Efficient generation of human primordial germ cell-like cells from pluripotent stem cells in a methylcellulose-based 3D system at large scale

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.

Evolving Role of RING1 and YY1 Binding Protein in the Regulation of Germ-Cell-Specific Transcription

“…Gametogenesis has been thoroughly studied in model organisms, and in some aspects, in vitro differentiation systems using embryonic stem (ES) cells [5,6] and induced pluripotent stem (iPS) cells [7,8]. By now, a lot of information has been gathered about the epigenetic regulation, the specific transcription factors, important signalization events, and key effectors necessary for commitment of stem cells to germline fate [9,10,11,12,13,14] from different model organism and by in vitro differentiation of stem cells to primordial germ-cell-like cells (PGCLCs) [9,10,11,12,13,14,15,16,17].…”

Advances in Female Germ Cell Induction from Pluripotent Stem Cells

“…Different stages correspond to distinct gene expressions and epigenetic regulations [ 26 ]. These distinct epigenetic regulatory networks of female GCs at sequential developmental phases could be studied through the genome-wide DNA methylation and chromatin accessibility using single-cell resolution [ 88 ]. These efforts on female GCs development mechanisms would contribute to a more efficient and stable female GC induction in vitro .…”

“…Although human PGCLCs had been recapitulated in vitro , U-bottom 96 plates or other similar plates used in these studies limited the scale production of human PGCLCs production [ 6 – 8 ]. Recently, a new modified system of methylcellulose-based 3D induction system combined with low-cell attachment plates was reported to produce human PGCLCs from human PSCs at a large scale, with similar gene expression and epigenetic modification profiles to human PGCs [ 88 ]. Besides the 3D induction system, 3D bioprosthetic ovaries were also confirmed to provide 3D support for oocyte cultivation.…”

Approaches and Technologies in Male Fertility Preservation

“…These PGCs can either be auto-transplanted into the testes (in vivo) or used in vitro in order to induce the development of gametes [113][114][115][116]. 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].…”

“…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].…”