Quiescent very small embryonic-like stem cells resist oncotherapy and can restore spermatogenesis in germ cell depleted mammalian testis.

: Adult mouse and human testes harbor relatively quiescent, pluripotent very small embryonic-like stem cells (VSELs), in addition to actively dividing spermatogonial stem cells (SSCs). Here we report that various oncotherapy regimens in human cancer patients (n=7) and busulphan treatment (25mg/Kg body weight) in eight weeks old male mice (n=15) selectively affects actively dividing SSCs, spermatogonia, haploid germ cells and somatic microenvironment resulting in germ cell aplasia, whereas VSELs are unaffected and persist in otherwise germ cell depleted testis. Testicular VSELs are 2-5 µm in size, have high nucleo-cytoplasmic ratio, SCA-1+/CD45-/LIN- (mice), CD133+/CD45-/LIN- (human survivors of childhood cancer) and express various pluripotent transcripts including OCT-4A. SCA-1 sorted cells from busulphan treated mice testes in vitro formed small clusters suggestive of self-renewal and differentiation into progenitors, which divide rapidly. Inter-tubular random injections of syngeneic Sertoli cells (105 cells per testis, n=14) or bone marrow derived mesenchymal cells (104 cells per testis, n=16) into the germ cell depleted busulphan treated mice testes, were able to restore spermatogenesis from persisting VSELs. Transplanted Sertoli or mesenchymal cells possibly were a source of growth factors essential for VSELs differentiation. Since sperm formation occurred in situ, various epigenetic concerns associated with the 'synthetic gametes' may be eliminated in our approach. Ability of mesenchymal cells to restore spermatogenesis may benefit existing azoospermic survivors of childhood cancer who were otherwise deprived of testicular tissue cryopreservation prior to oncotherapy. Further studies are warranted to delineate the underlying mechanisms and to study quality and potential of sperm generated by this approach.