Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system

Cell Death and Differentiation, Jan 2018

Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility.

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Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system

Abstract Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility. Introduction Infertility has currently become one of the most serious issues affecting human reproduction and health, due to genetic variants and segregating alleles [1, 2], environmental pollution, and epigenetic factors. It has been reported that about 15% of couples are infertile in the USA [3], and half of them result from male infertility [4]. In China, it has been estimated that 50 million of men are infertile, and notably, azoospermia comprise 25% of male infertility cases [5]. Therefore, it is of great interest to generate functional gametes for patients with male infertility especially for azoospermia. Spermatogenesis is a complex process by which spermatogonial stem cells (SSCs) self-renew and differentiate into haploid spermatids within the elaborated microenvironment or the niche in the seminiferous epithelium. Any errors that occur during spermatogenesis can lead to male infertility [6]. Owing to the destroyed niche of testes, azoospermic patients with SSCs are usually unable to produce functional spermatids. Thus, generating human male gametes in vitro has retained a key issue and central goal in the field of cell biology and reproductive medicine [3]. Production of spermatids in vitro would not only provide male gametes for azoospermic patients but also offers an excellent platform for investigating molecular mechanisms underlying human germ cell development and male infertility [7]. Various methods for the in vitro derivation of male germ cells have been developed, mostly based on the two-dimensional (2D) culture, the implementation of defined medium, and feeder cells [8,9,10]. These studies illustrate that spermatogenesis including meiosis can be initiated in vitro. However, the 2D culture cannot effectively mimic the microenvironment of testis due to the lack of relevant growth factors and the disruption of spatial structure, and thus its differentiation efficiency is relatively low. In contrast to conventional 2D culture, the three-dimensional (3D) culture could provide an ideally spatial environment for the cells. Moreover, the 3D culture technique can build a system in vitro, which is closed to the cell developmental microenvironment in vivo. The 3D culture can be utilized to probe the interactions among male germ cells, somatic cells (e.g., Sertoli cells and Leydig cells), and the extracellular matrix (ECM) during spermatogenesis [11,12,13,14]. In addition, the 3D culture system has been used to generate male germ cells in rodents. It has been shown that 3D culture using collagen matrix for mouse testicular cells promotes the differentiation of germ cells to spermatids [15]. In addition, testicular tissues of newborn mice and mouse SSCs can be induced to differentiate into functional spermatids in vitro using 3D culture system [16,17,18], but the efficiency is exceedingly low, usually <2%. Round spermatids can be derived in vitro from mouse spermatogonia [19]; nevertheless, the functionality of the spermatids has not been evaluated. Recently, mouse embryonic stem (ES) cells can be differentiated to germ cells, which can complete meiosis in vitro and give rise to spermatids with fertilization and healthy offspring [20]. However, generation of functional spermatids in vitro has not yet been achieved in humans but is highly anticipated. In the present study, we have for the first time reported the detailed information on a 3D-induced (3D-I) system as well as molecular and cellular evidence demonstrating efficient differentiation of human SSCs into functional spermatids in vitro. According to the “gold standard” criteria for the in vitro-derived gametes [3], phenotypic characteristics, DNA content, chromosome content, chromosomal synapsis and recombination, Y chromosome microdeletions, genetic and epigenetic imprinting, and fertilization and development capacity were detected in the (...truncated)


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Min Sun, Qingqing Yuan, Minghui Niu, Hong Wang, Liping Wen, Chencheng Yao, Jingmei Hou, Zheng Chen, Hongyong Fu, Fan Zhou, Chong Li, Shaorong Gao, Wei-Qiang Gao, Zheng Li, Zuping He. Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system, Cell Death and Differentiation, 2018, pp. 747-764, Issue: 25, DOI: 10.1038/s41418-017-0015-1