Characterisation and hierarchy of the spermatogonial stem cell compartment in human spermatogenesis by spectral...

About one in six couples experience fertility problems, and male infertility accounts for about half of these cases. Spermatogenesis originates from a small pool of spermatogonial stem cells (SSCs), which are of interest for the treatment of infertility but remain poorly characterised in humans. Using multiparametric spectral flow cytometric analysis with a 16-colours (16-C) panel of cell markers, we identify novel markers of SSCs and provide insights into unravelling and resolving the heterogeneity of the human spermatogonial cells. This 16-C panel of markers allowed the identification of a primitive SSCs state with the β2M^-CD51/61^-ITGA6⁺SSEA4⁺TSPAN33⁺THY1⁺CD9^medEPCAM^medCD155⁺CD148⁺CD47^highCD7^high phenotype, with a profile close to the most primitive SSCs states 0 and SSC1-B previously defined by sc-RNAseq approach. The hierarchy of events in the spermatogonial stem cell and progenitor compartment of human spermatogenesis can be delineated. This highlights the importance of a multi-parametric and spectral cytometry approach. The in-depth characterisation of testicular cells should help to overcome the lack of stem cell knowledge, that hinders the understanding of the regenerative potential of SSCs, and is a critical parameter for the successful development of new SSCs-based cell therapies.