Cohen Adam B, Nikmehr Banafsheh, Abdelaal Omar A, Escott Megan, Walker Stephen J, Atala Anthony, Sadri-Ardekani Hooman
Wake Forest Institute of Regenerative Medicine, Winston-Salem, NC 27101, USA.
Department of Urology, Atrium Health Wake Forest Baptist, Winston-Salem, NC 27157, USA.
Biomedicines. 2024 Aug 6;12(8):1774. doi: 10.3390/biomedicines12081774.
Spermatogenesis produces male gametes from spermatogonial stem cells (SSC), beginning at puberty. Modern-day laboratory techniques allow for the long-term culture of SSC and in vitro spermatogenesis. The specific biochemical processes that occur during spermatogenesis remain poorly understood. One particular element of spermatogenesis that has yet to be characterized is the role of microRNAs (miRNA), short, non-transcribed RNAs that act as post-translational regulators of gene activity. In this study, we seek to describe the presence of miRNA in a two-dimensional (2D) SSC culture and a 3D human testis organoid (HTO) system. Testicular cells were isolated from the frozen tissue of three brain-dead subjects, propagated in cultures for four to five weeks, and used to form 3D HTOs. Following organoid formation, differentiation of testicular cells was induced. RNA was isolated from the whole testis tissue (WT) showing in vivo conditions, HTO Day Zero (2D SSC culture), Day 2 HTOs, and Day 23 differentiated HTOs, then analyzed for changes in miRNA expression using the Nanostring nCounter miRNA panel. One hundred ninety-five miRNAs met the criteria for expression in WT, 186 in 2D culture, 190 in Day 2 HTOs, and 187 in differentiated HTOs. One hundred thirty-three miRNAs were common across all conditions, and 41, 17, 6, and 11 miRNAs were unique for WT, 2D culture, Day 2 HTOs, and differentiated HTOs, respectively. Twenty-two miRNAs were similar between WT and differentiated HTOS. We evaluated the miRNA expression profiles of progressively complex stages of testicular cell culture, culminating in a 3D organoid model capable of meiotic differentiation, and compared these to WT. We identified a great variance between the native tissue and the culture system; however, some miRNAs are preserved. These data may provide avenues for deeper understanding of spermatogenesis and the ability to improve this process in the laboratory. Research on miRNA continues to be an essential avenue for understanding human spermatogenesis.
精子发生从青春期开始，由精原干细胞（SSC）产生雄性配子。现代实验室技术允许对SSC进行长期培养以及体外精子发生。精子发生过程中发生的具体生化过程仍知之甚少。精子发生中一个尚未被表征的特定元素是微小RNA（miRNA）的作用，miRNA是短的非转录RNA，作为基因活性的翻译后调节因子。在本研究中，我们试图描述miRNA在二维（2D）SSC培养物和三维人睾丸类器官（HTO）系统中的存在情况。从三名脑死亡受试者的冷冻组织中分离出睾丸细胞，在培养物中传代培养四到五周，并用于形成三维HTO。类器官形成后，诱导睾丸细胞分化。从显示体内条件的全睾丸组织（WT）、HTO第零天（2D SSC培养物）、第2天的HTO和第23天分化的HTO中分离RNA，然后使用Nanostring nCounter miRNA面板分析miRNA表达的变化。195种miRNA符合在WT中表达的标准，2D培养物中有186种，第2天的HTO中有190种，分化的HTO中有187种。133种miRNA在所有条件下都很常见，WT、2D培养物、第2天的HTO和分化的HTO分别有41、17、6和11种独特的miRNA。WT和分化的HTO之间有22种miRNA相似。我们评估了睾丸细胞培养逐渐复杂阶段的miRNA表达谱，最终形成了一个能够进行减数分裂分化的三维类器官模型，并将其与WT进行比较。我们发现天然组织和培养系统之间存在很大差异；然而，一些miRNA得以保留。这些数据可能为更深入了解精子发生以及在实验室中改善这一过程的能力提供途径。对miRNA的研究仍然是理解人类精子发生的重要途径。
