Microbiomic association between the saliva and salivary stone in patients with sialolithiasis.

Salivary stones, known as sialoliths, form within the salivary ducts due to abnormal salivary composition and cause painful symptoms, for which surgical removal is the primary treatment. This study explored the role of the salivary microbial communities in the formation of sialoliths. We conducted a comparative analysis of microbial communities present in the saliva and salivary stones, and sequenced the 16S rRNA gene in samples obtained from patients with sialoliths and from healthy individuals. Although the diversity in the saliva was high, the essential features of the microbial environment in sialoliths were low diversity and evenness. The association of microbial abundance between stones and saliva revealed a positive correlation between Peptostreptococcus and Porphyromonas, and a negative correlation for Pseudomonas in saliva. The functional potential differences between saliva and stones Bacterial chemotaxis and the citrate cycle were negatively correlated with most genera found in salivary stone samples. However, the functions required for organic compound degradation did not differ between the saliva samples. Although some microbes were shared between the sialoliths and saliva, their compositions differed significantly. Our study presents a novel comparison between salivary stones and salivary microbiomes, suggesting potential preventive strategies against sialolithiasis.

The association of tonsillar microbiota with biochemical indices based on obesity and tonsillar hypertrophy in children.

The correlation between tonsil microbiome and tonsillar hypertrophy has not been well established. Given that oral dysbiosis is related to several metabolic diseases and that tonsillar hypertrophy leads to disordered breathing during sleep and obesity in children, it is necessary to investigate the relationship between the oral microbiome and tonsillar hypertrophy. After 16S rRNA amplicon sequencing of tonsillectomy samples, we evaluated the correlation between the tonsil microbiome and biochemical blood indices in pediatric patients who underwent tonsillectomy. Groups are classified into two categories: based on BMI, and grades 2, 3, and 4 based on tonsil size. Children with obesity and tonsillar hypertrophy have similar microbiome compositions and induce comparable changes in microbiome abundance and composition, confirming the association from a metagenomic perspective. In addition, obesity and tonsillar hypertrophy demonstrated a strong correlation with the Proteobacteria to Firmicutes (P/F) ratio, and among various biochemical indicators, alanine aminotransferase (ALT) levels increase with obesity and tonsillar hypertrophy, indicating a possible association of tonsil microbiome and liver metabolism. These novel findings demonstrate the significance of the tonsil microbiome and suggest the need for tonsil regulation, particularly during childhood.

Nitrosylation of ß2-Tubulin Promotes Microtubule Disassembly and Differentiated Cardiomyocyte Beating in Ischemic Mice.

BACKGROUND: Beating cardiomyocyte regeneration therapies have revealed as alternative therapeutics for heart transplantation. Nonetheless, the importance of nitric oxide (NO) in cardiomyocyte regeneration has been widely suggested, little has been reported concerning endogenous NO during cardiomyocyte differentiation. METHODS: Here, we used P19CL6 cells and a Myocardiac infarction (MI) model to confirm NO-induced protein modification and its role in cardiac beating. Two tyrosine (Tyr) residues of ß2-tubulin (Y106 and Y340) underwent nitrosylation (Tyr-NO) by endogenously generated NO during cardiomyocyte differentiation from pre-cardiomyocyte-like P19CL6 cells. RESULTS: Tyr-NO-ß2-tubulin mediated the interaction with Stathmin, which promotes microtubule disassembly, and was prominently observed in spontaneously beating cell clusters and mouse embryonic heart (E11.5d). In myocardial infarction mice, Tyr-NO-ß2-tubulin in transplanted cells was closely related with cardiac troponin-T expression with their functional recovery, reduced infarct size and thickened left ventricular wall. CONCLUSION: This is the first discovery of a new target molecule of NO, ß2-tubulin, that can promote normal cardiac beating and cardiomyocyte regeneration. Taken together, we suggest therapeutic potential of Tyr-NO-ß2-tubulin, for ischemic cardiomyocyte, which can reduce unexpected side effect of stem cell transplantation, arrhythmogenesis.

Therapeutic Effect of HDAC5 Binding and Cell Penetrating Peptide for the Treatment of Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is an incurable disease that negatively influences the quality of life of patients. Current and emerging therapies target proinflammatory cytokines and/or receptors to downregulate proinflammatory responses, but insufficient remission requires other therapeutic agents. Herein, we report that the synthetic anti-inflammatory peptide 15 (SAP15) is capable of cell penetration and anti-inflammatory activity in human macrophages. METHODS: SAP15 was labeled with fluorescence and administered to human leukemia monocytic cells (THP-1) cells for cell penetration analysis. Using biolayer interferometry analysis, the binding affinity of SAP15 with histone deacetylase 5 (HDAC5) was measured. SAP15-treated THP-1 cells were analyzed by protein phosphorylation assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In addition, in vivo analysis of the therapeutic effect on IBD was observed in a dextran sulfate sodium (DSS)-induced model. Samples from SAP15-treated mice were analyzed at both the macroscopic and microscopic levels using ELISA, myeloperoxidase (MPO) assays, and histological evaluations. RESULTS: SAP15 was internalized within the cytosol and nucleus of THP-1 cells and bound to the HDAC5 protein. SAP15-treated macrophages were assessed for protein phosphorylation and showed inhibited phosphorylation of HDAC5 and other immune-related proteins, which led to increased M2-like macrophage markers and decreased M1-like macrophage markers and tumor necrosis factor-α and interleukin-6 cytokine levels. The SAP15 treatment on IBD model showed significant recovery of colon length. Further histological analysis of colon demonstrated the therapeutic effect of SAP15 on mucosal layer. Moreover, proinflammatory cytokine levels and MPO activity from the plasma show that SAP15 is effective in reduced proinflammatory responses. CONCLUSION: These findings suggest that SAP15 is a novel peptide with a novel cell-penetrating peptide with anti-inflammatory property that can be used as a therapeutic agent for IBD and other inflammatory diseases.

Topical Delivery of Cell-Penetrating Peptide-Modified Human Growth Hormone for Enhanced Wound Healing.

Protein drugs have been emerging as a class of promising therapeutics. However, their topical application has been limited by their high molecular weight and poor permeability to the cell membrane. In this study, we aimed to enhance human growth hormone (hGH) permeability for topical application by conjugation of TAT peptide, a cell-penetrating peptide, to hGH via crosslinker. After TAT was conjugated to hGH, TAT-hGH was purified by affinity chromatography. TAT-hGH significantly increased cell proliferation compared with the control. Interestingly, the effect of TAT-hGH was higher than hGH at the same concentration. Furthermore, the conjugation of TAT to hGH enhanced the permeability of TAT-hGH across the cell membrane without affecting its biological activity in vitro. In vivo, the topical application of TAT-hGH into scar tissue markedly accelerated wound healing. Histological results showed that TAT-hGH dramatically promoted the re-epithelialization of wounds in the initial stage. These results demonstrate TAT-hGH as a new therapeutic potential drug for wound healing treatment. This study also provides a new method for topical protein application via enhancement of their permeability.

Distinction of Male and Female Trees of Ginkgo biloba Using LAMP.

Ginkgo biloba is utilized as food, medicine, wood, and street trees among other things. The objective of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for gender distinction of G. biloba. Male-specific SCAR gene can be utilized to identify G. biloba gender using LAMP. The optimized LAMP conditions, temperature 60 °C, 2-mM MgSO4, and [F3/B3]:[FIP/BIP] primer ratio of 1:4 were selected as final conditions. The G. biloba SCAR LAMP displayed a sensitivity of 10 ng when amplified by concentration under the optimum conditions. Additionally, it demonstrated a particular response in male with SYBR Green I in LAMP analysis that can be a more powerful tool for field and scale-up applications. Our work represents a first attempt to identify G. biloba gender using LAMP and offers an efficient and reliable tool for roadside landscaping.

Vacuoles isolated from Saccharomyces cerevisiae inhibit differentiation of 3T3-L1 adipocyte.

Yeast vacuoles contain various hydrolytic enzymes including lipase. They play important roles in intracellular signaling and metabolism. Using these characteristics, the aim of this study is to determine effects of yeast vacuoles on the triglyceride accumulation and differentiation of pre-adipocytes to adipocytes using 3T3-L1 cells. The accumulation of lipid droplets and triglyceride were reduced after treatment with vacuoles. As a result of not maintaining the expression of C/EBPß and C/EBPδ in vacuole-treated adipocytes, expression levels of C/EBPα and PPARγ in vacuole-treated adipocytes were significantly reduced. The expression of adiponectin in the late differentiation stage was increased compared to that in the control. By confirming that vacuolar enzymes also inhibit differentiation of adipocytes same as vacuoles, it can be concluded that the adipogenesis inhibitory effect of vacuoles is by lipase of vacuolar enzymes. Yeast-derived vacuoles could be an important source for inhibiting accumulation of lipids and obesity-related inflammation by suppressing adipogenesis.

Cyclin-Dependent Kinase 1 Inhibition Potentiates the Proliferation of Tonsil-Derived Mesenchymal Stem Cells by Delaying Cellular Senescence.

Mesenchymal stem cells (MSCs) have been widely used in tissue regeneration and stem cell therapy and are currently being tested in numerous clinical trials. Senescence-related changes in MSC properties have attracted considerable attention. Senescent MSCs exhibit a compromised potential for proliferation; senescence acts as a stress response that prevents the proliferation of dysfunctional cells by inducing an irreversible cell cycle arrest. Here, we established a senescent MSC model using senescence-associated ß-galactosidase, proliferation, and cell cycle assays. We further identified novel biomarker candidates for old, senescent tonsil-derived MSCs (TMSCs) using transcriptomics. A plot of the cellular senescence pathway showed cyclin-dependent kinase 1 (CDK1; +8-fold) and CDK2 (+2-fold), and transforming growth factor beta 2 (TGFB2; +2-fold) showed significantly higher expression in old TMSCs than in young TMSCs. The CDK family was shown to be related to cell cycle and proliferation, as confirmed by quantitative RT-PCR. As replicative senescence of TMSCs, the gene and protein expression of CDK1 was significantly increased, which was further validated by inhibiting CDK1 using an inhibitor and siRNA. Taken together, we suggest that the CDK1 can be used as a selective senescence biomarker of MSCs and broaden the research criteria for senescent mechanisms.

Optimization of skeletal muscle-derived fibroblast isolation and purification without the preplating method.

Most embryonic fibroblasts have been widely used as feeder cells to support stem cell cultures, and in the case of human embryonic stem cells, the manipulation with human embryonic stem cells is prohibited in most countries for ethical reasons. However, the importance of tissue origin is increasing because cell surface markers and extracellular matrix proteins are secreted differently depending on the tissue origin of fibroblasts. In particular, as fibroblasts and myoblasts are mixed in skeletal muscle tissue, it is necessary to selectively separate only fibroblasts. The preplating technique was used to isolate fibroblasts from mouse skeletal muscle tissue, and the morphological and functional characteristics were investigated to optimize the efficient purification method of isolated fibroblasts. Cell morphology and doubling time were not notably associated with preplating. The preplating method did not induce significant functional changes, including those in the expression of fibroblast-specific genes (Vim and Fsp1) and myoblast-specific genes (Myod and Myog), until passage number 5. Moreover, skeletal muscle-derived fibroblasts before and after cryopreservation retained the morphological and functional properties until passage 5 after thawing. Based on the comprehensive results, the characteristics of skeletal muscle-derived fibroblasts were maintained up to passage 5 regardless of preplating, and fibroblast-specific properties were maintained even after cryopreservation. In this study, we optimized the isolation and purification methods for skeletal muscle-derived fibroblasts. These methods are expected to be used in various applications for tissue engineering.

Tonsil-derived mesenchymal stem cells incorporated in reactive oxygen species-releasing hydrogel promote bone formation by increasing the translocation of cell surface GRP78.

Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated ß-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.

Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts.

Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.

Engineered synthetic cell penetrating peptide with intracellular anti-inflammatory bioactivity: An in vitro and in vivo study.

Various biomaterials have been used for bone and cartilage regeneration, and inflammation associated with biomaterial implantation is also increased. A 15-mer synthetic anti-inflammatory peptide (SAP15) was designed from human ß-defensin 3 to penetrate cells and induce intracellular downregulation of inflammation. The downregulation of inflammation was achieved by the binding of SAP15 to intracellular histone deacetylase (HDAC5). SAP15-mediated inhibition of inflammation was examined in vitro and in vivo using murine macrophages, human articular chondrocytes, and a collagen-induced arthritis (CIA) rat model. Surface plasmon resonance and immunoprecipitation assays indicated that SAP15 binds to HDAC5. SAP15 inhibited the lipopolysaccharide (LPS)-induced phosphorylation of intracellular HDAC5 and NF-κB p65 in murine macrophages. SAP15 treatment increased aggrecan and type II collagen expression and decreased osteocalcin expression in LPS-induced chondrocytes. Subcutaneous injection of SAP15-loaded sodium hyaluronic acid (HA) solution significantly decreased hind paw swelling, joint inflammation, and serum cytokine levels in CIA rats compared with the effects of sodium HA solution alone. The SAP15-loaded HA group exhibited preservation of cartilage and bone structure in CIA rat joints. Moreover, a more robust anti-inflammatory effect of the SAP15 loaded HA was observed than that of etanercept (an anti-tumor necrosis factor-alpha [TNF-α] antibody)-loaded HA. These findings suggest that SAP15 has an anti-inflammatory effect that is not controlled by sodium HA and is mediated by inhibiting HDAC5, unlike the anti-inflammatory mechanism of etanercept. These results demonstrate that SAP15 is useful as an inflammatory regulator of biomaterials and can be developed as a therapeutic for the treatment of inflammation.

Octanoyl glycol chitosan enhances the proliferation and differentiation of tonsil-derived mesenchymal stem cells.

Biofunctional polymers have been widely used to enhance the proliferation and functionality of stem cells. Here, we report the development of a new biofunctional polymer, octanoyl glycol chitosan (OGC), and demonstrate its effects on the cell cycle and stem cell function using tonsil-derived mesenchymal stem cells (TMSCs). OGC treatment (100 µg/mL) significantly increased the proliferation of TMSCs, which could be attributed to cyclin D1 up-regulation in the G1 phase of the cell cycle. Additionally, OGC enhanced the ability of TMSCs to differentiate into adipocytes, chondrocytes, and osteoblasts. Taken together, this new biofunctional polymer, OGC, can promote stemness and osteogenesis, as well as induce stem cell proliferation by enhancing the intracellular metabolic rate and regulating the cell cycle. Thus, in the future, OGC could be a potential therapeutic additive for improving stem cell function.

Association between the microbiomes of tonsil and saliva samples isolated from pediatric patients subjected to tonsillectomy for the treatment of tonsillar hyperplasia.

Oral microbes have the capacity to spread throughout the gastrointestinal system and are strongly associated with multiple diseases. Given that tonsils are located between the oral cavity and the laryngopharynx at the gateway of the alimentary and respiratory tracts, tonsillar tissue may also be affected by microbiota from both the oral cavity (saliva) and the alimentary tract. Here, we analyzed the distribution and association of the microbial communities in the saliva and tonsils of Korean children subjected to tonsillectomy because of tonsil hyperplasia (n = 29). The microbiome profiles of saliva and tonsils were established via 16S rRNA gene sequencing. Based on the alpha diversity indices, the microbial communities of the two groups showed high similarities. According to Spearman's ranking correlation analysis, the distribution of Treponema, the causative bacterium of periodontitis, in saliva and tonsils was found to have a significant positive correlation. Two representative microbes, Prevotella in saliva and Alloprevotella in tonsils, were negatively correlated, while Treponema 2 showed a strong positive correlation between saliva and tonsils. Taken together, strong similarities in the microbial communities of the tonsils and saliva are evident in terms of diversity and composition. The saliva microbiome is expected to significantly affect the tonsil microbiome. Furthermore, we suggest that our study creates an opportunity for tonsillar microbiome research to facilitate the development of novel microbiome-based therapeutic strategies.

A transcriptomic analysis of serial-cultured, tonsil-derived mesenchymal stem cells reveals decreased integrin α3 protein as a potential biomarker of senescent cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have been widely used for stem cell therapy, and serial passage of stem cells is often required to obtain sufficient cell numbers for practical applications in regenerative medicine. A long-term serial cell expansion can potentially induce replicative senescence, which leads to a progressive decline in stem cell function and stemness, losing multipotent characteristics. To improve the therapeutic efficiency of stem cell therapy, it would be important to identify specific biomarkers for senescent cells. METHODS: Tonsil-derived mesenchymal stem cells (TMSCs) with 20-25 passages were designated as culture-aged TMSCs, and their mesodermal differentiation potentials as well as markers of senescence and stemness were compared with the control TMSCs passaged up to 8 times at the most (designated as young). A whole-genome analysis was used to identify novel regulatory factors that distinguish between the culture-aged and control TMSCs. The identified markers of replicative senescence were validated using Western blot analyses. RESULTS: The culture-aged TMSCs showed longer doubling time compared to control TMSCs and had higher expression of senescence-associated (SA)-ß-gal staining but lower expression of the stemness protein markers, including Nanog, Oct4, and Sox2 with decreased adipogenic, osteogenic, and chondrogenic differentiation potentials. Microarray analyses identified a total of 18,614 differentially expressed genes between the culture-aged and control TMSCs. The differentially expressed genes were classified into the Gene Ontology categories of cellular component (CC), functional component (FC), and biological process (BP) using KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. This analysis revealed that those genes associated with CC and BP showed the most significant difference between the culture-aged and control TMSCs. The genes related to extracellular matrix-receptor interactions were also shown to be significantly different (p < 0.001). We also found that culture-aged TMSCs had decreased expressions of integrin α3 (ITGA3) and phosphorylated AKT protein (p-AKT-Ser473) compared to the control TMSCs. CONCLUSIONS: Our data suggest that activation of ECM-receptor signaling, specifically involved with integrin family-mediated activation of the intracellular cell survival-signaling molecule AKT, can regulate stem cell senescence in TMSCs. Among these identified factors, ITGA3 was found to be a representative biomarker of the senescent TMSCs. Exclusion of the TMSCs with the senescent TMSC markers in this study could potentially increase the therapeutic efficacy of TMSCs in clinical applications.

A Synthetic Cell-Penetrating Heparin-Binding Peptide Derived from BMP4 with Anti-Inflammatory and Chondrogenic Functions for the Treatment of Arthritis.

We report dual therapeutic effects of a synthetic heparin-binding peptide (HBP) corresponding to residues 15-24 of the heparin binding site in BMP4 in a collagen-induced rheumatic arthritis model (CIA) for the first time. The cell penetrating capacity of HBP led to improved cartilage recovery and anti-inflammatory effects via down-regulation of the iNOS-IFNγ-IL6 signaling pathway in inflamed RAW264.7 cells. Both arthritis and paw swelling scores were significantly improved following HBP injection into CIA model mice. Anti-rheumatic effects were accelerated upon combined treatment with Enbrel® and HBP. Serum IFNγ and IL6 concentrations were markedly reduced following intraperitoneal HBP injection in CIA mice. The anti-rheumatic effects of HBP in mice were similar to those of Enbrel®. Furthermore, the combination of Enbrel® and HBP induced similar anti-rheumatic and anti-inflammatory effects as Enbrel®. We further investigated the effect of HBP on damaged chondrocytes in CIA mice. Regenerative capacity of HBP was confirmed based on increased expression of chondrocyte biomarker genes, including aggrecan, collagen type II and TNFα, in adult human knee chondrocytes. These findings collectively support the utility of our cell-permeable bifunctional HBP with anti-inflammatory and chondrogenic properties as a potential source of therapeutic agents for degenerative inflammatory diseases.

Optimization of Microenvironments Inducing Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Endothelial Cell-Like Cells.

Background: Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments. Methods: TMSCs were preconditioned with Dulbecco's Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4 days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers. Results: Preconditioning TMSCs in EGM for 4 days and then replating them on Matrigel induced the formation of a capillary-like network in 3 h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells. Conclusion: This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4 days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.

Application of Tonsil-Derived Mesenchymal Stem Cells in Tissue Regeneration: Concise Review.

Since the discovery of stem cells and multipotency characteristics of mesenchymal stem cells (MSCs), there has been tremendous development in regenerative medicine. MSCs derived from bone marrow have been widely used in various research applications, yet there are limitations such as invasiveness of obtaining samples, low yield and proliferation rate, and questions regarding their practicality in clinical applications. Some have suggested that MSCs from other sources, specifically those derived from palatine tonsil tissues, that is, tonsil-derived MSCs (TMSCs), could be considered as a new potential therapeutic tool in regenerative medicine due to their superior proliferation rate and differentiation capabilities with low immunogenicity and ease of obtaining. Several studies have determined that TMSCs have differentiation potential not only into the mesodermal lineage but also into the endodermal as well as ectodermal lineages, expanding their potential usage and placing them as an appealing option to consider for future studies in regenerative medicine. In this review, the differentiation capacities of TMSCs and their therapeutic competencies from past studies are addressed. Stem Cells 2019;37:1252-1260.

Identification of cell-penetrating osteogenic peptide from copine-7 protein and its delivery system for enhanced bone formation.

Peptide and proteins are recognized as highly selective and therapeutically active biomaterials, as well as relatively safe in clinical application. A calcium phospholipid-binding protein, copine 7 (CPNE7), has been recently identified to induce hard tissue regeneration, including bone and dentin by internalizing into the cells. However, the clinical application of the full length of CPNE7 has limited due to its large size with short half-life. Herein, as an alternative to CPNE7, six bioactive synthetic peptides are designed from CPNE7 (CPNE7-derived peptides, CDP1-CDP6) and investigated their osteogenic potential. Among the CDPs, CDP4 have the highest level of cell-penetrating activity as well as osteogenic efficiency in dental pulp stem cells (DPSCs). CDP4 increased the expression of osteogenesis-related genes and proteins, which was comparable to that by BMP-2. The cell penetration capacity of CDP4 may synergistically induce the osteogenic potential of DPSCs. Moreover, the implantation of the mixture of CDP4 with injectable collagen gel increased bone formation with recovery in the mouse calvarial defect model, comparable to full-length CPNE7 and even BMP-2. In conclusion, these results suggest that our synthetic peptide, CDP4, can be applied in combination with biomaterial to provide high osteogenic efficacy in the field of bone tissue engineering.