDs using patient-derived cells.

Main Methods:

  • Development of differentiation protocols for mouse and human pluripotent stem cells towards gonadal progenitors.
  • Transcriptomic analysis to characterize in vitro-derived murine gonadal cells and compare them to in vivo counterparts.
  • Generation and characterization of Sertoli-like cells from 46,XY human induced pluripotent stem cells (hiPSCs), including those with DSD-associated variants.
  • Utilizing CRISPR-Cas9 gene editing to correct genetic variants in hiPSCs and assess phenotypic rescue.

Main Results:

  • In vitro-derived murine gonadal cells demonstrated equivalence to embryonic day 11.5 in vivo progenitors based on transcriptomic analysis.
  • Human iPSCs from 46,XY individuals differentiated into Sertoli-like cells exhibiting testis-specific gene expression, anti-Müllerian hormone secretion, migration, and tubule formation.
  • Cells derived from 46,XY DSD female hiPSCs with an NR5A1 variant showed abnormal gene expression and failed tubule formation.
  • CRISPR-Cas9 correction of the NR5A1 variant in hiPSCs rescued the observed cellular phenotype.

Conclusions:

  • The developed protocols provide a robust in vitro system for generating gonadal progenitor cells from pluripotent stem cells.
  • This system effectively models key aspects of male sex determination and allows for the study of DSDs.
  • The successful correction of a DSD phenotype via CRISPR-Cas9 highlights the utility of this model for understanding genetic contributions to DSDs and for potential therapeutic strategies.