R-Spondin 2 Induces Odontogenic Differentiation of Dental Pulp Stem/Progenitor Cells via Regulation of Wnt/β-Catenin Signaling

Abstract

Odontoblast cells generated from human dental pulp stem/progenitor cells (hDPSCs) secrete reparative dentin in responds to an injury. Endogenous Wnt signaling is also activated during this process, and these Wnt-activated cells are responsible for the following repair response. R-spondin 2 (Rspo2) is a potent stem cell growth factor, which strongly potentiates Wnt/β-catenin signaling and plays a vital role in cell differentiation and regeneration. However, the role of Rspo2 during odontoblast differentiation in hDPSCs has not yet been completely understood. This study investigated the effects of Rspo2 on hDPSCs to provide therapeutic insight into dentin regeneration and reparative dentin formation. HDPSCs were extracted from human molars or premolars. Immunofluorescence staining and flow cytometric analysis were used to detect the mesenchymal stem cell markers in hDPSCs. EdU assay and Cell Counting Kit-8 (CCK-8) were performed to explore cell proliferation. The odontogenic differentiation levels were determined by measuring the mRNA and protein expression of DSPP, DMP-1, ALP, and BSP. Immunofluorescence staining was performed to detect the localization of β-catenin. The biological effects of Rspo2 on hDPSCs was investigated using the Lentivirus-based Rspo2 shRNA and recombined human Rspo2 (rhRspo2). Recombined human DKK-1 (rhDKK-1) and recombined human Wnt3a (rhWnt3a) were used for further investigation. The cells generated from human dental pulp expressed mesenchymal stem cell markers Vimentin, Stro-1, Nestin, C-kit, CD90, and CD73, while were negative for CD3, CD31, and CD34. The mRNA expression levels of the odontogenic-related genes DSPP, DMP-1, ALP, and BSP were upregulated in the rhRspo2 treated cells. Silencing Rspo2 suppressed the proliferation and differentiation of the hDPSCs. Blockade of Wnt signaling with DKK-1 inhibited Rspo2-induced activation of Wnt/β-catenin signaling and cell differentiation. The combined use of rhWnt3a and rhRspo2 created a synergistic effect to improve the activation of Wnt/β-catenin signaling. Rspo2 promoted the proliferation and odontogenic differentiation of hDPSCs by regulating the Wnt/β-catenin signaling pathway.

MiR-148a-3p Regulates the Invasion and Odontoblastic Differentiation of Human Dental Pulp Stem Cells via the Wnt1/β-Catenin Pathway

“…1A , on the day 7, the primary cells adhered to each other and exhibited long spindle shape, showing the characteristics of hDPSCs ( 2 ). Then, the hDPSCs were identified by flow cytometry for cell surface markers ( 20 , 21 ), and 96.77% of the cells were CD44-positive, 98.74% were CD73-positive, and 90.19% were CD29-positive; whereas 0.11% of the cells were CD31-negative, 0.13% were CD34-negative, and 0.44% were CD45-negative ( Fig. 1B ).…”

“…It has been reported that stathmin promotes the proliferation and odontoblastic/osteogenic differentiation of hDPSCs through activating the Wnt/β-catenin signaling pathway ( 38 ). The activation of Wnt/β-catenin signaling pathway promotes the odontogenic differentiation of dental pulp stem cells ( 21 , 39 ). However, Scheller et al ( 40 ) reported that Wnt/β-catenin signaling pathway inhibits the differentiation of DPSCs.…”

Comparative Analysis of Dental Pulp and Periodontal Stem Cells: Differences in Morphology, Functionality, Osteogenic Differentiation and Proteome

“…We observe odontoblastic markers only in samples differentiated from DPSC. DPSC are known to be capable of odontoblastic differentiation [ 14 , 15 , 55 ] and are also responsible for tertiary dentin formation [ 12 ] and denticle biomineralization [ 35 ]. PDLSC mechanism of osteogenic differentiation is not the same as in DPSC.…”

“…They have three MSC ‘classical’ differentiation capacities (osteogenic, adipogenic, and chondrogenic) along with the cranial neural crest progenitor cells ones: they give rise to neurons, myoblasts, and odontoblasts [ 1 , 4 , 10 , 11 , 12 , 13 ]. It has turned out that dental stem cells are able to differentiate rather into odontoblasts than into osteoblasts under the influence of osteogenic factors [ 11 , 14 , 15 , 16 ]. The ability for odontoblastic differentiation is one of the most important features of pulp stem cells.…”

N-Cadherin Regulates the Odontogenic Differentiation of Dental Pulp Stem Cells via β-Catenin Activity

“…This could be further supported by previous research reporting that the expression of β-catenin was significantly upregulated during odontogenic differentiation of DPSCs, and knockdown of β-catenin disrupted odontogenic differentiation, which could be reversed by the lithium chloride-induced accumulation of β-catenin ( Han et al, 2014 ). Besides, mounting evidence shows that numerous regulators, including but not limited to enhancer of zeste homolog 2, Stathmin, vacuolar protein sorting 4B, R-Spondin 2, and lncRNA DANCR , regulate the odontogenic differentiation of DPSCs through the Wnt/β-catenin pathway ( Chen et al, 2016 ; Li et al, 2018 ; Pan et al, 2019 ; Zhang et al, 2019 ; Gong et al, 2020 ).…”