Osteoclasts adapt to physioxia perturbation through DNA demethylation

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.Nishikawa K., Seno S., Yoshihara T., et al. Osteoclasts adapt to physioxia perturbation through DNA demethylation. EMBO Reports 22, e53035 (2021); https://doi.org/10.15252/embr.202153035

Efficient and Scalable Purification of Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells by VCAM1 Surface Expression

RATIONALE: Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs. METHOD AND RESULT: We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10×10(5) VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines. CONCLUSION: We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy

Biological modification of tooth surface by laser-based apatite coating techniques

Background: Development of new clinical regenerative procedures is needed for the reconstruction of the connective tissue attachment lost to periodontal disease. Apatite coating on the affected root surfaces could improve root surface biocompatibility and promote the reestablishment of connective tissue attachment. Highlight: We developed two novel techniques that use laser light for coating the tooth surface with apatite. In the laser-assisted biomimetic (LAB) process, a tooth substrate was placed in a supersaturated calcium phosphate solution and irradiated for 30 min with low-energy pulsed laser light. Due to the laser-assisted pseudo-biomineralization, a submicron-thick apatite film was created on the laser-irradiated tooth surface. Furthermore, we created a fluoride-incorporated apatite film on the tooth surface using the LAB process and demonstrated its antibacterial activity against Streptococcus mutans. In the laser-induced forward transfer with optical stamp (LIFTOP) process, a thin apatite film loaded with the cell-adhesion protein, fibronectin, was prepared beforehand as a raw material on the optical stamp (carbon- and polydimethylsiloxane-coated support) by a conventional biomimetic process. After irradiation with a single laser pulse, the film (microchip) was transferred onto a tooth substrate via laser ablation of the carbon sacrificial layer. The LIFTOP process requires only a short processing time and has a minimal heat effect on the film; thus, the film exhibits cell adhesion activity even after the LIFTOP process. Conclusion: The LAB and LIFTOP processes have the potential as novel tools for tooth surface modification in the treatment of periodontal disease

Laser-Induced Forward Transfer with Optical Stamp of a Protein-Immobilized Calcium Phosphate Film Prepared by Biomimetic Process to a Human Dentin

The rapid and area-specific printing of calcium phosphate with superior biocompatibility and osteoconductivity is a useful technique for the surface functionalization of biomedical devices. We recently demonstrated the laser-induced forward transfer (LIFT) of a brittle calcium phosphate film onto a soft and shock-absorbing polydimethylsiloxane (PDMS) substrate. In this work, a new LIFT using an optically transparent PDMS-coated stamp, which we hereafter call LIFT with optical stamp (LIFTOP), was introduced to achieve the transfer of brittle films to harder substrates. Cell adhesion protein fibronectin-immobilized calcium phosphate films (Fn-CaP) were prepared on the optical stamp through a biomimetic process. Then, the irradiation of a single laser pulse transferred the Fn-CaP film from the optical stamp onto relatively hard substrates, polyethylene terephthalate and human dentin. As a result of this LIFTOP process, Fn-CaP microchips with a shape corresponding to the laser beam spot were printed on the substrates. Cross-sectional observation of the interface between the Fn-CaP microchip and the dentin substrate revealed good attachment between them without obvious gaps for the most part

High Immobilization Efficiency of Basic Protein within Heparin-Immobilized Calcium Phosphate Nanoparticles

Previously, we achieved one-pot fabrication of heparin-immobilized calcium phosphate (CaP) nanoparticles with high dispersibility by a precipitation process in a highly supersaturated reaction solution. In this study, we revealed that the heparin-immobilized CaP nanoparticles have a greater co-immobilizing capacity for basic proteins than for acidic proteins. In this process, heparin acted as not only a particle-dispersing agent but also as an immobilizing agent for basic proteins; it remarkably (approximately three-fold) improved the immobilization efficiency of cytochrome C (a model basic protein) within the CaP nanoparticles. The content of cytochrome C immobilized within the nanoparticles was increased with an increase in cytochrome C concentration in the reaction solution and by aging the nanoparticles. The obtained nanoparticles were dispersed well in water owing to their large negative zeta potentials derived from heparin, irrespective of the content of cytochrome C. Similar results were obtained also for another basic protein, lysozyme, but not for an acidic protein, albumin; the immobilization efficiency of albumin within the nanoparticles was decreased by heparin. These findings provide new insights into the co-immobilization strategy of proteins within heparin-immobilized CaP nanoparticles and will be useful in the design and fabrication of nanocarriers for protein delivery applications

Bioactive micropatterning of apatite immobilizing cell adhesion protein by laser-induced forward transfer with a shock absorber

The additive patterning of apatite with good biocompatibility and osteoconductivity is a useful technique for the production and surface functionalization of biomaterials. We developed this technique through our laser-induced forward transfer (LIFT) process using a laser-absorbing sacrificial layer in combination with a shock-absorbing polydimethylsiloxane (PDMS) receiver. With the PDMS shock-absorbing function, even the brittle apatite and that immobilizing the cell adhesion protein fibronectin (Fn-apatite) were successfully transferred and micropatterned while maintaining their dense, filmy state. The laser pulse energy effect was investigated, leading to the optimum energy range just above the transfer threshold. The apatite and Fn-apatite micropatterns exhibited superior cytocompatibility compared to the PDMS surface, and could potentially be used for cellular micromanipulation. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen