Abstract
Recently, Leydig cell (LC) transplantation has been revealed as a promising strategy for treating male hypogonadism; however, the key problem restricting the application of LC transplantation is a severe lack of seed cells. It seems that targeted activation of endogenous genes may provide a potential alternative. Therefore, the aim of this study was to determine whether targeted activation of Nr5a1, Gata4 and Dmrt1 (NGD) via the CRISPR/dCas9 synergistic activation mediator system could convert human foreskin fibroblasts (HFFs) into functional Leydig‐like cells. We first constructed the stable Hsd3b‐dCas9‐MPH‐HFF cell line using the Hsd3b‐EGFP, dCas9‐VP64 and MS2‐P65‐HSF1 lentiviral vectors and then infected it with single guide RNAs. Next, we evaluated the reprogrammed cells for their reprogramming efficiency, testosterone production characteristics and expression levels of Leydig steroidogenic markers by quantitative real‐time polymerase chain reaction or Western blotting. Our results showed that the reprogramming efficiency was close to 10% and that the reprogrammed Leydig‐like cells secreted testosterone rapidly and, more importantly, responded effectively to stimulation with human chorionic gonadotropin and expressed Leydig steroidogenic markers. Our findings demonstrate that simultaneous targeted activation of the endogenous NGD genes directly reprograms HFFs into functional Leydig‐like cells, providing an innovative technology that may have promising potential for the treatment of male androgen deficiency diseases.
Highlights
Leydig cells (LCs), which are distributed in the interstitial tissue of the testis, are the primary source of androgens and play an essential role in spermatogenesis and male development.[1,2] Male hypogonadism may be associated with symptoms related to low production of testosterone, such as changes in body composition, increased fatigue, sexual dysfunction, depressed mood and decreased bone mineral density.[3,4,5] Traditional testosterone replacement therapy has a good effect on male hypogonadism
We examined the expression levels of genes including Nr5a1, Gata[4] and Dmrt[1] by quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis at the indicated time points, and the relative quantitative data revealed that the mRNA levels in cells treated with sgNGD were higher than those in cells treated with the sgMock control (Figure 5A)
We further evaluated the kinetics of the expression of downstream Leydig steroidogenic markers including Cyp11a1, Cyp17a1, Hsd3b, Hsd17b, Star and Lhcgr and the up‐regulation of these specific markers was subsequently validated by qRT‐ PCR in sgNGD‐treated cells (Figure 5B)
Summary
Leydig cells (LCs), which are distributed in the interstitial tissue of the testis, are the primary source of androgens and play an essential role in spermatogenesis and male development.[1,2] Male hypogonadism may be associated with symptoms related to low production of testosterone, such as changes in body composition, increased fatigue, sexual dysfunction, depressed mood and decreased bone mineral density.[3,4,5] Traditional testosterone replacement therapy has a good effect on male hypogonadism. LC transplantation could result in the production of testosterone for a longer time period and maintain physiological patterns of the hormone.[7] the real problem restricting the application of LC transplantation is that there is a severe lack of seed cells.[1] In past decade, stem cells from different sources have been induced to differentiate into LCs.[8,9,10,11,12] Our previous studies have demonstrated that umbilical cord‐derived mesenchymal stem cells (MSCs) can by differentiated into LCs by appropriate inducing factors.[13,14] MSCs may present a low differentiation efficiency, ethical concerns and even tumorigenic risk. The identification of an alternative source of LCs would be of extraordinary significance for clinical applications as well as basic research
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