{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:52:39Z","timestamp":1782283959135,"version":"3.54.5"},"reference-count":38,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2024,4,26]],"date-time":"2024-04-26T00:00:00Z","timestamp":1714089600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 (n = 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\u2014notably the presence of serum\u2014on 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.<\/jats:p>","DOI":"10.3390\/ijms25094731","type":"journal-article","created":{"date-parts":[[2024,4,26]],"date-time":"2024-04-26T08:18:27Z","timestamp":1714119507000},"page":"4731","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Revealing Genetic Dynamics: scRNA-seq Unravels Modifications in Human PDL Cells across In Vivo and In Vitro Environments"],"prefix":"10.3390","volume":"25","author":[{"given":"Ali T.","family":"Abdallah","sequence":"first","affiliation":[{"name":"Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany"},{"name":"Institute of Medical Statistics and Computational Biology, Faculty of Medicine, University of Cologne, 50923 Cologne, Germany"},{"name":"Interdisciplinary Center for Clinical Research, University Hospital RWTH, 52074 Aachen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Peitz","sequence":"additional","affiliation":[{"name":"Institute of Reconstructive Neurobiology, Life and Brain Center, University Hospital Bonn, 53105 Bonn, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0266-6095","authenticated-orcid":false,"given":"Anna","family":"Konermann","sequence":"additional","affiliation":[{"name":"Department of Orthodontics, University Hospital Bonn, 53111 Bonn, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1111\/j.1600-0757.2005.00135.x","article-title":"Proteomics for the periodontium: Current strategies and future promise","volume":"40","author":"McCulloch","year":"2006","journal-title":"Periodontology 2000"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1002\/(SICI)1097-0185(199606)245:2<327::AID-AR15>3.0.CO;2-R","article-title":"Periodontal ligament cell population: The central role of fibroblasts in creating a unique tissue","volume":"245","author":"Lekic","year":"1996","journal-title":"Anat. Rec."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1034\/j.1600-0757.2000.2240104.x","article-title":"Role of physical forces in regulating the form and function of the periodontal ligament","volume":"24","author":"McCulloch","year":"2000","journal-title":"Periodontology 2000"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1111\/j.1600-0757.1997.tb00094.x","article-title":"The periodontal ligament: A unique, multifunctional connective tissue","volume":"13","author":"Beertsen","year":"1997","journal-title":"Periodontology 2000"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1177\/154405910208100609","article-title":"Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis","volume":"81","author":"Han","year":"2002","journal-title":"J. Dent. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1902\/jop.2001.72.3.324","article-title":"Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model","volume":"72","author":"Oates","year":"2001","journal-title":"J. Periodontol."},{"key":"ref_7","first-page":"24","article-title":"Oral stem cells, decoding and mapping the resident cells populations","volume":"3","author":"Zhang","year":"2022","journal-title":"Biomater. Transl."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1007\/s13577-017-0161-2","article-title":"Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: Implications for regenerative medicine","volume":"30","author":"Murabayashi","year":"2017","journal-title":"Hum. Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"859025","DOI":"10.3389\/fvets.2022.859025","article-title":"A Review of Fetal Bovine Serum in the Culture of Mesenchymal Stromal Cells and Potential Alternatives for Veterinary Medicine","volume":"9","author":"Pilgrim","year":"2022","journal-title":"Front. Vet. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1016\/j.archoralbio.2011.03.003","article-title":"Implications of cultured periodontal ligament cells for the clinical and experimental setting: A review","volume":"56","author":"Marchesan","year":"2011","journal-title":"Arch. Oral Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/nmeth.1315","article-title":"mRNA-Seq whole-transcriptome analysis of a single cell","volume":"6","author":"Tang","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"a026898","DOI":"10.1101\/cshperspect.a026898","article-title":"Single-Cell Applications of Next-Generation Sequencing","volume":"9","author":"Anaparthy","year":"2019","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1038\/nature21350","article-title":"Scaling single-cell genomics from phenomenology to mechanism","volume":"541","author":"Tanay","year":"2017","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1093\/bfgp\/elx035","article-title":"Experimental design for single-cell RNA sequencing","volume":"17","author":"Chandra","year":"2018","journal-title":"Brief. Funct. Genom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1038\/s41419-019-2049-4","article-title":"SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP\/TGF-\u03b2 cell signaling pathway","volume":"10","author":"Wang","year":"2019","journal-title":"Cell Death Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"650","DOI":"10.1038\/s41467-018-08247-x","article-title":"Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds","volume":"10","author":"Dedhia","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5086","DOI":"10.1002\/jcp.27312","article-title":"YAP regulates periodontal ligament cell differentiation into myofibroblast interacted with RhoA\/ROCK pathway","volume":"234","author":"He","year":"2019","journal-title":"J. Cell. Physiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"912","DOI":"10.2319\/101609-578.1","article-title":"Orthodontic mechanical tension effects on the myofibroblast expression of alpha-smooth muscle actin","volume":"80","author":"Meng","year":"2010","journal-title":"Angle Orthod."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1324","DOI":"10.1902\/jop.2010.100054","article-title":"Periodontal ligament fibroblasts sustain destructive immune modulators of chronic periodontitis","volume":"81","author":"Messer","year":"2010","journal-title":"J. Periodontol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Inomata, M., Amano, S., Abe, M., Hayashi, T., and Sakagami, H. (2022). Innate immune response of human periodontal ligament fibroblasts via the Dectin-1\/Syk pathway. J. Med. Microbiol., 71.","DOI":"10.1099\/jmm.0.001627"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e81370","DOI":"10.7554\/eLife.81370","article-title":"Transcriptional drifts associated with environmental changes in endothelial cells","volume":"12","author":"Afshar","year":"2023","journal-title":"Elife"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.1091\/mbc.e04-10-0949","article-title":"Isolation and transcription profiling of purified uncultured human stromal stem cells: Alteration of gene expression after in vitro cell culture","volume":"16","author":"Boquest","year":"2005","journal-title":"Mol. Biol. Cell"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1007\/s10616-013-9633-8","article-title":"A plea to reduce or replace fetal bovine serum in cell culture media","volume":"65","author":"Gstraunthaler","year":"2013","journal-title":"Cytotechnology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1634\/stemcells.2005-0094","article-title":"In vitro expansion of human mesenchymal stem cells: Choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability","volume":"23","author":"Shahdadfar","year":"2005","journal-title":"Stem Cells"},{"key":"ref_25","first-page":"32","article-title":"Cyclic stretch enhances reorientation and differentiation of 3-D culture model of human airway smooth muscle","volume":"16","author":"Asano","year":"2018","journal-title":"Biochem. Biophys. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"9656","DOI":"10.1096\/fj.201900479R","article-title":"Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function","volume":"33","author":"Ortega","year":"2019","journal-title":"FASEB J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1007\/s10787-020-00690-x","article-title":"Selenium and selenoproteins: It\u2019s role in regulation of inflammation","volume":"28","author":"Hariharan","year":"2020","journal-title":"Inflammopharmacology"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.jdermsci.2012.06.003","article-title":"Role of superoxide dismutase 3 in skin inflammation","volume":"67","author":"Kwon","year":"2012","journal-title":"J. Dermatol. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1515\/biol-2022-0045","article-title":"SOX4 promotes high-glucose-induced inflammation and angiogenesis of retinal endothelial cells by activating NF-\u03baB signaling pathway","volume":"17","author":"Wei","year":"2022","journal-title":"Open Life Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"109213","DOI":"10.1016\/j.exer.2022.109213","article-title":"Roles of CCL2 and CCL3 in intraocular inflammation during Bacillus endophthalmitis","volume":"224","author":"Mursalin","year":"2022","journal-title":"Exp. Eye Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1007\/s00011-016-0995-1","article-title":"The biological function and significance of CD74 in immune diseases","volume":"66","author":"Su","year":"2017","journal-title":"Inflamm. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1307","DOI":"10.1111\/1346-8138.13397","article-title":"Variation at HLA-DPB1 is associated with dermatomyositis in Chinese population","volume":"43","author":"Zhang","year":"2016","journal-title":"J. Dermatol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.12688\/f1000research.15809.1","article-title":"Comparison of clustering tools in R for medium-sized 10x Genomics single-cell RNA-sequencing data","volume":"7","author":"Freytag","year":"2018","journal-title":"F1000Research"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"151517","DOI":"10.1016\/j.aanat.2020.151517","article-title":"A novel serum-free medium for the isolation, expansion and maintenance of stemness and tissue-specific markers of primary human periodontal ligament cells","volume":"231","author":"Heim","year":"2020","journal-title":"Ann. Anat."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1038\/nbt.4096","article-title":"Integrating single-cell transcriptomic data across different conditions, technologies, and species","volume":"36","author":"Butler","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Stoeckius, M., Zheng, S., Houck-Loomis, B., Hao, S., Yeung, B.Z., Mauck, W.M., Smibert, P., and Satija, R. (2018). Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol., 19.","DOI":"10.1186\/s13059-018-1603-1"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Ibrahim, M.M., and Kramann, R. (2019). genesorteR: Feature Ranking in Clustered Single Cell Data. bioRxiv.","DOI":"10.1101\/676379"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/j.cels.2015.12.004","article-title":"The Molecular Signatures Database (MSigDB) hallmark gene set collection","volume":"1","author":"Liberzon","year":"2015","journal-title":"Cell Syst."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/9\/4731\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:33:58Z","timestamp":1760106838000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/9\/4731"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,26]]},"references-count":38,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2024,5]]}},"alternative-id":["ijms25094731"],"URL":"https:\/\/doi.org\/10.3390\/ijms25094731","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,26]]}}}