Plap-1/Aspn lineage tracing and single-cell transcriptomics reveals cellular dynamics in the periodontal ligament

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated Protein-1 (Plap-1/Aspn) as a PDL-specific extracellular matrix. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiated into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1-Ibsp+Sparcl1+ and Plap-1-Ibsp+Col11a2+, respectively. Other populations such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells were also identified. RNA velocity analysis suggested that Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during the periodontal injury model showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.


Introduction 1
Mammalian teeth are supported by periodontal tissue, which consists of the periodontal 2 ligament (PDL), gingiva, alveolar bone, and cementum. As periodontal tissue is highly 3 adaptive to normal and excess occlusal and orthodontic forces, it undergoes extensive 4 remodeling (Beertsen et al. 1997). Periodontal tissue also possesses some regenerative 5 capacity, as demonstrated in human and animal models (Sallum et   The PDL expresses a large number of extracellular matrix (ECM) components, 33 including collagen (Naveh et al. 2018) and proteoglycans (Chen et al. 2021). Among these, 34 several genes are candidate PDL-specific genes (Takimoto et al. 2015;Horiuchi et al. 1999). 35 We previously identified periodontal ligament associated Protein-1 (Plap-1/Aspn) from a 36 addition, we investigated whether a stromal cell marker CD51, also known as integrin αV, 23 can label stromal cells including Plap-1 + cells. CD51 + cells included most GFP + cells, with an 24 average of 30% single-positive cells. GFP + cells consistently accounted for approximately 25 30% of the population. We also tested the workflow using a Col1a1-GFP mouse line that 26 expresses GFP in both fibroblastic and cemento-/osteoblastic cells in periodontal tissue (Fig.  27   S3F). As expected, more GFP + cells were observed in the Col1a1-GFP PDL than in the 28 Plap-1-GFP PDL (Fig. S3G). A comparison of gingiva with the PDL revealed that 29 Col1a1-GFP expression levels were higher in the PDL than in gingiva, consistent with a 30 previous report that collagen turnover is more active in the PDL than in gingiva (Sodek 31 1977). Additionally, we examined the culture conditions suitable for PDLCs and developed a 32 method that enabled stable PDL cell expansion in vitro under hypoxic conditions (Fig. 3E). 33 GFP + cells exhibited significantly higher CFU-F formation compared to all cells from the 34 PDL-enriched population (Fig. 3F) and demonstrated osteogenic differentiation capacity in 7 vitro (Fig. 3G), confirming that PDLCs contained stem/progenitor cells. However, cellular 1 composition and heterogeneity of PDL were not determined from the analysis. 2 3 Generation of PDL atlas using single-cell transcriptome analysis 4 After establishing an efficient PDLC isolation technique, we obtained cell suspensions from 5 20 adult wild-type mice. All viable cells in the PDL were collected by cell sorting and 6 subjected to single-cell transcriptome analysis to generate a "PDL single-cell atlas" (Fig. 4A,  7 4B, and S4A). After the removal of low-quality cells, 17 clusters were identified from the PDL 8 scRNA-seq data (7,318 cells) (Fig. 4C). The cell types in each cluster were annotated using 9 known cell marker genes (Fig. 4D). The most abundant populations were stromal cells 10 expressing collagens, matrix metalloproteinases, and platelet-derived growth factor 11 receptors ( Fig. 4D and S4B). Consistent with the histological analysis ( Fig. 1F), 12 collagen-expressing stromal cells were mutually exclusively defined by the expression of 13 Plap-1/Aspn or Ibsp (Fig. 4E). The stromal cell cluster was subjected to the in-depth 14 analysis (Fig. 5). Non-stromal cell clusters, including immune, epithelial, and vascular 15 endothelial cells, were also identified ( Fig. 4D, S4C, and S4E). Flow cytometry analysis of 16 isolated immune cells from PDL showed the presence of macrophages, proerythroblasts, B 17 cells, and T cells in PDL and no overlap with Plap-1-GFP + cells (Fig. S4D). Two epithelial 18 cell clusters were distinguished by differential expression of Keratin 15 (Krt15) and Keratin 19 17 (Krt17). FISH analysis confirmed that Krt17-positive epithelial cells were derived from the 20 inner and outer epithelia, while Krt15-positive epithelial cells were derived from the 21 basement membrane (Fig. S4F). Notably, a cluster of Plap-1 + cells exhibited distinct gene To identify the heterogeneity in the PDL that maintains tissue homeostasis, 3,675 stromal 35 cells expressing collagens, matrix metalloproteinases, and platelet-derived growth factor 36 and flow cytometry analysis showed they were 5.4 % of the PDL live singlets (Fig. S5D). 23 Lepr-, Gli1-, Axin2-, and Acta2-positive cells have been implicated in stem/progenitor cells in 24 the PDL. However, in this dataset, they were not associated with a specific cluster, with the 25 exception of Lepr + cells that were enriched in cluster 5 (Fig. S5E). Crucially, none of these 26 cells were observed at the top of the lineage hierarchy (Fig. 5D). Among Ibsp + clusters, 27 Col11a2 expression was specific to cluster 2 (Fig. 5G), and this gene was exclusively 28 expressed in osteoblasts but not in cementoblasts (Fig. 5H). Compared to the osteoblast 29 cluster, the other Ibsp + cluster (cluster 6) expressed higher levels of Sparcl1 (Fig. 5I). 30 Sparcl1 expression was observed in cementoblasts and cementocytes, but not in 31 osteoblasts ( Fig. 5J and S5F). However, the cluster did not specifically express recently 32 reported markers (Nagata et al. 2021), including Parathyroid hormone-related protein 33 (Pthlh), Class III β-tubulin (Tubb3), and Wnt inhibitory factor 1 (Wif1) (Fig. S5G). To assess 34 the putative differentiation pathways, we also performed a pseudotime analysis to clusters 0, 35 2, 4, and 6. Gene expression kinetics of Plap-1, Ibsp, Col3a1, Col1a1, Sparcl1, Col11a2, 1 Lrrc15, and Ly6a showed unique transient changes during differentiation ( Figure S6A-F). 2 3 Plap-1 + PDLCs directly contribute to periodontal tissue repair 4 To investigate the role of PDLCs in the repair of injured periodontal tissue, we utilized a 5 ligature-induced periodontitis model (Fig. 6A). In this widely used model (Abe and 6 Hajishengallis 2013), a suture is placed around the second molar of the maxilla for 7 days to 7 induce periodontal tissue loss. Upon ligature removal, the periodontal tissue can be repaired 8 to the baseline level 7-14 days after removal. On day 1, after ligature removal, a layer of 9 cementum on the surface remained intact, although alveolar bone resorption was observed 10

Mural cell population in the PDL is quiescent cells 29
Long-term lineage tracing analysis with Nes-Cre revealed that Nes + mural cells were 30 quiescent and remained perivascular for up to a year without expansion during periodontal 31 tissue homeostasis (Fig. S7A). Based on this observation, we performed a ligature 32 experiment with Nes-Cre lineage tracing to determine if Plap-1 + Nes + cells contribute to 33 periodontal tissue repair (Fig. S7B). On day 7, after ligature removal, we did not detect the 34 Nes lineage cells in repaired PDL, alveolar bone, and cementum (Fig. S7C). These results 35 suggest that Nes + mural cells in the PDL did not contribute to the periodontal tissue repair in 1 a cell-autonomous manner. Periodontitis is a chronic inflammation of periodontal tissues caused by bacterial biofilms, 5 which eventually cause irreversible destruction of periodontal tissues. Periodontitis has a 6 high prevalence worldwide and is the most common cause of tooth loss in adults in 7 developed countries. In addition to attaching teeth to the alveolar bone, the PDL interferes 8 with and senses the occlusal force and transmits it to the central nervous system (Beertsen 9 et al. 1997). Dental implants, however, lack these functions because they form a direct heterogeneous cell types. Notably, the stromal compartment was collectively divided into 23

Plap-1 + fibroblastic cells or Ibsp + osteo-/cementoblastic cells in a mutually exclusive manner. 24
Plap-1 is also highly expressed in the human PDL and used as a cultured PDLC marker 25 In conclusion, our work identified Plap-1 as a PDLC-specific molecule, and cell 28 lineage analysis using knock-in mice confirmed that PDLCs differentiated into osteoblasts 29 and cementoblasts and played a role in periodontal tissue homeostasis. In addition, we 30 developed a novel cell isolation method and performed scRNA-seq to generate a PDL 31 single-cell atlas that clearly defined osteoblasts and cementoblasts. Furthermore, lineage 32 tracing revealed that injured periodontal tissues were repaired by PDLCs. These findings 33 will contribute to the development of efficient regenerative therapy for periodontitis.

Declaration of interests 17
The authors declare the following financial interests/personal relationships that may be 18 Rosa26-tdTomato mice were generated, and tamoxifen was administered. In the system, a 18 fusion protein of a mutated estrogen receptor and Cre recombinase is expressed in Plap-1 + 19 cells, and the Plap-1 + cells are labeled with tdTomato once tamoxifen is administrated. The 20 mark is heritable and permanent and thus transmitted to all the descendants of Plap-1 + cells. The yellow arrowheads indicate PDL tissue in the left panel. In contrast, PDL tissue is 4 absent but intact pulpal tissue is evident in the right panel. The difference was statistically significant (p < 0.001, two tailed unpaired t-test, n= 3-6 mice,

Lead contact 30
Further information and requests for resources and reagents should be directed and will be 31 fulfilled by the lead contact, Tomoaki Iwayama (iwayama.tomoaki.dent@osaka-u.ac.jp) 32 33

Materials availability 34
Unique materials generated in this study are available from the lead contact without 35 restriction. 36 1 Data and code availability 2 • Single-cell RNA sequencing data were accessed from GEO: GSE197828. 3 • All data needed to evaluate the conclusions in the paper are presented in the paper 4 and/or Supplementary Information. 5 • Additional data related to this study can be requested from the authors. 6 7 Experimental model and subject details 8

Mice 9
All animal experiments in this study were approved by the Animal Experimentation

ES cell targeting and vector design 22
A targeting vector harboring the PGK-DTA-long homology 23 arm-GFP-2A-CreER-WPRE-pA-short homology arm sequence was generated. Correctly 24 targeted ES cell clones were identified by PCR and confirmed by Southern blotting using the 25 primers and probes listed in Supplementary Table S1. Chimeric males were generated using 26

standard aggregation of clumped ES cells with eight-cell embryos and transplantation 27
procedures and were used to establish germline transmission. 28 29

Isolation of PDL-derived cells 11
After euthanization with CO 2 gas, the mice were perfused with PBS, and the maxilla and analyses. In some experiments, PDL was scraped from the surface of the mesial root of the 28 first molar using micro-instruments, including a micro-curette (0.5 mm) (Fine Science Tools). 29 Scraped tissue was digested as described above.  Cb.