Leydig-like cells derived from reprogrammed human foreskin fibroblasts by CRISPR/dCas9 increase the level of serum testosterone in castrated male rats.

In the past few years, Leydig cell (LC) transplantation has been regarded as an effective strategy for providing physiological patterns of testosterone in vivo. Recently, we have successfully converted human foreskin fibroblasts (HFFs) into functional Leydig-like cells (iLCs) in vitro by using the CRISPR/dCas9 system, which shows promising potential for seed cells. However, it is not known whether the reprogrammed iLCs can survive or restore serum testosterone levels in vivo. Therefore, in this study, we evaluate whether reprogrammed iLCs can restore the serum testosterone levels of castrated rats when they are transplanted into the fibrous capsule. We first developed the castrated Sprague Dawley rat model through bilateral orchiectomy and subsequently injected extracellular matrix gel containing transplanted cells into the fibrous capsule of castrated rats. Finally, we evaluated dynamic serum levels of testosterone and luteinizing hormone (LH) in castrated rats, the survival of implanted iLCs, and the expression levels of Leydig steroidogenic enzymes by immunofluorescence staining and Western blotting. Our results demonstrated that implanted iLCs could partially restore the serum testosterone level of castrated rats, weakly mimic the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis for a short period, and survive and secrete testosterone, through 6 weeks after transplantation. Therefore, this study may be valuable for treating male hypogonadism in the future.