Characterisation and hierarchy of the spermatogonial stem cell compartment in human spermatogenesis by spectral cytometry using a 16-colors panel

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.