Improvement of Germ Stem Cell Pool by Leptin Supplementation and Gene Editing for Male Infertility

Abstract

Infertility affects up to 12-15% of sexually active couples, and male factors contribute about 30-55% of these cases. Infertility can be caused by injuries, genetic factors, exposure to toxicants, immune-suppressive and anti-cancer treatments, but a large number are unexplained (idiopathic). Impairment of fertility is a major concern for the pediatric cancer survivors (PCSs). Freezing of testicular tissue containing spermatogonial stem cell (SSCs) is the only option to preserve the fertility. Sperm collection or culture of SSCs are not available in azoospermic patients with MCM8 gene disorders. For these infertile men, somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into germ cells after correction of mutation via gene editing. Culture is critical to expand the number SSCs from small biopsies obtained from the testes of prepubertal patients prior to transplantation and to enable gene editing if SSCs or iPSCs for patients with genetic infertility. We tested the hypothesis that leptin supplementation may induce proliferation of neonatal mouse SSCs in vitro via ERK and/or STAT3 pathways. We also hypothesized that Mcm8 mutation may be corrected by CRISPR/Cas9 to produce gene edited primordial germ cells like cells (PGCLCs) that could be transplanted to restore fertility in Mcm8-/- infertile mouse. C57BL/6 neonatal SSCs were cultured and treated with leptin (0-200 ng/ml). 100 ng/ml leptin supplementation enhances proliferation SSC cultures via ERK and STAT3 pathways. Two novel Mcm8-/- mouse iPSC lines (591 and 574) were generated from Mcm8-/- mice fibroblasts. A validated Mcm8-/- iPSC (591-14) clone was gene-edited via CRISPR/Cas9 and identified with one Mcm8 allele corrected back to the wild type by DNA sequencing. Gene-corrected (Mcm8gc/-) clone 14 was differentiated to PGCLCs that exhibited a SSEA1+/CD61+ phenotype. These studies may allow the expansion of SSC pool, in vitro, prior to auto-transplantation to regenerate spermatogenesis in cancer survivors and correction of single gene mutation causing infertility by CRISPR/Cas9 tool in infertile male.