Pendergraft Samuel S, Sadri-Ardekani Hooman, Atala Anthony, Bishop Colin E
Wake Forest Institute for Regenerative Medicine, Winston-Salem, North Carolina, USA.
Department of Urology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
Biol Reprod. 2017 Mar 1;96(3):720-732. doi: 10.1095/biolreprod.116.143446.
Existing methods for evaluating the potential gonadotoxicity of environmental agents and pharmaceutical compounds rely heavily on animal studies. The current gold standard in vivo functional assays in animals are limited in their human predictive capacity. In addition, existing human two-dimensional in vitro models of testicular toxicity do not accurately reflect the in vivo situation. A more reliable testicular in vitro model system is needed to better assess the gonadotoxic potential of drugs prior to progression into clinical trials. The overall goal of this study was to develop a three-dimensional (3D) in vitro human testis organoid culture system for use as both a predictive first tier drug-screening tool and as a model of human testicular function. Multicellular human testicular organoids composed of Spermatogonial Stem Cells, Sertoli, Leydig and peritubular cells were created and evaluated over time for morphology, viability, androgen production and ability to support germ cell differentiation. Enzyme-linked immunosorbent assay measurements confirmed that the organoids produced testosterone continuously with and without hCG stimulation. Upregulation of postmeiotic genes including PRM1 and Acrosin, detected by quantitative-PCR, digital PCR and Immunofluorescence, indicated the transition of a small percentage of diploid to haploid germ cells. As a novel screening tool for reproductive toxicity, 3D organoids were exposed to four chemotherapeutic drugs, and they responded in a dose-dependent manner and maintained IC50 values significantly higher than 2D cultures. This 3D human testis organoid system has the potential to be used as a novel testicular toxicity-screening tool and in vitro model for human spermatogenesis.
评估环境因子和药物化合物潜在性腺毒性的现有方法在很大程度上依赖于动物研究。当前动物体内功能测定的金标准在预测人类情况方面存在局限性。此外，现有的人类睾丸毒性二维体外模型不能准确反映体内情况。需要一个更可靠的睾丸体外模型系统，以便在进入临床试验之前更好地评估药物的性腺毒性潜力。本研究的总体目标是开发一种三维（3D）体外人类睾丸类器官培养系统，用作预测性的一级药物筛选工具和人类睾丸功能模型。创建了由精原干细胞、支持细胞、间质细胞和睾丸周细胞组成的多细胞人类睾丸类器官，并随时间评估其形态、活力、雄激素产生以及支持生殖细胞分化的能力。酶联免疫吸附测定结果证实，无论有无hCG刺激，类器官都能持续产生睾酮。通过定量PCR、数字PCR和免疫荧光检测到减数分裂后基因（包括PRM1和顶体蛋白酶）的上调，表明一小部分二倍体生殖细胞向单倍体生殖细胞转变。作为一种新型的生殖毒性筛选工具，3D类器官暴露于四种化疗药物中，它们呈剂量依赖性反应，且维持的IC50值显著高于二维培养物。这种3D人类睾丸类器官系统有潜力用作新型睾丸毒性筛选工具和人类精子发生的体外模型。
