Engineering of 3D substrates with maximum similarity to seminiferous tubules would help to produce functional sperm cells in vitro from stem cells. Here, we present a 3D electrospun gelatin (EG) substrate seeded with Sertoli cells and determine its potential for guided differentiation of embryonic stem cells (ESCs) toward germline cells. The EG was fabricated by electrospinning, and its morphology under SEM, as well as cytobiocompatibility for Sertoli cells and ESCs, was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and cell attachment assay. Embryoid bodies (EBs) were formed from ESCs and co-cultured with Sertoli cells, induced with BMP4 for 3 and 7 consecutive days to induce the differentiation of EBs toward germline cells. The differentiation was investigated by immunocytochemistry (ICC), flow cytometry, and RT-PCR in four experimental groups of EBs (EBs cultured in gelatin-coated cell culture plates); Scaffold/EB (EBs cultured on EG); ESCs/Ser (EBs and Sertoli cells co-cultured on gelatin-coated cell culture plates without EG); and Scaffold/EB/Ser (EBs and Sertoli cells co-cultured on EG). All experimental groups exhibited a significantly increased MVH (germline-specific marker) and decreased c-KIT (stemness marker) expression when compared with the EB group. ICC and flow cytometry revealed that Scaffold/EB/Ser had the highest level of MVH and the lowest c-KIT expression at both 3 and 7 days postdifferentiation compared with other groups. RT-PCR results showed a significant increase in the germline marker (Dazl) and a significant decrease in the ESC stemness marker (Nanog) in Scaffold/EB compared to the EB group. The germline markers Gcna, Stella, Mvh, Stra8, Piwil2, and Dazl were significantly increased in Scaffold/EB/Ser compared to the Scaffold/EB group. Our findings revealed that the EG scaffold can provide an excellent substrate biomimicking the micro/nanostructure of native seminiferous tubules and a platform for Sertoli cell–EB communication required for growth and differentiation of ESCs into germline cells.

中文翻译：

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明胶电纺垫作为潜在的共培养系统，用于从胚胎干细胞体外生产精子细胞

与生精小管最大相似性的3D底物工程将有助于体外产生功能性精子细胞从干细胞。在这里，我们介绍了接种Sertoli细胞的3D电纺明胶（EG）底物，并确定了其指导胚胎干细胞（ESC）向种系细胞分化的潜力。EG是通过静电纺丝法制备的，并通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑和细胞粘附证实了其在SEM下的形态以及支持细胞和ESC的细胞生物相容性。分析。由ESC形成胚状体（EB），并与Sertoli细胞共培养，并连续3天和7天用BMP4诱导以诱导EB向生殖系细胞的分化。通过免疫细胞化学（ICC），流式细胞仪和RT-PCR在四个实验组的EB（在明胶包被的细胞培养板上培养的EB）中研究了这种分化。支架/ EB（在EG上培养的EB）；ESCs / Ser（EB和Sertoli细胞在不含EG的明胶包被的细胞培养板上共培养）；和Scaffold / EB / Ser（EB和Sertoli细胞在EG上共培养）。与EB组相比，所有实验组均表现出显着增加的MVH（生殖细胞特异性标记）表达和降低的c-KIT（干性标记）表达。ICC和流式细胞仪显示，与其他组相比，Scaffold / EB / Ser在分化后3天和7天均具有最高的MVH水平和最低的c-KIT表达。RT-PCR结果显示种系标记显着增加（与EB组相比，所有实验组均表现出显着增加的MVH（生殖细胞特异性标记）表达和降低的c-KIT（干性标记）表达。ICC和流式细胞仪显示，与其他组相比，Scaffold / EB / Ser在分化后3天和7天均具有最高的MVH水平和最低的c-KIT表达。RT-PCR结果显示种系标记显着增加（与EB组相比，所有实验组均表现出显着增加的MVH（生殖细胞特异性标记）表达和降低的c-KIT（干性标记）表达。ICC和流式细胞仪显示，与其他组相比，Scaffold / EB / Ser在分化后3天和7天均具有最高的MVH水平和最低的c-KIT表达。RT-PCR结果显示种系标记显着增加（DAZL）和ESC干性标记（一个显著下降的Nanog）在支架/ EB比EB组。生殖系标记Gcna，斯特拉，MVH，STRA8，PIWIL2和DAZL进行了比较，脚手架/ EB组支架/ EB /丝氨酸显著上升。我们的发现表明，EG支架可以提供模仿天然生精小管的微/纳米结构的出色底物，并为ESCs生长和分化为种系细胞所需的Sertoli细胞-EB通讯平台。