Generation of fertile offspring from Kitw/Kitwv mice through differentiation of gene corrected nuclear transfer embryonic stem cells

Abstract

Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit w /Kit wv mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kit w /Kit wv somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kit w /Kit wv infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.

Restore natural fertility of Kitw/Kitwv mouse with nonobstructive azoospermia through gene editing on SSCs mediated by CRISPR-Cas9

“…This strategy only includes SSC isolation, in vitro gene editing, and transplantation, three main steps which are greatly simplified compared to a previous report [9]. Besides, autologous transplantation is more ethical and immunotoxic reactions are removed.…”

“…Gamete-deficient NOA due to germ cells’ genetic mutations, such as RBMY [5], KLHL10 [6], and SYCP3 [7], has been incurable by assisted reproductive technologies and difficult to bear for patients [8]. Cell therapies are considered to be one of the most promising strategies to rescue this type of infertility [9].…”

“…c-Kit is expressed in germ cells and controls germ cell differentiation in mammalian testis [10]. Heterozygous mutant mice with W and WV mutation of this gene, the Kit w /Kit wv mice, whose spermatogonia are considerably reduced or exhausted, have been used as an excellent animal model to develop therapeutic strategies for gamete-deficient patients due to genetic mutations by Yuan et al [9]. They isolated tail-tip fibroblasts from adult Kit w /Kit wv mice and then derived embryonic stem cells from these cloned blastocysts obtained by somatic cell nuclear transfer.…”

Approaches and Technologies in Male Fertility Preservation

“…Recently, iPSCs were developed from Klinefelter and NOA patients [117,118]. It was shown that iPSC from rodents, monkeys, and humans can be differentiated into male germ cells [4,85,112,[118][119][120][121][122][123][124][125][126][127][128]. The addition of bone morphogenetic proteins (BMPs) to human iPSC induced their differentiation into primordial germ and meiotic cells [129].…”

干细胞与再生医学

“…此外, 人卵母细胞的形态分 级 [44] 及异种克隆的尝试 [45] , 都促进了治疗性克隆的 发展. 最近, Kit w /Kit wv 基因型 ntESCs 经基因修饰及 体内分化, 使 Kit w /Kit wv 不育雄性小鼠获得了正常可 育子代 [46] , 以及小鼠 ESCs 体外诱导分化为可育精细 胞样细胞(spermatid-like cells) [47] , 为再生医学治疗男 性不育指明了方向 [46] . 提高核移植重编程效率是促进治疗性克隆发展 的潜在动力, 关于如何提高核移植效率的文献层出 不穷.…”