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+CD9medEPCAMmedCD155+CD148+CD47highCD7high 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.