Background: Differentiation of the gonads in men is closely dependent on Sertoli cells maturation. Differences of sex development (DSD) are caused by variations in this process. The study of the mechanisms underlying these complex conditions is crucial for optimal clinical management and Sertoli cells would be an ideal model for this purpose. However, there are two main obstacles for the study of human Sertoli cells. Firstly, mature human Sertoli cells lose their proliferation abilities in culture. Secondly, the currently available models (human NT2D1 and mouse TM4 cells) demonstrated to have limitations due to their origin as mouse or tumor cells.
Objective and Hypothesis: To establish a more suitable model to study human testis formation, we differentiated human fibroblasts-derived induced pluripotent stem cells (iPSCs) into human Sertoli-like cells.
Methods: We reprogrammed human fibroblasts into iPSCs by lentivirus transduction of reprograming factors (Oct4, SOX2, NANOG, LIN28, KLF4 and C-MYC). Subsequently, we guided the differentiation of iPSCs into SLCs by growth factors and characterized this new model by new generation sequencing techniques including 44.946 genes expression analysis. In a more detailed analysis, we selected 20 gene markers for the different stages of Sertoli cell development including SRY-Related HMG-Box 9 (SOX9), vimentin (VIM), Cytochrome P450 Retinoid Metabolizing Protein (CYP26B1) and Proto-Oncogene Tyrosine-Protein Kinase Src (SRC). We additionally tested whether SLC are able to create three-dimensional structures in gel matrix and the expression of claudin-11 (CLDN-11) in tight junctions.
Results: This approach revealed that SLCs expressed Sertoli cell markers such as SOX9 and VIM, When compared the other current models (NT2D1 and TM4 cells), SLCs showed a reduction of the germ cell markers SOX2, POU5F1, DPPA2, DPPA4 and NANOG and an increased expression of Sertoli cell markers CYP26B1, SCF and SRC (P<0.05 for all). We additionally demonstrated the ability of SLCs to form three-dimensional structures when grown in extracellular matrix gel and expressed CLDN-11 in the tight junctions as human Sertoli cells.
Conclusion: Harnessing the power of iPSCs we were able to generate Sertoli-like cells that show genetic and functional similarities to human Sertoli cells. Thanks to this novel approach, Sertoli-like cells may become an alternative source of patient-specific Sertoli cells models that may boost the understanding of the individual complexities of DSD patients.
27 Sep 2018 - 29 Sep 2018