This study investigated the role of Forkhead box Q1 (FOXQ1) in the osteogenic differentiation of bone mesenchymal stem cells.
Mouse bone mesenchymal stem cells (mBMSCs) were transfected with ...lentivirus to generate Foxq1-overexpressing mBMSCs, Foxq1-suppressed mBMSCs, and mBMSC controls. The activity of osteogenic differentiation was evaluated with alizarin red staining, alkaline phosphatase activity assay, and RT-qPCR. Wnt/β-catenin signaling activities were compared among groups by TOPFlash/FOPFlash assay, immunofluorescence staining, and western blot assay of beta-catenin (CTNNB1). Coimmunoprecipitation mass spectrometry was also carried out to identify proteins binding with FOXQ1.
Our data showed that FOXQ1 expression was positively correlated with the osteogenic differentiation of the mBMSCs. FOXQ1 also promoted the nuclear translocation of CTNNB1 in the mBMSCs, enhancing Wnt/β-catenin signaling, which was also shown to be essential for the osteogenic differentiation-promoting effect of FOXQ1 in the mBMSCs. Annexin A2 (ANXA2) was bound with FOXQ1, and its depletion reversed the promoting effect of FOXQ1 on Wnt/β-catenin signaling.
These results showed that FOXQ1 binds with ANXA2, promoting Wnt/β-catenin signaling in bone mesenchymal stem cells, which subsequently promotes osteogenic differentiation.
Objective. This study is aimed at evaluating the effects of platelet-rich plasma (PRP) on proliferation, viability, and odontogenic differentiation of neural crest stem-like cells (NCSCs) derived ...from human dental apical papilla. Materials and Methods. Cells from apical papillae were obtained and then induced to form neural spheres. The expression of NCSC markers p75NTR and HNK-1 in neural sphere cells was detected by immunofluorescence staining. Human PRP was prepared by a 2-step centrifugation method and activated by CaCl2 and thrombin. The concentrations of PDGF-BB and TGF-β1 in whole blood and PRP were measured by an ELISA kit. PRP in five different concentrations (0%, 2.5%, 5%, 10%, and 25%) was applied to culture NCSCs. On the 1st, 3rd, 5th, and 7th days, cell proliferation was evaluated by CCK8. Cell viability was tested by a live/dead staining kit. mRNA and protein expression of DSPP and BMP4 were analyzed by RT-qPCR and western blot, respectively. Statistical analysis was performed by a one-way analysis of variance (ANOVA) test or t-test. Results. Dental apical papilla cells formed neural spheres, from which cells displayed positive expression of p75NTR and HNK-1. The concentrations of PDGF-BB and TGF-β1 in PRP were about 3.5-fold higher than those in whole blood. 5% and 10% PRP significantly promoted proliferation of NCSCs, while 25% and 50% PRP inhibited cell proliferation from Day 3 to Day 7. Low-concentration (2.5%, 5%, and 10%) PRP slightly improved viability of NCSCs on Day 7. On the other hand, high-concentration (25% and 50%) PRP significantly inhibited viability of NCSCs from Day 3 to Day 7. RT-qPCR and western blot results indicated that 10% PRP could promote odontogenic differentiation of NCSCs on Day 7. mRNA and protein expression of DSPP and BMP4 were significantly upregulated in the 10% PRP group compared to those in the control group (P<0.05). Conclusions. PRP is a simply acquirable blood derivative which contains high concentration of growth factors like PDGF-BB and TGF-β1. PRP in a proper concentration could promote proliferation, viability, and odontogenic differentiation of NCSCs derived from human dental apical papilla.
Tooth development, as one of the major mineralized tissues in the body, require fine-tuning of mineralization micro-environment. The interaction between dental epithelium and mesenchyme plays a ...decisive role in this process. With epithelium-mesenchyme dissociation study, we found interesting expression pattern of insulin-like growth factor binding protein 3 (IGFBP3) in response to disruption of dental epithelium-mesenchyme interaction. Its action and related mechanisms as regulator of mineralization micro-environment during tooth development are investigated.
Expressions of osteogenic markers at early stage of tooth development are significantly lower than those at later stage. BMP2 treatment further confirmed a high mineralization micro-environment is disruptive at early stage, but beneficial at later stage of tooth development. In contrast, IGFBP3's expression increased gradually from E14.5, peaked at P5, and decreased afterwards, demonstrating an inverse correlation with osteogenic markers. RNA-Seq and Co-immunoprecipitation showed that IGFBP3 regulates the Wnt/beta-catenin signaling pathway activity by enhancing DKK1 expression and direct protein-protein interaction. The suppression of the mineralization microenvironment effectuated by IGFBP3 could be reversed by the DKK1 inhibitor WAY-262611, further demonstrating that IGFBP3 exerted its influence via DKK1.
A deeper understanding of tooth development mechanisms is essential for tooth regeneration, which have great implications for dental care. The current study demonstrated that the IGFBP3 expression is regulated in accordance with the needs of the mineralization microenvironment during tooth development, and IGFBP3 exerts its modulating action on osteogenic/odontogenic differentiation of hDPSCs by DKK1-Wnt/ beta-catenin axis.
Each year ~5.4 million children and adolescents in the United States suffer from dental infections, leading to pulp necrosis, arrested tooth-root development and tooth loss. Apical revascularization, ...adopted by the American Dental Association for its perceived ability to enable postoperative tooth-root growth, is being accepted worldwide. The objective of the present study is to perform a meta-analysis on apical revascularization. Literature search yielded 22 studies following PRISMA with pre-defined inclusion and exclusion criteria. Intraclass correlation coefficient was calculated to account for inter-examiner variation. Following apical revascularization with 6- to 66-month recalls, root apices remained open in 13.9% cases (types I), whereas apical calcification bridge formed in 47.2% (type II) and apical closure (type III) in 38.9% cases. Tooth-root lengths lacked significant postoperative gain among all subjects (p = 0.3472) or in subgroups. Root-dentin area showed significant increases in type III, but not in types I or II cases. Root apices narrowed significantly in types II and III, but not in type I patients. Thus, apical revascularization facilitates tooth-root development but lacks consistency in promoting root lengthening, widening or apical closure. Post-operative tooth-root development in immature permanent teeth represents a generalized challenge to regenerate diseased pediatric tissues that must grow to avoid organ defects.
Hematopoietic stem cells (HSCs) in the endosteum of mesoderm-derived appendicular bones have been extensively studied. Neural crest-derived bones differ from appendicular bones in developmental ...origin, mode of bone formation and pathological bone resorption. Whether neural crest-derived bones harbor HSCs is elusive. Here, we discovered HSC-like cells in postnatal murine mandible, and benchmarked them with donor-matched, mesoderm-derived femur/tibia HSCs, including clonogenic assay and long-term culture. Mandibular CD34 negative, LSK cells proliferated similarly to appendicular HSCs, and differentiated into all hematopoietic lineages. Mandibular HSCs showed a consistent deficiency in lymphoid differentiation, including significantly fewer CD229 + fractions, PreProB, ProB, PreB and B220 + slgM cells. Remarkably, mandibular HSCs reconstituted irradiated hematopoietic bone marrow in vivo, just as appendicular HSCs. Genomic profiling of osteoblasts from mandibular and femur/tibia bone marrow revealed deficiencies in several HSC niche regulators among mandibular osteoblasts including Cxcl12. Neural crest derived bone harbors HSCs that function similarly to appendicular HSCs but are deficient in the lymphoid lineage. Thus, lymphoid deficiency of mandibular HSCs may be accounted by putative niche regulating genes. HSCs in craniofacial bones have functional implications in homeostasis, osteoclastogenesis, immune functions, tumor metastasis and infections such as osteonecrosis of the jaw.
Focal adipose deficiency, such as lipoatrophy, lumpectomy or facial trauma, is a formidable challenge in reconstructive medicine, and yet scarcely investigated in experimental studies. Here, we ...report that Pyrintegrin (Ptn), a 2,4-disubstituted pyrimidine known to promote embryonic stem cells survival, is robustly adipogenic and induces postnatal adipose tissue formation in vivo of transplanted adipose stem/progenitor cells (ASCs) and recruited endogenous cells. In vitro, Ptn stimulated human adipose tissue derived ASCs to differentiate into lipid-laden adipocytes by upregulating peroxisome proliferator-activated receptor (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), with differentiated cells increasingly secreting adiponectin, leptin, glycerol and total triglycerides. Ptn-primed human ASCs seeded in 3D-bioprinted biomaterial scaffolds yielded newly formed adipose tissue that expressed human PPARγ, when transplanted into the dorsum of athymic mice. Remarkably, Ptn-adsorbed 3D scaffolds implanted in the inguinal fat pad had enhanced adipose tissue formation, suggesting Ptn's ability to induce in situ adipogenesis of endogenous cells. Ptn promoted adipogenesis by upregulating PPARγ and C/EBPα not only in adipogenesis induction medium, but also in chemically defined medium specifically for osteogenesis, and concurrently attenuated Runx2 and Osx via BMP-mediated SMAD1/5 phosphorylation. These findings suggest Ptn's novel role as an adipogenesis inducer with a therapeutic potential in soft tissue reconstruction and augmentation.
Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, ...major glands, and teeth. Despite myriad literature on cell–cell interactions and ligand–receptor binding, the roles of extracellular vesicles in epithelium–mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that ∼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27aash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/β-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium–mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.
This study aimed to investigate the role for Foxq1 in proliferation activity regulation of dental pulp stem cells (DPSCs). Proliferation of DPSC was induced by calcium hydroxide, then expression ...alteration of Foxq1 was evaluated. Lentivirus was employed to manipulate Foxq1 level in DPSC, and proliferation activities were evaluated. To look into mechanism regulating Foxq1 level after calcium hydroxide stimulation, expressions of various microRNAs were evaluated, then bioinformatics study and dual-luciferase study were carried out to confirm targeting relationship between microRNA and Foxq1. The result of our study indicated that proliferation activities of DPSCs were enhanced after calcium hydroxide stimulation, during which expression of Foxq1 was also up-regulated. Cell viability and progression from G1 to S phase were both improved with overexpression of Foxq1, and microRNAs profiling study and dual-luciferase result suggested miR-320b contributed to the up-regulation of Foxq1 after calcium hydroxide stimulation. These results suggested that miR-320b mediated Foxq1 up-regulation promote proliferation of dental pulp stem cells.
•Foxq1 was found to be up-regulated in dental pulp stem cell after calcium hydroxide stimulation.•Overexpression of Foxq1 promote proliferation of dental pulp stem cell, while inhibition of Foxq1 repress its proliferation.•MiR-320b depression after calcium hydroxide stimulation ease its inhibition on Foxq1, thus promote Foxq1 expression.
Background This study investigated the role of Forkhead box Q1 (FOXQ1) in the osteogenic differentiation of bone mesenchymal stem cells. Methods Mouse bone mesenchymal stem cells (mBMSCs) were ...transfected with lentivirus to generate Foxq1-overexpressing mBMSCs, Foxq1-suppressed mBMSCs, and mBMSC controls. The activity of osteogenic differentiation was evaluated with alizarin red staining, alkaline phosphatase activity assay, and RT-qPCR. Wnt/beta-catenin signaling activities were compared among groups by TOPFlash/FOPFlash assay, immunofluorescence staining, and western blot assay of beta-catenin (CTNNB1). Coimmunoprecipitation mass spectrometry was also carried out to identify proteins binding with FOXQ1. Results Our data showed that FOXQ1 expression was positively correlated with the osteogenic differentiation of the mBMSCs. FOXQ1 also promoted the nuclear translocation of CTNNB1 in the mBMSCs, enhancing Wnt/beta-catenin signaling, which was also shown to be essential for the osteogenic differentiation-promoting effect of FOXQ1 in the mBMSCs. Annexin A2 (ANXA2) was bound with FOXQ1, and its depletion reversed the promoting effect of FOXQ1 on Wnt/beta-catenin signaling. Conclusion These results showed that FOXQ1 binds with ANXA2, promoting Wnt/beta-catenin signaling in bone mesenchymal stem cells, which subsequently promotes osteogenic differentiation. Keywords: Forkhead box Q1, Bone mesenchymal stem cells, Osteogenic differentiation, Wnt/beta-catenin, Annexin A2
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