Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany.
Institute of Medical Statistics and Computational Biology, Faculty of Medicine, University of Cologne, 50923 Cologne, Germany.
Int J Mol Sci. 2024 Apr 26;25(9):4731. doi: 10.3390/ijms25094731.
The periodontal ligament (PDL) is a highly specialized fibrous tissue comprising heterogeneous cell populations of an intricate nature. These complexities, along with challenges due to cell culture, impede a comprehensive understanding of periodontal pathophysiology. This study aims to address this gap, employing single-cell RNA sequencing (scRNA-seq) technology to analyze the genetic intricacies of PDL both in vivo and in vitro. Primary human PDL samples ( = 7) were split for direct in vivo analysis and cell culture under serum-containing and serum-free conditions. Cell hashing and sorting, scRNA-seq library preparation using the 10x Genomics protocol, and Illumina sequencing were conducted. Primary analysis was performed using Cellranger, with downstream analysis via the R packages Seurat and SCORPIUS. Seven distinct PDL cell clusters were identified comprising different cellular subsets, each characterized by unique genetic profiles, with some showing donor-specific patterns in representation and distribution. Formation of these cellular clusters was influenced by culture conditions, particularly serum presence. Furthermore, certain cell populations were found to be inherent to the PDL tissue, while others exhibited variability across donors. This study elucidates specific genes and cell clusters within the PDL, revealing both inherent and context-driven subpopulations. The impact of culture conditions-notably the presence of serum-on cell cluster formation highlights the critical need for refining culture protocols, as comprehending these influences can drive the creation of superior culture systems vital for advancing research in PDL biology and regenerative therapies. These discoveries not only deepen our comprehension of PDL biology but also open avenues for future investigations into uncovering underlying mechanisms.
牙周膜 (PDL) 是一种高度特化的纤维组织，包含具有复杂性质的异质细胞群体。这些复杂性以及由于细胞培养而带来的挑战，阻碍了对牙周病理生理学的全面理解。本研究旨在通过单细胞 RNA 测序 (scRNA-seq) 技术来分析体内和体外牙周膜的遗传复杂性，从而解决这一差距。对 7 个原发性人牙周膜样本进行了拆分，分别用于直接体内分析和在含有血清和无血清条件下的细胞培养。进行了细胞哈希和分选、使用 10x Genomics 方案制备 scRNA-seq 文库以及 Illumina 测序。使用 Cellranger 进行初步分析，然后通过 R 包 Seurat 和 SCORPIUS 进行下游分析。鉴定出 7 个不同的 PDL 细胞簇，包含不同的细胞亚群，每个亚群都具有独特的遗传特征，其中一些在表达和分布上表现出供体特异性模式。这些细胞簇的形成受到培养条件的影响，特别是血清的存在。此外，某些细胞群体被发现是牙周膜组织固有的，而其他群体则表现出供体间的变异性。本研究阐明了牙周膜中的特定基因和细胞簇，揭示了固有和受环境驱动的亚群。培养条件的影响——特别是血清的存在——对细胞簇形成的影响突出表明需要改进培养方案，因为理解这些影响可以推动创建更优越的培养系统，这对于推进牙周膜生物学和再生治疗的研究至关重要。这些发现不仅加深了我们对牙周膜生物学的理解，也为未来揭示潜在机制的研究开辟了道路。
