Abstract
BackgroundThe mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation.MethodsThe efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage.ResultsIn the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system.DiscussionThe in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.
Highlights
In mammals, including humans, the earliest germ cells are primordial germ cells (PGCs), which are derived from early embryos
Methylcellulose is widely used in 3D culture for cell suspension and it could significantly increase the yield of target cells and generate more mature cells or tissue types (Jiang et al, 2015; Kothapalli & Kamm, 2013; Lee & Park, 2016; Otsuji et al, 2014)
We aimed to test the efficiency of a MC-based 3D hPGCLC induction system
Summary
In mammals, including humans, the earliest germ cells are primordial germ cells (PGCs), which are derived from early embryos. An effective in vitro human germ cell induction system or disease model is in high demand and will facilitate an understanding of human germline development and provide a platform for treating disorders arising from human germ cell defects (Nagamatsu & Hayashi, 2017). The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. An effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage
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