Zbp1-positive cells are osteogenic progenitors in periodontal ligament

Abstract

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Although Anatomically Micrometers Apart: Human Periodontal Ligament Cells Are Slightly More Active in Bone Remodeling Than Alveolar Bone Derived Cells

“…Another explanation could be that the outgrowth of this PDL contained mainly stem cells that were osteogenesis negative. Recently, Ueda et al (2021) described cell clones isolated from PDL that were highly osteogenic versus clones that were very lowly osteogenic.…”

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

“…To decipher the cell types in the PDL that contributes to the homeostasis and repair of periodontal tissue, an efficient method for isolation and analysis of PDL cells is required. We previously established a method for isolating PDL-derived cells by scraping the root surface with a PDL explant (Ueda et al 2021). However, this method can only be used to analyze migratory cells.…”

“…In this study, we used GFP expression in Plap-1-GFP-2A-CreER mice to evaluate the efficacy of PDL cell isolation. We adopted our previous protocol in which the gingiva was removed from the maxilla before tooth extraction to avoid any contamination from the gingival cells to extracted molars (Ueda et al 2021). We first digested the scraped PDL with enzymes.…”

“…Its transcript levels in the maxilla, including the PDL, have been reported to be more than 10-fold greater than levels in adipose tissue, brain, heart, lung, liver, stomach, intestine, kidney, spleen, bone marrow, and muscle (Sakashita et al 2021). Compared to dissected gingiva, dissected PDLs contain 60 times more transcripts (Ueda et al 2021). Nevertheless, the expression of Plap-1 in PDL at the single-cell resolution has yet to be investigated.…”

Periodontal tissue stem cells and mesenchymal stem cells in the periodontal ligament

“…1 ). In order to understand the cell populations in PDL at the single-cell level, Ueda et al performed single-cell cloning of cells migrated from the mouse PDL and established single cell-derived cell lines with various degrees of osteogenic differentiation potential [32] . When each clone was characterized, there were no significant differences in their morphology, surface antigens, or proliferative capacity, despite their apparently different osteogenic potential.…”