Comprehensive analysis of transcription factors involved in odontoblast differentiation mechanism.

Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6 J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2 days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.

Inhibition of NLRP3 inflammasome activity by MCC950 leads to exacerbation of SjÓ§gren's syndrome pathologies in non-obese diabetic mice.

SjÓ§gren's syndrome (SS) is an autoimmune inflammatory disease characterized by chronic inflammation and dysfunction of exocrine glands and causes dry mouth, dry eyes and various systemic health problems. The objective of this study is to define the in vivo actions of the endogenous NLRP3 inflammasome, a key initiator and mediator of various immune and inflammatory conditions, in newly established SS disease. MCC950, a highly specific small-molecule inhibitor of NLRP3 inflammasome formation and activation, was intraperitoneally administered to the female non-obese diabetic (NOD) mice aged 11 weeks, which have newly established SS-like hyposalivation and pathologies. The injection was conducted three times weekly for three consecutive weeks and mice were analysed for characteristic SS pathologies at the end of the treatments. MCC950 treatment resulted in a marked reduction in salivary secretion and an exacerbation of leukocyte infiltration of submandibular glands. The disease-worsening effect of MCC950 treatment was accompanied by increased T and B cell numbers, enhanced T helper 1 response and reduced aquaporin 5 expression in submandibular glands. Hence, ablation of endogenous NLRP3 inflammasome activity by MCC950 with established autoimmune exocrinopathy exacerbates salivary gland dysfunction and inflammation, indicating a disease-alleviating and inflammation-dampening action of the endogenous NLRP3 inflammasome activity during established SS disease in the non-obese diabetic mouse model.

Mechanical stress-induced FGF-2 promotes proliferation and consequently induces osteoblast differentiation in mesenchymal stem cells.

Mechanical stimuli serve as crucial regulators of bone mass, promoting bone formation. However, the molecular mechanisms governing how mesenchymal stem cells (MSCs) respond to mechanical cues during their differentiation into osteogenic cells remain elusive. In this study, we found that cyclic stretching enhances MSC proliferation but does not increase the expression of osteoblast-related genes. We further revealed that this proliferative effect is mediated by fibroblast growth factor 2 (FGF-2), synthesized by MSCs in response to mechanical stress. Cell proliferation induced by cyclic stretching was inhibited upon the addition of either U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), or early growth response 1 (EGR1)-targeting small-hairpin RNA (shRNA), indicating the involvement of the extracellular signal-regulated kinase (ERK)/EGR1 signaling pathway. Osteoblast differentiation, evaluated through ALP activity, osteoblast-related gene expression, and mineralization, was stimulated by recombinant human FGF-2 (rhFGF-2) when applied during the proliferation phase, but not when applied during the differentiation stage alone. Our results suggest that FGF-2 indirectly promotes osteoblast differentiation as a downstream effect of stimulating cell proliferation. For the first time, we demonstrate that cyclic stretching induces MSCs to produce FGF-2, which in turn encourages cell proliferation through an autocrine/paracrine mechanism, consequently leading to osteoblast differentiation.

Hedgehog-Related Mutation Causes Bone Malformations with or without Hereditary Gene Mutations.

The hedgehog (Hh) family consists of numerous signaling mediators that play important roles at various stages of development. Thus, the Hh pathway is essential for bone tissue development and tumorigenesis. Gorlin syndrome is a skeletal and tumorigenic disorder caused by gain-of-function mutations in Hh signaling. In this review, we first present the phenotype of Gorlin syndrome and the relationship between genotype and phenotype in bone and craniofacial tissues, including the causative gene as well as other Hh-related genes. Next, the importance of new diagnostic methods using next-generation sequencing and multiple gene panels will be discussed. We summarize Hh-related genetic disorders, including cilia disease, and the genetics of Hh-related bone diseases.

FGF4 and FGF9 have synergistic effects on odontoblast differentiation.

The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.

Three-dimensional Culture Using Atelocollagen Sponge and Self-assembling Peptide Hydrogel.

This study aimed to assess the combined application of two biomaterials, a selfassembling peptide hydrogel (SPH) and an atelocollagen sponge (ACS). The ACS was combined with SPH (PuraMatrixⓇ or PanaceaGelⓇ) and its osteogenic effects on mouse osteoblastic cell line MC3T3 then evaluated. Each type of SPH was successfully incorporated into the ACS. The MC3T3 cells showed uniform distribution within the scaffold. No necrotic cells were observed throughout the experimental procedures. When the SPH was combined with the ACS, the MC3T3 cells differentiated toward the osteo-lineage, expressing Alp, Runx2, Osx, Bsp, and Oc. PanaceaGelⓇ exhibited a stronger osteogenic effect on the cells than PuraMatrixⓇ.

Interleukin-22 Exerts Detrimental Effects on Salivary Gland Integrity and Function.

Interleukin-22 (IL-22) affects epithelial tissue function and integrity in a context-dependent manner. IL-22 levels are elevated in salivary glands of Sjögren's syndrome (SS) patients, but its role in the pathogenesis of this disease remains unclear. The objective of this study is to elucidate the impact of IL-22 on salivary gland tissue integrity and function in murine models. We showed that IL-22 levels in sera and salivary glands increased progressively in female non-obese diabetic (NOD) mice, accompanying the development of SS. Administration of IL-22 to the submandibular glands of NOD mice prior to the disease onset reduced salivary secretion and induced caspase-3 activation in salivary gland tissues, which were accompanied by alterations in multiple genes controlling tissue integrity and inflammation. Similarly, IL-22 administration to submandibular glands of C57BL/6 mice also induced hyposalivation and caspase-3 activation, whereas blockade of endogenous IL-22 in C57BL/6 mice treated with anti-CD3 antibody mitigated hyposalivation and caspase-3 activation. Finally, IL-22 treatment reduced the number of viable C57BL/6 mouse submandibular gland epithelial cells cultured in vitro, indicating a direct impact of this cytokine on these cells. We conclude that IL-22 exerts a detrimental impact on salivary gland tissues.

Alternate-Day Fasting Ameliorates Newly Established Sjögren's Syndrome-like Sialadenitis in Non-Obese Diabetic Mice.

Intermittent fasting confers protections to various diseases including autoimmune disorders, but the specific effects of intermittent fasting on Sjögren's syndrome (SS) remains inconclusive. The present study was undertaken to determine the specific impact of alternate-day fasting (ADF) on newly established SS-like sialadenitis using non-obese diabetic (NOD) mice. Female NOD mice were deprived of food every other day from 10 to 13 weeks of age, the early stage of established SS, and then analyzed for the disease characteristics. Mice in the ADF group had higher salivary flow rate and attenuated submandibular gland (SMG) inflammation, compared to the control mice fed with standard chow ad libitum. The improvements were accompanied with a decrease in the total leukocytes, T and B lymphocytes and activated CD4 and CD8 T cells, and a down-regulation of pro-inflammatory cytokines IFN-γ and IL-17, chemokine receptor CXCR3 and its ligands CXCL9 and CXCL11 in the SMGs. ADF also led to elevated mRNA levels of water channel protein aquaporin 5 and tight junction protein claudin-1, two factors crucial for normal salivary secretion in the SMGs. In addition, ADF reduced the proportion of IFN-γ- and IL-17- expressing CD4 T cells and diminished mRNA levels of IFN-γ, TNF-α, and IL-17 in the total submandibular draining lymph node cells. Taken together, ADF is effective in ameliorating newly established SS-associated salivary gland exocrinopathy in NOD mice.

The effect of BMP4, FGF8 and WNT3a on mouse iPS cells differentiating to odontoblast-like cells.

We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14 days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells.

The concurrent stimulation of Wnt and FGF8 signaling induce differentiation of dental mesenchymal cells into odontoblast-like cells.

Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/ß-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3ß inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/ß-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice.

Induced pluripotent stem cells from homozygous Runx2-deficient mice show poor response to vitamin D during osteoblastic differentiation.

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.

Effects of KnockOut Serum Replacement on Differentiation of Mouse-Induced Pluripotent Stem Cells into Odontoblasts.

Serum serves as a source of rich nutrients during in vitro cell culture, facilitating cell adhesion, growth, and differentiation. When culturing stem cells for transplantation, however, it must be remembered that such culture medium may contain substances potentially harmful to the proposed recipient and may even induce cellular damage. The purpose of this study was to determine whether KnockOut Serum Replacement (KSR), a chemically defined medium supplement, enhanced in vitro differentiation of induced pluripotent stem cells into odontoblasts. Cranial neural crest cells, precursors of odontoblasts, were generated from mouse-induced pluripotent stem cells. They were then cultured in serum-free Dulbecco's modified Eagle's/F12 medium containing fibroblast growth factor 8 with or without KSR. The cells cultured with KSR showed strong proliferation, acquired a spindle-like morphology, and connected with the surrounding cells. KnockOut Serum Replacement also boosted expression of odontoblast markers as measured by qRT-PCR, and increased dentin sialoprotein as assessed by immunostaining. These results confirmed that mouse-induced pluripotent stem cells differentiated into odontoblasts under serum-free conditions, and that KSR enhanced the efficiency of this process.

Keratinocytes from Gorlin Syndrome-induced pluripotent stem cells are resistant against UV radiation.

Gorlin syndrome (GS) is an autosomal dominant genetic disorder involving Patched 1 (PTCH1) mutations. The PTCH1 is a receptor as well as an inhibitor of hedgehog (Hh) to sequester downstream Hh pathway molecules called Smoothened (SMO). PTCH1 mutations causes a variety of GS conditions including falx calcification, odontogenic keratocytes and basal cell carcinomas (BCC). Because PTCH1 is a major driver gene of sporadic BCC, GS patients are characteristically prone to BCC. In order to elucidate the pathological mechanism of BCC-prone GS patients, we investigated keratinocytes derived from GS patient specific iPS cells (G-OFiPSCs) which were generated and reported previously. We found that keratinocytes derived from G-OFiPSCs (GKCs) have increased expression of Hh target molecules. GKCs were irradiated and those cells showed high resistance to UV induced apoptosis. BCL2, known as anti-apoptotic molecule as well as Hh target, significantly increased in GKCs. Several molecules involved in DNA repair, cell cycle control, senescence, and genotoxic stress such as TP53, BRCA1 and GADD45A increased only in GKCs. GKCs are indicated to be resistant to UV irradiation by upregulating molecules which control DNA repair and genotoxic even under DNA damage caused by UV. The anti-apoptotic properties of GKCs may contribute BCC.

A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors.

A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H-mutated (GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.

Effects of intermittent treatment with parathyroid hormone (PTH) on osteoblastic differentiation and mineralization of mouse induced pluripotent stem cells in a 3D culture model.

BACKGROUND/OBJECTIVES: PTH plays an important role in bone remodeling, and different actions have been reported depending on its administration method. iPSCs are promising as a cell source for regeneration of periodontal tissue due to their ability of proliferation and pluripotency. However, the effects of PTH on iPSCs remain mostly unknown. The purpose of this study was to investigate in vitro effects of parathyroid hormone (PTH) on osteoblastic differentiation of induced pluripotent stem cells (iPSCs) in a 3D culture model. MATERIALS AND METHODS: Following embryoid body (EB) induction from mouse iPSCs (miPSCs), dissociated cells (miPS-EB-derived cells) were seeded onto atelocollagen sponge (ACS) in osteoblast differentiation medium (OBM). Cell-ACS constructs were divided into three groups: continuous treatment with human recombinant PTH (1-34) (PTH-C), intermittent PTH treatment (PTH-I) or OBM control. To confirm the expression of PTH receptor-1(PTH1R), the expression of Pth1r and cAMP production over time were assessed. Real-time PCR was used to assess the expression of genes encoding osterix (Sp7), runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1), and osteocalcin (Bglap) at different time points. Mineralization was assessed by von Kossa staining. Histochemical staining was used to analyze alkaline phosphatase (ALP) activity, and immunolocalization of SP7 and BGLAP was analyzed by confocal laser scanning microscopy (CLSM). RESULTS: On days 7 and 14, expression of the Pth1r in miPS-EB-derived cells was increased in all groups. Production of cAMP, the second messenger of the PTH1R, tended to increase in the PTH-I group compared with PTH-C group on day 14. Expression of Col1a1 in the PTH-I group on day 14 was significantly higher than other groups. There was a time-dependent increase in the expression of Sp7 in all groups. On day 14, the expression level of Sp7 in the PTH-I group was significantly higher than other groups. In von Kossa staining, the PTH-I group showed higher level of staining compared with other groups on day 14, whereas the level was slightly attenuated in the PTH-C group. In histochemical staining, ALP-positive cells were significantly increased in the PTH-I group compared with other groups on day 14. In CLSM analysis, the numbers of SP7- and BGLAP-positive cells showed a gradual increase over time, and on day 14, a significantly greater SP7 expression was observed in the PTH-I group than other groups. CONCLUSION: These results suggested that the intermittent PTH treatment promotes osteoblastic differentiation and mineralization of miPSCs in the ACS scaffold.

Gorlin Syndrome: Recent Advances in Genetic Testing and Molecular and Cellular Biological Research.

Gorlin syndrome is a skeletal disorder caused by a gain of function mutation in Hedgehog (Hh) signaling. The Hh family comprises of many signaling mediators, which, through complex mechanisms, play several important roles in various stages of development. The Hh information pathway is essential for bone tissue development. It is also the major driver gene in the development of basal cell carcinoma and medulloblastoma. In this review, we first present the recent advances in Gorlin syndrome research, in particular, the signaling mediators of the Hh pathway and their functions at the genetic level. Then, we discuss the phenotypes of mutant mice and Hh signaling-related molecules in humans revealed by studies using induced pluripotent stem cells.

Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells.

Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequencing experiments to analyze gene expression profiles of cNCCs at different time points after induction. cNCCs expressed several neural crest specifier genes; however, some previously reported specifier genes such as paired box 3 and Forkhead box D3, which are essential for embryonic neural crest development, were not expressed. Moreover, ETS proto-oncogene 1, transcription factor and sex-determining region Y-box 10 were only expressed after 14 days of induction. Finally, cNCCs expressed multiple protocadherins and a disintegrin and metalloproteinase with thrombospondin motifs enzymes, which may be crucial for their migration.

Stem cell-based modeling and single-cell multiomics reveal gene-regulatory mechanisms underlying human skeletal development.

Although the skeleton is essential for locomotion, endocrine functions, and hematopoiesis, the molecular mechanisms of human skeletal development remain to be elucidated. Here, we introduce an integrative method to model human skeletal development by combining in vitro sclerotome induction from human pluripotent stem cells and in vivo endochondral bone formation by implanting the sclerotome beneath the renal capsules of immunodeficient mice. Histological and scRNA-seq analyses reveal that the induced bones recapitulate endochondral ossification and are composed of human skeletal cells and mouse circulatory cells. The skeletal cell types and their trajectories are similar to those of human embryos. Single-cell multiome analysis reveals dynamic changes in chromatin accessibility associated with multiple transcription factors constituting cell-type-specific gene-regulatory networks (GRNs). We further identify ZEB2, which may regulate the GRNs in human osteogenesis. Collectively, these results identify components of GRNs in human skeletal development and provide a valuable model for its investigation.

Bone regeneration of induced pluripotent stem cells derived from peripheral blood cells in collagen sponge scaffolds.

OBJECTIVE: Stem cell-based regeneration therapy offers new therapeutic options for patients with bone defects because of significant advances in stem cell research. Although bone marrow mesenchymal stem cells are the ideal material for bone regeneration therapy using stem cell, they are difficult to obtain. Induced pluripotent stem cells (iPSCs) are now considered an attractive tool in bone tissue engineering. Recently, the efficiency of establishing iPSCs has been improved by the use of the Sendai virus vector, and it has become easier to establish iPSCs from several type of somatic cells. In our previous study, we reported a method to purify osteogenic cells from iPSCs.This study aimed to evaluate the osteogenic ability of iPSCs derived from peripheral blood cells. METHODOLOGY: Mononuclear cells (MNCs) were obtained from human peripheral blood. Subsequently, T cells were selectively obtained from these MNCs and iPSCs were established using Sendai virus vectors. Established iPSCs were evaluated by the expression of undifferentiated markers and teratoma formation assays. Osteoblasts were induced from these iPSCs and evaluated by the expression of osteoblast markers. Additionally, the induced osteoblasts were transplanted into rat critical size calvaria bone defect models with collagen sponge scaffolds. Samples were evaluated by radiographical and histological assessments. RESULTS: Induced osteoblasts expressed several osteoblast-specific markers. The results of radiographical and histological assessments revealed that the cell transplant group had bone formations superior to those of the control group. CONCLUSIONS: This study suggests that peripheral blood MNCs have the potential to differentiate into osteoblasts. Although there are some hurdles in iPSC transplantation, osteoblasts obtained from MNC-iPSCs could be applied to bone regeneration therapy in the future.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

The transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.