Molecular gatekeepers balancing differentiation and maintenance of human male germline stem cells uncovered by single cell resolution analysis

Human male germline homeostasis depends on a balance between differentiation and maintenance of spermatogonial stem cells. To unveil the molecular regulation of this balance we compared human testicular tissues with severely perturbed germ cell differentiation with the normal situation. By performing single cell RNA sequencing of close to 30.000 cells and histomorphometric analysis we demonstrate that the proportion of spermatogonia remains remarkably stable despite the dwindling numbers of differentiated germ cells. In spite of this, these samples displayed altered stem cell dynamics with a push of reserve stem cells towards differentiation. This shift in dynamics was associated with the activation of gene networks regulated by four main gatekeeping transcription factors: ASCL2, EGR4, HOXC9, and DLX5. We provide invaluable information on the pathways regulating human germline stem cell dynamics along with evidence for a striking depletion of the reserve stem cell pool when these are disturbed. Overall design: single-cell RNA-Seq of testicular tissue of patients with normal spermatogenesis (Normal; n=3) and patients with cryptozoospermia (Crypto; n=3)