Generation Of Fertile Offspring From Kit^w/Kit^wv Mice Through Differentiation Of Gene Corrected Nuclear Transfer Embryonic Stem Cells

Primordial germ cells(PGCs) are the earliest source of both male and female germ cells. Spermatogenesis is a complex process of cell specification in male sexual reproduction, which contains three main stages, including proliferation of spermatogonial stem cells and mitosis, meiosis of spermatocytes and sperm metamorphosis. The process that spherical sperm metamorphosed into elongated spermatids is known as sperm formation. And germ cells’ abnormalities will lead to a reduction of postnatal germ cells formation in the gonads or even the disapperance, and spermatogenesis disorders will lead to little, weak, abnormal sperm and azoospermia. Numerous reasons can cause the formation disorder of germ cells, whereas gene mutation is one of the common reasons.According to the World Health Organization(WHO) assessment, infertility has been a threat to about 15 percent of reproductive age couples in the world, which means that one of every six to seven couples undergo reproductive disorders. In our country, recent surveys claims 10 percent of married couples bears infertility, and the incidence is rising. Male infertility accounts for about half of human infertility. There are many reasons in male sterility diseases, such as the formation of germ cells, abnormal spermatogenesis and so on. Genetic mutations can cause both the problems, which lead to defective sperm function, thereby causing male infertility. Though assisted reproductive technology can solve some infertility problems, it’s still inability to azoospermia patients, in whose testes functional gametes are no available. Therefore patients could not produce progeny even with assisted reproduction technologies such as in vitro fertilization(IVF) and ICSI. Additionally, many gene mutations from parent could be passed to the next generation, which still affecting fertility. It is a major challenge to restore the fertility of patients with azoospermia due to genetic mutations. In our study, using Kitw/Kitwv mouse model, we manage to investigate the feasibility of generating functional sperm from gamete deficient patients by combining the reprogramming and gene correction technologies.1. Derivation of nuclear transfer embryonic stem cells from the Kitw/Kitwv fibroblastsThe Kitw/Kitwv mutation ntESCs were derived from cloned embryos which are created by nuclear transfer of Kitw/Kitwv somatic cells. “all-ES” mice were produced by tetraploid complementation with Kitw/Kitwv mutation ntESCs, which certified that Kitw/Kitwv mutation ntESCs were authentic pluripotent stem cells.2. W point mutation correction via TALENsFor gene correction, mutation nuclear transferred embryonic stem cells(ntESCs) were co-transfected with TALEN expression vectors and the targeting vector. Then, drug-resistant clones were picked out after neomycin and ganciclovir selection, then removed the piggyBac-flanked selection cassette. No excess exogenous gene are left, so it does not affect the genome integrity, and it is easy to operate, even more specifical.3. Germ cells specification from repaired ntESCs in vitroThe corrected ntESCs were further differentiated into primordial germ cell- like cells(PGCLCs) that could be purified by the surface markers of PGCs, SSEA1 and Integrin β3 after 6 days’ differentiation according to the protocol described previously. Unrepaired ntESCs could not produce PGCLCs via in vitro differentiation.4. PGCLCs’ transplantation into Kitw/Kitwv or busulfan-treated mouse testes.Firstly repaired PGCLCs were directly transplanted into Kitw/Kitwv testes. In order to take advantage of busulfan-treated testes as receptors, a red fluorescent protein(RFP) introducted into repaired ntESCs as a tracer. So as to mimic the clinical application, repaired PGCLCs were co-transplanted with enhanced green fluorescent protein(EGFP) labeled testicular somatic cells. Two months later, transplanted testes were isolated for slice analysis, and these three methods were all able to establish complete seminiferous epithelium and accomplish spermatogenesis.5. Generation of fertile offspring with gametes derived from PGCLCsSeminiferous tubules were separated and digested into single cells for sorting haploid cells which were used for intracytoplasmic sperm injection(ICSI),and healthy individuals were generated from transplanted by virtue of pesudo-pregnant mice.In this study, we combined the somatic cell reprogramming, gene correction and germ cell differentiation from embryonic stem cell to turn the adult somatic cells to PGCLCs successfully in vitro, and then achieved functional sperm which could get healthy offspring via the in vivo environment. The usage of mouse model provided the possibility to improve the feasibility of our technic, meanwhile, our researches also lay a solid foundation for the clinic treatment of female infertility caused by mutation.