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

Hua Huang,Jie Sun,Liang Zhong,Yanping Hou,Jin Zhou,Xiaoyu Xing,Zhiyuan Zhang

doi:10.1111/jcmm.15018

Hua Huang, Jie Sun + Show 5 more

Open Access

https://doi.org/10.1111/jcmm.15018

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Abstract

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.

Highlights

H.E staining showed that transplanted human foreskin fibroblasts (HFFs) and into functional Leydig-like cells (iLCs) were capable of surviving and distributed in the Matrigel just as Leydig cells localize in the interstitium of the testis, and the nuclei of HFFs were arranged in a spindle shape, but the nuclei of iLCs were arranged in a round-like shape (Figure 3B,C)
We successfully converted human foreskin fibroblasts into functional iLCs by simultaneous activation of the endogenous target genes Nr5a1, Gata[4] and Dmrt[1], and to our knowledge, this is the first study of reprogrammed iLCs based on the CRISPR/dCas[9] SAM system[12]; importantly, the iLCs showed some characteristics of adult LCs, including the ability to secrete testosterone, respond to hCG in vitro and express Leydig steroidogenic enzymes
We demonstrated that reprogrammed iLCs based on the CRISPR/dCas[9] SAM system could survive for over 6 weeks and partially recover the serum testosterone level of castrated rats when they were transplanted into the fibrous capsule

Summary

| INTRODUCTION

Male hypogonadism due to testosterone deficiency is common in men aged 40-79 years, approximately 20% of whom are suffering from the disease, and incidence increases with age.[1,2] Several studies show that hypogonadism can lead to arteriosclerosis, decreased bone mineral density and depressed mood as well as fatigue and sexual dysfunction.[3,4,5] Testosterone replacement therapy (TRT) is. We successfully reprogrammed human foreskin fibroblasts into functional iLCs by using the CRISPR/dCas[9] SAM system for simultaneous activation of the endogenous target genes Nr5a1, Gata[4] and Dmrt[1], and importantly, the iLCs possessed some characteristics of mature LCs, including the ability to secrete testosterone, respond to hCG in vitro and express Leydig steroidogenic enzymes.[12] it is not known whether the reprogrammed iLCs can restore serum testosterone levels in vivo or mimic the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis. We demonstrated that the transplanted iLCs could partially restore the serum testosterone levels of castrated rats, mimic weakly the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis for a short period, and survive and secrete testosterone, though weakly, at 6 weeks after transplantation. This study may be valuable for male hypogonadism in future

| MATERIALS AND METHODS

Findings

| DISCUSSION