The molecular signature of human testicular peritubular cells revealed by single-cell analysis

Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. It contrast to rodents, it is composed of several cell layers and extracellular matrix. Peritubular cells are crucial for sperm transport and emerge as contributors to the spermatogonial stem cell niche, yet testicular peritubular cells are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs) isolated from testicular samples of donors with normal spermatogenesis and compared our data with recently published ex vivo data and, if available, with immunohistochemical data of the Human Protein Atlas. We observed a significant overlap between our results and genes expressed in vivo. Based on expression of a set of smooth muscle markers, HTPCs can be classified as smooth muscle cells. Small differences between in vivo/in vitro expressed genes may be due to plasticity/phenotypic switching. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities four cellular states were observed. Characteristics of mesenchymal stromal cells and Leydig stem cells were noted, yet differentiation attempts into fat cells, bone cells or mature Leydig cells were not successful. Further analyses confirmed known stem cell niche-relevant factors (e.g. GDNF) and identified unknown functions e.g. the ability to produce retinoic acid. The results reveal that HTPCs upon isolation and culture are a highly adequate cellular model as they retain major characteristics. They therefore allow us to define the signature and functions of human testicular peritubular cells. The data may serve as a resource for future studies to better understand male (in)fertility. Single-cell RNA-seq of in vitro cultured human testicular peritubular cells (HTPCs).

人类睾丸的管周细胞（peritubular cells）可形成环绕生精小管（seminiferous tubules）的微型腔室。与啮齿类动物不同，该腔室由多层细胞及细胞外基质共同构成。管周细胞在精子运输过程中发挥关键作用，且现已被证实为精原干细胞龛（spermatogonial stem cell niche）的组成成分；然而睾丸管周细胞仍是目前人体中研究程度最低的细胞类群之一。本研究针对从正常生精功能供体的睾丸样本中分离、并经体外培养的人类睾丸管周细胞（human testicular peritubular cells, HTPCs）开展单细胞RNA测序（single-cell RNA sequencing），并将测序所得数据与已发表的离体实验数据，以及可获取的人类蛋白质图谱（Human Protein Atlas）免疫组织化学数据进行比对分析。分析结果显示，本研究的数据与体内表达的基因存在显著重合。基于一组平滑肌标志物的表达特征，HTPCs可被归类为平滑肌细胞。体内与体外培养环境下基因表达的微小差异，可能源于细胞可塑性与表型转换（plasticity/phenotypic switching）。向培养基中添加胎牛血清（FCS）后，同样可观察到细胞可塑性现象。基于转录组相似性分析，本研究鉴定出四种细胞状态。研究发现HTPCs兼具间充质基质细胞（mesenchymal stromal cells）与睾丸间质干细胞（Leydig stem cells）的特征，但尝试将其定向分化为脂肪细胞、成骨细胞或成熟睾丸间质细胞均未获得成功。进一步分析证实了已知的干细胞龛相关因子（如胶质细胞源性神经营养因子GDNF），同时发现了此前未被报道的新功能，例如合成视黄酸（retinoic acid）的能力。研究结果表明，经分离与体外培养的HTPCs是一套高度适配的细胞模型，因其保留了人类睾丸管周细胞的核心生物学特征；借此可明确人类睾丸管周细胞的表达特征与功能。本研究数据可为未来深入探究男性生育与不育相关机制的研究提供宝贵的参考资源。本数据集为体外培养人类睾丸管周细胞（HTPCs）的单细胞RNA测序数据。