Male sex is determined by expression of the Y chromosome gene SRY in the week 6 human XY embryonic gonad specifying its development into a testis; in the absence of SRY, the XX gonad develops into an ovary. Sex determination is a useful model to study cell fate decisions. The embryonic gonads are bipotential, and SRY differentiates the somatic cells into Sertoli cells that would otherwise become ovarian granulosa cells.
In about 1% of live births intersex conditions occur where babies are born with gonads typical of neither sex. The underlying molecular basis of intersex remains unknown in many forms1, in part due to the lack of manipulatable models that recapitulate human sex determination. Mouse genetic models cannot be relied upon exclusively to recapitulate human intersex conditions, and show limitations such as functional redundancy, gene dosage or genetic buffering (alternative pathways existing for the same functional outcome) often resulting in no phenotypic consequence2. Furthermore, differences of gene expression thresholds and genetic robustness between humans and mice are becoming apparent3.
A human in vitro model that can be used to model Sertoli cell function is NT2/D1, a multipotent clonal cell line derived from a testicular tumour4. NT2/D1 cells can model a variety of human developmental processes. Undifferentiated, they model early events in male sex determination, showing SRY activation of endogenous SOX9. Differentiated, they model neuronal development5 or smooth muscle development6 under different treatments.
We have established an NT2/D1-Cas9 cell line, and characterised these cells via a suite of cell phenotyping assays including xCELLigence® RTCA and HoloMonitor® live cell imaging. We will discuss their application to validate candidate intersex genes, deleted to assess their individual gene contributions to ‘Sertoli cell like’ characteristics such as cell adhesion, proliferation and migration, tight junction formation and germ cell maintenance.