目的:使用新型三维(3D)培养系统获得能够诱导完全植入前胚泡发育的从头雄性配子。
方法:将小鼠胚胎干细胞(mESCs)通过在海藻酸钠中加入氯化钙进行球化,描绘模拟生精管的3D环境。作为一种控制,使用在二维板上培养的mESC。将含有来自两种方法的mESC的平板和球体暴露于活化素A,bFGF,和KSR,然后暴露于BMP4,LIF,SCF,和EGF促进分化为雄性生殖样细胞。
方法:评估细胞的VASA,DAZL,和BOULE在第3天和第10天。随后将细胞注射到活化的卵母细胞中,并在第15、22、29和36天使用延时成像进行监测。还通过延时成像监测了使用成熟附睾精子产生的对照概念。
结果:在第3天,在平板上分化的细胞表达1%的VASA和29%的DAZL。在球体中,VASA的表达率为15%,DAZL的表达率为45%(P<.001)。在第10天,在平板上分化的细胞具有7%的VASA表达,DAZL的23%,和BOULE只有0.5%。分化为球体的细胞以20%的比率表达VASA,DAZL为43%,和BOULE为10%(P<.001)。在第3天球中的随后分化表现出43%的DAZL(在精原细胞中表达)表达和15%的VASA(进一步的精子发生进程)表达。在第10天,重新评估DAZL和VASA表达,并增加到45%和18%,分别。BOULE,一种仅在减数分裂后精母细胞中表达的标记,以8%表示,而顶体酶在精子细胞中以2%表达。在第15天,VASA表达稳定在17%,BOULE的峰值为10%,和顶体酶达到5%。在第22天,VASA的表达增加到19%,BOULE下降到8%,顶体蛋白达到7%的峰值。在第29天,VASA表达达到20%的峰值,BOULE下降到2%,顶体酶稳定在7%。在第36天,VASA表达保持在13%,而BOULE和顶体蛋白表达下降到0%和1%,分别。对照组获得88.4%的受精率和76.9%的囊胚率。从头配子的受精率达到35.0%,61.1%,81.8%,在第15、22、29和36天分别为50.0%。新生胚细胞产生的囊胚率为5.0%,16.7%,36.4%,第15、22、29和36天分别为8.3%。
结论:我们的新型3D分化模型可以产生功能性配子,旨在消除同种异体/异种移植的需要。总体标志物表达降低和胚泡发育减少表明,在大约30天,球内生殖细胞分化与小鼠精子发生的长度相关。将进行未来的实验以确认我们发现的可重复性和后代的最终生成。
To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system.
Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells.
Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging.
On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas
acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and
acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and
acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and
acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and
acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively.
Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.