Application of Dental Pulp Stem Cells for Bone and Neural Tissue Regeneration in Oral and Maxillofacial Region.

In the oral and maxillofacial region, the treatment of severe bone defects, caused by fractures, cancers, congenital abnormalities, etc., remains a great challenge. In addition, neurological disorders are frequently accompanied by these bone defects or the treatments for them. Therefore, novel bone regenerative techniques and methods to repair nerve injury are eagerly sought. Among them, strategies using dental pulp stem cells (DPSCs) are promising options. Human DPSCs can be collected easily from extracted teeth and are now considered a type of mesenchymal stem cell with higher clonogenic and proliferative potential. DPSCs have been getting attention as a cell source for bone and nerve regeneration. In this article, we reviewed the latest studies on osteogenic or neural differentiation of DPSCs as well as bone or neural regeneration methods using DPSCs and discussed the potential of DPSCs for bone and nerve tissue regeneration.

Effect of Conventional Treatment on Dental Complications and Ectopic Ossifications Among 30 Adults With XLH.

CONTEXT: Conventional treatment of X-linked hypophosphatemia (XLH) was reported to prevent dental complications, but whether the preventive effect was different among different types of teeth, including anterior teeth and molar teeth, is uncertain. Evidence of the preventive effect of conventional treatment on ectopic ossifications is also limited. OBJECTIVE: To compare dental complications and ectopic ossifications among adults with XLH with early (<5 years old) or late (≥5 years old) conventional treatment. METHODS: This retrospective observational study included a total of 30 adults with XLH; orthopantomograms, spinal computed tomography scans, and X-rays of hip/knee joints were studied. Dental complications, including the decayed, missing, filled (DMF) index and devitalized teeth, apical periodontitis, and periodontitis, were evaluated. The ossification of the anterior/posterior longitudinal ligament and yellow ligament indexes (OA/OP/OY indexes) and the sum of the OA/OP/OY indexes (OS index) were utilized to evaluate the severity of spinal ligament ossification. The severity of the hip/knee osteophytes was evaluated using the Kellgren-Lawrence (KL) classification. RESULTS: The number of sound teeth was significantly lower and the DMF index was significantly higher in patients with late treatment. The severity of dental complications in the anterior tooth and molar tooth, OA/OP/OY/OS index, and KL grade were not significantly different among patients with early treatment and those with late treatment. CONCLUSION: Early treatment could prevent dental complications but did not prevent ectopic ossification in adult patients with XLH. The difference in the preventive effect was not observed among different types of teeth.

VCAM-1 and GFPT-2: Predictive markers of osteoblast differentiation in human dental pulp stem cells.

INTRODUCTION: Dental pulp stem cells (DPSCs) have high proliferative and multilineage differentiation potential in mesenchymal stem cells. However, several studies have indicated that there are individual differences in the potential for osteogenic differentiation of DPSCs, and the factors determining these differences are unknown. OBJECTIVE: To identify the genes responsible for the individual differences in the osteogenic differentiation ability of DPSCs. METHODS: We divided DPSCs into high and low osteogenic differentiation ability groups (HG or LG) with ALP and von Kossa stain, and compared the gene expression patterns using RNA-seq. In addition, genes that may affect osteogenic differentiation were knocked down using small interfering RNA (siRNA) and their effects were investigated. RESULTS: The RNA-seq patterns revealed that VCAM1 and GFPT2 were significantly expressed at higher levels in the HG than in the LG. The results of siRNA analysis showed that VCAM1 and GFPT2 knockdown significantly reduced the expression of osteogenic markers. Furthermore, we analyzed the involvement of these two genes in cell signaling in DPSC differentiation. The results indicated that the VCAM1-mediated Ras-MEK-Erk and PI3K/Akt pathways are involved in the osteogenic differentiation of DPSCs, and that GFPT2-mediated HBP signaling influences the osteogenic differentiation of DPSCs. CONCLUSIONS: These findings indicate that DPSCs that highly express VCAM1 and GFPT2 have a high capacity for osteogenic differentiation. Evaluation of VCAM1 and GFPT2 expression in undifferentiated DPSCs may predict the outcome of bone regenerative therapy using DPSCs. Moreover, the expression levels of VCAM1 and GFPT2 in DPSCs may be useful in setting criteria for selecting donors for allogeneic cell transplantation for bone regeneration.

LDL uptake-dependent phosphatidylethanolamine translocation to the cell surface promotes fusion of osteoclast-like cells.

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.

Combinatorial effects of inorganic ions on adhesion and proliferation of osteoblast-like cells.

Combinatorial effects of three ions, namely silicate (Si), calcium (Ca), and magnesium (Mg) ions, on the adhesion and proliferation of MC3T3-E1 mouse osteoblast-like cells were evaluated. The cells were cultured in single-, dual-, or triple-ion-conditioned culture media with systematically changed ion concentrations. The ranges of Si, Ca, and Mg ion concentrations were set as 10-70, 80-400, and 25-500 ppm, respectively. The numbers of adherent live cells were measured after culturing for 3 h and for 1, 3, and 5 days to examine cell adhesion and proliferation, respectively. Mg ions predominantly enhanced cell adhesion in both the dual-ion (xSi-zMg and yCa-zMg) and triple-ion (xSi-yCa-zMg) systems but had no effect when they acted individually in the single-ion system. Conversely, Si ions predominantly enhanced cell proliferation in most single- and triple-ion-conditioned media. Evaluation of the combinatorial effects of the three ions on cell adhesion and proliferation revealed that the dual- and triple-ion-conditioned media mainly conferred synergistic effects on adhesion but antagonistic effects on proliferation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1042-1051, 2019.

Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

Runx1 and Runx3 Are Downstream Effectors of Nanog in Promoting Osteogenic Differentiation of the Mouse Mesenchymal Cell Line C3H10T1/2.

Previously, we reported that the transcription factor Nanog, which maintains the self-renewal of embryonic stem cells (ESCs), promotes the osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 through a genome reprogramming process. In the present study, to clarify the mechanism underlying the multipotency of mesenchymal stem cells (MSCs) and to develop a novel approach to bone regenerative medicine, we attempted to identify the downstream effectors of Nanog in promoting osteogenic differentiation of mouse mesenchymal cells. We demonstrated that Runx1 and Runx3 are the downstream effectors of Nanog, especially in the early and intermediate osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2.

Lectin-like oxidized low-density lipoprotein receptor-1 abrogation causes resistance to inflammatory bone destruction in mice, despite promoting osteoclastogenesis in the steady state.

Inflammatory bone diseases have been attributed to increased bone resorption by augmented and activated bone-resorbing osteoclasts in response to inflammation. Although the production of diverse proinflammatory cytokines is induced at the inflamed sites, the inflammation also generates reactive oxygen species that modify many biological compounds, including lipids. Among the oxidized low-density lipoprotein (LDL) receptors, lectin-like oxidized LDL receptor-1 (LOX-1), which is a key molecule in the pathogenesis of multifactorial inflammatory atherosclerosis, was downregulated with osteoclast differentiation. Here, we demonstrate that LOX-1 negatively regulates osteoclast differentiation by basically suppressing the cell-cell fusion of preosteoclasts. The LOX-1-deleted (LOX-1(-/-)) mice consistently decreased the trabecular bone mass because of elevated bone resorption during the growing phase. In contrast, when the calvaria was inflamed by a local lipopolysaccharide-injection, the inflammation-induced bone destruction accompanied by the elevated expression of osteoclastogenesis-related genes was reduced by LOX-1 deficiency. Moreover, the expression of receptor activator of NF-κB ligand (RANKL), a trigger molecule for osteoclast differentiation, evoked by the inflammation was also abrogated in the LOX-1(-/-) mice. Osteoblasts, the major producers of RANKL, also expressed LOX-1 in response to proinflammatory agents, interleukin-1ß and prostaglandin E2. In the co-culture of LOX-1(-/-) osteoblasts and wild-type osteoclast precursors, the osteoclastogenesis induced by interleukin-1ß and prostaglandin E2 decreased; this process occurred in parallel with the downregulation of osteoblastic RANKL expression. Collectively, LOX-1 abrogation results in resistance to inflammatory bone destruction, despite promoting osteoclastogenesis in the steady state. Our findings indicate the novel involvement of LOX-1 in physiological bone homeostasis and inflammatory bone diseases.

Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control green fluorescent protein (GFP) gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process.

Evaluation of the implant type tissue-engineered cartilage by scanning acoustic microscopy.

The tissue-engineered cartilages after implantation were nonuniform tissues which were mingling with biodegradable polymers, regeneration cartilage and others. It is a hard task to evaluate the biodegradation of polymers or the maturation of regenerated tissues in the transplants by the conventional examination. Otherwise, scanning acoustic microscopy (SAM) system specially developed to measure the tissue acoustic properties at a microscopic level. In this study, we examined acoustic properties of the tissue-engineered cartilage using SAM, and discuss the usefulness of this devise in the field of tissue engineering. We administered chondrocytes/atelocollagen mixture into the scaffolds of various polymers, and transplanted the constructs in the subcutaneous areas of nude mice for 2 months. We harvested them and examined the sound speed and the attenuation in the section of each construct by the SAM. As the results, images mapping the sound speed exhibited homogenous patterns mainly colored in blue, in all the tissue-engineered cartilage constructs. Contrarily, the images of the attenuation by SAM showed the variation of color ranged between blue and red. The low attenuation area colored in red, which meant hard materials, were corresponding to the polymer remnant in the toluidine blue images. The localizations of blue were almost similar with the metachromatic areas in the histology. In conclusion, the SAM is regarded as a useful tool to provide the information on acoustic properties and their localizations in the transplants that consist of heterogeneous tissues with various components.

BMP-2 embedded atelocollagen scaffold for tissue-engineered cartilage cultured in the medium containing insulin and triiodothyronine--a new protocol for three-dimensional in vitro culture of human chondrocytes.

When the chondrocytes are isolated from the native cartilage and proliferate in vitro, they soon lose their original ability to express glycosaminoglycan (GAG) and type II collagen, which is termed dedifferentiation, or decrease cell viability. We first examined in vitro cartilage regeneration of tissue-engineered pellets that consisted of human auricular chondrocytes and atelocollagen and that were incubated in vitro under stimulation with bone morphogenetic protein-2 (BMP-2), insulin, and T(3). We then examined the administration of those growth factors into the scaffold or in the medium and explored the possibility that the atelocollagen, the hydrogel scaffold of the chondrocytes, may function for drug delivery of the factors. BMP-2 in the atelocollagen with the supplement of insulin and T3 in the medium could not only produce a greater GAG matrix in a shorter period but also sustain cell viability with lower mortality. The insulin in the medium could be better administered only for 2 weeks, rather than 3 weeks, which would save time and cost, hence shortening the in vitro culture of chondrocytes. Our protocol of mixing BMP-2 into the atelocollagen with the supplement of insulin and T3 hormone might provide a new insight into the development of tissue engineering in chondrogenesis.

Administration of the insulin into the scaffold atelocollagen for tissue-engineered cartilage.

Three-dimensional culture of the tissue-engineered cartilage constructs may increase the matrix production, but central necrosis must occur if the construct becomes large. To increase the cell viability in the middle part of constructs and to enhance the in vivo cartilage regeneration, we attempted to administer the insulin into the scaffold. Insulin is known to strongly enhance the matrix production in the chondrocytes. The pellets of human auricular chondrocytes with atelocollagen hydrogel were 3D-cultured in the medium. The comparison among three groups (insulin mixed in the atelocollagen, insulin added to the medium, and control group, i.e.; insulin in neither atelocollagen nor medium) revealed that both insulin mixed in the atelocollagen and that in the medium could effectively promoted the cell viability and matrix synthesis of the chondrocytes. The daily assay also showed the gradual release of insulin from the atelocollagen hydrogel, suggesting that this material may work as a control release of insulin. We actually transplanted the poly-L-lactide porous scaffolds carrying the chondrocytes and the atelocollagen mixed with or without insulin, into the nude mice, showing that glycosaminoglycan accumulation was evident in the group with insulin although less without insulin. We thus showed the possibility to enhance the in vivo cartilage regeneration, when administered insulin into the atelocollagen hydrogel.

Effects of fructo-oligosaccharide on DSS-induced colitis differ in mice fed nonpurified and purified diets.

We investigated whether feeding a purified compared with nonpurified diet supplemented with or without fructo-oligosaccharide (FOS; 50 g/kg diet) altered the response of C57BL/6 mice to DSS-induced diarrhea. In Expt. 1, we examined disease severity in mice receiving DSS (2% in drinking water) for 5 d. In Expt. 2, we measured cecal organic acid concentrations and fecal water-holding capacity (WHC). In Expts. 3 and 4, we tested whether polycarbophil calcium (PC), a water-absorbing polymer, altered fecal WHC and disease severity. FOS exacerbated diarrhea and weight loss in mice fed the purified diet and reduced fecal bleeding in mice fed the nonpurified diet (P < 0.05). Without DSS administration, cecal acetate and butyrate concentrations were higher in mice fed the nonpurified diet than in mice fed the purified diet (P < 0.05). Fecal WHC was higher in mice fed the nonpurified diet than in mice fed the purified diet (P < 0.05). One day after starting DSS administration, cecal succinate concentrations were higher in mice fed the FOS-supplemented purified diet than in mice fed the other 3 diets, whereas SCFA concentrations were higher in mice fed the nonpurified diet than in mice fed the purified diet (P < 0.05). PC supplementation increased fecal WHC and prevented FOS exacerbation of diarrhea in mice fed the purified diet (P < 0.05). We conclude that the effects of FOS on DSS-induced diarrhea differ in mice fed the purified and nonpurified diets. The protective effect of nonpurified diet was associated with increased production of organic acids and WHC in the intestinal contents.

The optimization of porous polymeric scaffolds for chondrocyte/atelocollagen based tissue-engineered cartilage.

To broaden the clinical application of cartilage regenerative medicine, we should develop an implant-type tissue-engineered cartilage with firmness and 3-D structure. For that, we attempted to use a porous biodegradable polymer scaffold in the combination with atelocollagen hydrogel, and optimized the structure and composition of porous scaffold. We administered chondrocytes/atelocollagen mixture into the scaffolds with various kinds of porosities (80-95%) and pore sizes (0.3-2.0 mm), consisting of PLLA or related polymers (PDLA, PLA/CL and PLGA), and transplanted the constructs in the subcutaneous areas of nude mice. The constructs using scaffolds of excessively large pore sizes (>1 mm) broke out on the skin and impaired the host tissue. The scaffold with the porosity of 95% and pore size of 0.3 mm could effectively retain the cells/gel mixture and indicated a fair cartilage regeneration. Regarding the composition, the tissue-engineered cartilage was superior in PLGA and PLLA to that in PLA/CA and PDLA. The latter two showed the dense accumulation of macrophages, which may deteriorate the cartilage regeneration. Although PLGA or PLLA has been currently recommended for the scaffold of cartilage, the polymer for which biodegradation was exactly synchronized to the cartilage regeneration would improve the quality of the tissue-engineered cartilage.

Tumor-induced osteomalacia associated with a maxillofacial tumor producing fibroblast growth factor 23: report of a case and review of the literature.

Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disease characterized by renal phosphate wasting and hypophosphatemia. Recently, it was reported that tumors associated with TIO produce fibroblast growth factor (FGF) 23, identified as the last member of the FGF family and of which excessive action causes several hypophosphatemic diseases whereas deficient FGF23 activity results in hyperphosphatemic tumoral calcinosis. In this case, although it was difficult to locate the associated tumor, an abnormal mass in the left maxilla was detected by imaging. The tumor was removed by partial resection of the left maxillary alveolar region. Thereafter, serum level of FGF23 rapidly decreased, hypophosphatemia improved, and the clinical symptoms greatly improved. Histopathologic diagnosis of the tumor was phosphaturic mesenchymal tumor, mixed connective tissue variant. Immunohistochemical findings confirmed that the removed tumor produced FGF23. These results indicate that development of osteomalacia in this patient was related to the maxillary tumor, which overexpressed FGF23.

Comparison of gut microbiota and allergic reactions in BALB/c mice fed different cultivars of rice.

Our preliminary clinical trial showed that consumption of cooked rice of a Japanese common cultivar Yukihikari improved atopic dermatitis associated with a suspected rice allergy, although the underlying mechanisms remain unclear. We hypothesised that the ameliorating effect of Yukihikari on atopic dermatitis is associated with the gut microbiota. BALB/c mice were fed a synthetic diet supplemented with uncooked and polished white rice powder prepared from one of four different cultivars: Yukihikari, rice A (common rice), rice B (brewery rice) and rice C (waxy rice). Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments showed that the composition of faecal microbiota was different between mice fed Yukihikari and those fed rice A. Analysis of the 16S rRNA clone library and species-specific real-time PCR showed that the abundance of Akkermansia muciniphila, a mucin degrader, tended to be lower in mice fed Yukihikari. The incidence of allergic diarrhoea induced by oral administration of ovalbumin in systemically immunised mice was lower in mice fed Yukihikari, albeit with no difference in serum antibodies specific to ovalbumin. In a separate experiment, serum antibody levels specific to orally administered ovalbumin were lower in mice fed Yukihikari. Additionally, the translocation of horseradish peroxidase in isolated segments of ileum and colon tended to be lower in mice fed Yukihikari, suggesting a reduction in gut permeability in mice fed Yukihikari. These data indicate that changes in the gut microbiota of mice fed Yukihikari could be advantageous in the prevention of food allergy.

Role of intestinal Bifidobacterium pseudolongum in dietary fructo-oligosaccharide inhibition of 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice.

Strategies to manipulate the gut microbiota have been explored for preventing allergy development. We previously showed that dietary supplementation with fructo-oligosaccharide (FOS) reduced 2, 4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS) in BALB/c mice. Because the CHS response was negatively correlated with the number of faecal bifidobacteria, particularly Bifidobacterium pseudolongum, the present study aimed to examine whether oral administration of B. pseudolongum affects CHS response. Viable B. pseudolongum was successfully isolated from mouse faeces. Female BALB/c mice were fed a synthetic diet with or without FOS supplementation, and B. pseudolongum (2 x 10(7) cells) was administered daily throughout the experimental period. Two weeks after starting the test diets, mice received DNFB on the ear auricle twice at 7-d intervals. Conventional cultivation and molecular biological analyses based on 16S rRNA gene sequences showed that administration of FOS and B. pseudolongum resulted in higher excretion of viable bifidobacteria, mainly B. pseudolongum. Although dietary FOS reduced the CHS response as demonstrated by ear swelling, B. pseudolongum administration resulted in a reduction in the initial phase only of the CHS response. B. pseudolongum administration increased hapten-specific IgG1, while dietary FOS decreased IgG2a in sera. Administration of FOS and B. pseudolongum decreased interferon-gamma production and increased IL-10 production in cervical lymph node cells restimulated with hapten in vitro. We conclude that B. pseudolongum proliferation in the intestinal tract is partially responsible for the reduction in DNFB-induced CHS response by dietary supplementation with FOS in mice, which may be mediated by the modulation of antigen-induced cytokine production.

Screening of chondrogenic factors with a real-time fluorescence-monitoring cell line ATDC5-C2ER: identification of sorting nexin 19 as a novel factor.

OBJECTIVE: To establish a cell culture system for noninvasive and real-time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors. METHODS: The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP-2), or transforming growth factor beta (TGFbeta) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene. RESULTS: We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5-C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP-2, or TGFbeta, showing good correspondence to the chondrogenic markers. Screening using the ATDC5-C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain-of-function and loss-of-function analyses revealed a potent chondrogenic activity of SNX19. CONCLUSION: We established the ATDC5-C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.