Transcriptome profile of highly osteoblastic/cementoblastic periodontal ligament cell clones

Abstract Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including “anatomical structure development” and “cell adhesion.” Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process “anatomical structure development,” Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies.


Introduction
Periodontitis is a polymicrobial, infection-induced inflammatory disease in the periodontium characterized by connective attachment loss and alveolar bone destruction. Epidemiological studies indicate that periodontitis is still a globally prevalent disease.
This periodontal disease may lead to functionally compromised dentition, which affects the quality of life of many subjects. 1 In the last decade, several attempts have been made to regenerate the tissues impaired due to periodontitis, including bone replacement grafts, guided tissue regeneration, enamel matrix derivative, and combined therapy. and by having stem cell-like properties, including the capacity for self-renewal and multipotency. [2][3][4][5][6] The periodontium is a complex structure composed of mineralized (cementum and alveolar bone) and non-mineralized tissues (PDL). Consequently, the regeneration of the periodontium requires a wellcoordinated process of cell differentiation. However, a detailed understanding of periodontal-derived cells, which is crucial for these emerging approaches, remains unclear. 2, [6][7] It is known that PDL is constituted by heterogeneous cell populations. 2,6,8 However, the molecular profile that distinguishes cells committed to osteo/cementoblastic (O/C) or fibroblastic phenotypes in PDL is still not fully understood. 2,6,9 To date, some studies have suggested that cathepsin K is involved in PDL tissue homeostasis through stimulation of collagen fiber accumulation and inhibition of osteoblast differentiation of human PDL cells. 7 Additionally, evidence suggests that the activation of the canonical Wnt signaling pathway enhances in vitro cementoblast differentiation of human PDL cells. 9 Emerging methods using high-throughput sequencing technologies (such as the massive parallelization of RNA-seq) have broadened our view of the extent and complexity of the PDL transcriptome. 10 For instance, RNA-seq analysis allows the detection and quantification of a broad range of transcripts and their splice-forms without requiring target specification, which leads to an unbiased and systematic approach to produce insights into important biological pathways and molecular mechanisms for cell regulation in a hypothesis-neutral environment. 10-12 In our study, CD105-enriched PDL cell clones with osteoblastic/ cementoblastic or fibroblastic potential were purified and had their transcriptomes compared after highthroughput RNA sequencing. Our hypothesis is that a comprehensive analysis of periodontium cells may shed light on how to promote an optimal microenvironment for periodontal mineralized and non-mineralized tissue formation. Finally, we expect that our results help in the development of more predictable outcomes for future regenerative approaches.

Cell Metabolic Activity Assay
For the metabolic analysis, cell clones were seeded (5 × 10 3 cells/well) in a 96-well plate (Corning Costar, USA) using standard medium and incubated in a humidified incubator at 37°C and 5% CO 2 for 24 h to allow cell adhesion to the discs. Thenr, the medium was changed for DMEM supplemented with 2% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ mL). This time point was considered as the baseline (time 0h) for the metabolic assay. The media was then replaced on days 3 and 7, and the metabolic activity of the cell on the experimental groups was evaluated at days 1, 3, 7, and 10, as previously described 15 using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT, Life Technologies, USA) assay.
RNA isolation, RNA-seq, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) Each cell clone was seeded, and RNA extraction was performed as previously described. 8    For other experiments, data were expressed as mean ± standard deviation (SD). T-test was used to analyze differences between two groups, and one-way or two-way analysis of variance (ANOVA) (α=0.05) was used to analyze differences among three or factorial analyses, respectively.

Results
Since we aimed to isolate clones with distinct O/C differentiation potentials from a PDL-CD105 + -enriched population, the biomineralization potential of this population was evaluated and compared to the total pool of cells obtained from PDL from third molars.
The PDL-CD105 + -enriched population exhibited a high proportion of cells that expressed mesenchymal stem cell (MSC)-related markers ( Figure 1A). However, the capacity for biomineralization of the PDL-CD105 +enriched population was not statistically higher than the unsorted population (PDL) (Figures 1B and 1C).

PDL clones present distinct ability to form a mineralized matrix in vitro
We obtained a total of 46 cell clones from the PDL-CD105 + mesenchymal progenitor population. According to the AR-S assay, two of the 46 clones showed a significantly higher potential to form a mineralized matrix in vitro under OM induction, namely G13 and G48, and were defined as clones of O/C phenotype (C-O) (Figures 2A and 2B). The remaining 44 clones showed a lower ability to form a mineralized matrix in vitro and were classified as clones of the fibroblastic phenotype (C-F). In the C-F group, two clones that rapidly expanded during clonal expansion were selected to represent the C-F group, namely clones G16 and G23 ( Figure 2A). The C-F and C-O groups were evaluated for their metabolic activity and showed no significant difference at any time point ( Figure 2C). Both clone groups showed increased metabolic activity on day 3 ( Figure 2C). that ~84% of the reads were at optimum quality and aligned to the human genome. Only aligned reads were retained for further analyses. As the heatmap shows, C-O clones presented a higher transcriptional activity   Figure   4B). Moreover, the biological process "cell adhesion" (GO:0007155) included the gene PCDHGA10, which is related to Cadherin pathway, and BMP4, which is linked to TGFβ/BMP pathway ( Figure 4C).

WNT2, WNT16, and WIF1 were validated to be upregulated by RT-qPCR
The significantly upregulated genes WNT2, WNT2B, WNT16, WIF1, PCDHGA10, and BMP4, found after RNA-seq analyses, were then selected for RT-qPCR validation. The data showed that C-O clones presented significantly higher expression of WNT2, WNT16, and WIF1 (Figures 5A-C). Although a trend of higher expression in C-O clones was observed, the genes WNT2B, PCDHGA10, and BMP4 presented no significant difference of expression between C-O and C-F clones ( Figures 5D-F).    The Cadherin superfamily was significantly overrepresented in clones with the potential to and WIF1 (C) was significantly higher in C-O clones than in C-F clones. The expression of WNT2B (D), PCDHGA10 (E) and BMP4 (F) was not statistically significantly up-regulated in C-O clones. Bars represent mean ± standard deviation (SD) of three independent experiments. *Statistical significance intergroup was determined using t-test (p<0.05) J Appl Oral Sci. 2020;28:e20200242 10/11 differentiate into the O/C phenotype. Studies have reported that the cross-talk between Cadherin and Wnt signaling regulates the mechanism underlining osteoblast differentiation. 37,38 Cadherins are suggested to bind to β-catenin, hindering its translocation to the nucleus, 38 thus reducing canonical Wnt signaling.
Cadherins also interact with Wnt co-receptor lipoprotein receptor-related protein 5 (LRP5), 37 which is essential to regulate bone mass. 39 In consistence with these findings, our study showed that C-O clone cells presented nine upregulated genes common between Cadherin and Wnt pathways, suggesting an interaction of these two pathways in the regulation of O/C cell lineage commitment localized into the periodontal dental ligament.
In short, we provided a comprehensive assessment of the transcriptome of human PDL progenitor cell clones with high O/C differentiation potential using a next-generation sequencing technology (RNA-seq).

Conflit of Interest
The authors declare no potential conflicts of interest to the authorship and/or publication of this article