Shuntaro Yamada

Dental pulp stem cells (DPSCs), a subset of mesenchymal stem/progenitor cells, exhibit remarkable self‐renewal and multipotent differentiation capacity, making them promising candidates for regenerative applications. While their in vitro characteristics have been extensively studied, the in vivo dynamics and molecular regulation during development are much less known. This study examined the embryonic/prenatal (E16.5) and early postnatal (P3.5 and P7.5) stages of mouse molar development to trace the temporospatial expression and overlap of five key molecules associated with DPSC: a trio of positive markers (CD73/ Nt5e , CD90/ Thy1 , and CD105/ Eng ) and two negative markers (CD34/ Cd34 and CD45/ Ptprc ). Reanalysis of single‐cell RNA sequencing data revealed a triple‐positive and double‐negative population overlapping in the dental mesenchyme by P7.5. The results were confirmed by multiple immunofluorescence‐based in situ labeling. These findings contribute to the understanding of the spatiotemporal in vivo expression landscape and dynamics of DPSC markers, thus bridging the gap between in vitro characterization and the complex developmental reality of the dental pulp.

Background: Mesenchymal stromal cell-derived conditioned medium (MSC-CM) represents a promising cell-free approach for regenerative medicine; however, variability in production conditions remains a major challenge for standardization. This study investigated how serum-free conditioning duration influences the proteomic composition and regenerative activity of human dental pulp stem cell-conditioned medium (DPSC-CM). Methods: DPSC-CM was collected after 24, 48, and 72 h of serum-free conditioning and characterized by label-free LC-MS/MS proteomics using donor-blocked differential abundance analysis. DPSC viability and apoptosis were assessed by Annexin V/PI flow cytometry. The angiogenic activity of CM 48 h and CM 72 h was evaluated using the ex ovo chick chorioallantoic membrane (CAM) assay, while all CM groups were assessed for their effects on human exfoliated deciduous teeth (SHED) metabolic activity and osteogenic differentiation. Osteogenesis was evaluated by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. PI3K/Akt signaling was investigated for CM 48 h by Western blotting. Results: Serum-free conditioning for up to 72 h did not significantly affect DPSC viability or apoptosis. Proteomic analysis identified 1562 proteins. Differential abundance analysis identified 914 differentially abundant proteins (DAPs) between CM 48 h and CM 24 h and 745 between CM 72 h and CM 24 h, whereas no DAPs were detected between CM 72 h and CM 48 h. Enriched biological processes included extracellular matrix organization, cell-substrate adhesion, cytoskeletal organization, protein folding, and angiogenesis-related pathways. In the CAM assay, CM 48 h in-creased total vessel area, whereas CM 72 h increased total vessel length. CM 48 h en-hanced SHED metabolic activity at day 3. Although ALP staining did not differ significantly among groups, CM 48 h produced greater matrix mineralization than CM 24 h. PI3K inhibition reduced Akt phosphorylation, whereas CM 48 h had no significant effect on the p-Akt/Akt ratio. Conclusion: Serum-free conditioning duration influences DPSC-CM composition and biological activity, with the major proteomic transition occurring between 24 and 48 h. Among the conditioning periods evaluated, 48 h demonstrated the most consistent combination of proteomic and functional effects, supporting further investigation as a candidate conditioning duration for DPSC-CM production.

Dental stem cells represent an accessible adult stem cell source with neuronal affinity due to their cranial neural crest origin, yet dental-derived neural-like spheroid models often suffer from heterogeneous differentiation, limited organization, and poor reproducibility. Here, we established a stepwise workflow to induce neurogenic differentiation of dental pulp stem cells (DPSCs) and organize them into spatially controlled, depolarization-responsive neural-like spheroids using a micropatterned culture platform. Seven-day neurogenic induction generated Neuro-DPSCs with marked morphological remodeling, neurite-like processes, and increased expression of neural lineage markers including TUJ1, NSE, MAP2, NeuN, and NEFM. In 3D culture, Neuro-DPSCs self-assembled into spheroidal aggregates, while substrate wettability strongly influenced aggregation and outgrowth. Hydrophobic surfaces promoted spheroid formation but limited long-term maintenance due to free-floating aggregation. To improve spatial control, a micropatterned platform was applied to define adhesive contact points, constrain spheroid positioning, and guide inter-spheroid neurite-like network formation. The resulting spheroids showed enrichment of gene networks associated with neurite-like organization, axon guidance, synaptic vesicle biology, neurotransmitter-related pathways, and receptor signaling, together with KCl-induced depolarization-responsive calcium activity. These findings demonstrate that neurogenic priming combined with micropattern-guided assembly provides a controllable workflow for generating spatially organized neural-like DPSC spheroids.

Estrogen receptor positive (ER+) breast cancer is primarily treated with endocrine therapies targeting ERα signaling. Although endocrine therapy has substantially improved survival in ER+ breast cancer, metastatic disease remains largely incurable, underscoring the need to elucidate additional mechanisms driving growth and proliferation. Here, we show that the homeobox protein IRX3 is selectively overexpressed in ER+ breast cancer and define the molecular function of IRX3 in ER+ breast cancer using an integrated combination of in vitro , in vivo and in silico approaches. We uncover a previously uncharacterized distal regulatory region that controls IRX3 transcription via ERα and associated steroid receptor coactivators. Consistent with this regulatory axis, anti-estrogen treatment resulted in marked downregulation of cellular IRX3 levels. Functionally, depletion of IRX3 suppresses proliferation of the human ER+ breast cancer cells in vitro, but paradoxically promotes tumor growth and metastatic dissemination in orthotopic xenografts in vivo by stimulating enhanced tumor vascularization. Finally, low tumor expression of IRX3 correlates with poorer survival outcomes in patients with ER+ breast cancer. Collectively, these findings establish IRX3 as an important regulator of ER+ breast tumor biology and reveal an ERα-dependent role for IRX3 in modulating proliferative and vascular programs in tumor progression. Significance: By identifying a novel ERα-dependent regulatory pathway, this work refines our understanding of how hormone signaling shapes both breast tumor growth and the surrounding microenvironment.

Functional scaffolds, especially decorated with nanoparticles advance bone tissue engineering. While the immune system plays an instrumental role in regulating the outcome of bone regeneration, the effects of differently functionalized nanoparticles on immune cells are poorly understood. In this study, we investigate the effect of biocompatible surface functionalizations and scaffold immobilization of nanodiamond particles (nDP), a promising biomaterial for bone tissue engineering. Six different nDP were tested on human monocyte-derived macrophages (Mo-Mø) to promote osteo-immunomodulatory and angiogenic responses for bone regeneration. Our results showed that nDP surface functionalization modulated macrophage polarization diversely, demonstrating the influence of charged surface groups, H-bonding motifs and terminal groups on the interaction of nDP. Specifically, charged nDP derivatives inducing a M1-like (pro-inflammatory) secretome profile showed pronounced osteogenic effects, whereas biotin-nDP showed an angiogenic effect, and a reduced inflammatory profile. When incorporated onto PCL scaffolds, the immunomodulatory properties of functionalized nDP were preserved, albeit with attenuated intensity. The distinct effects of different surface moieties can be used for tunable scaffold designs with spatiotemporally distributed modes of action for a next generation of biomaterials for bone tissue engineering

Aim: This study aimed to evaluate whether human dental pulp stem cells (DPSCs), after long‐term biobanking (7–8 years), retain their pro‐angiogenic properties and can be used to engineer vascularised tissues, addressing their potential for clinical translation in regenerative dentistry. Methodology: Cryopreserved DPSCs from adolescent donors were recovered from biobanking and characterised for chromosomal integrity, MSC immunophenotype and multipotency. After conditioning in pro‐angiogenic conditions in vitro, gene and protein expression were analysed by RT‐qPCR array, flow cytometry and high‐throughput immunophenotyping. Functional angiogenic capacity was assessed via in vitro tube formation, ex ovo CAM implantation assay, organ‐on‐chip perfusion model and long‐term culture (45 days) in clinical‐grade GelMA hydrogels, with and without HUVECs. Results: Biobanked DPSCs retained MSC identity and multi‐lineage differentiation potential. Pro‐angiogenic/endothelial conditioning enhanced the expression of angiogenic/endothelial genes (PECAM1, VEGFR2, NRP1, ACE), yet most cells maintained a pericyte‐like phenotype. Both naive and endothelial‐conditioned DPSCs (i.e., naiveDPSCs and endoDPSCs, respectively) significantly enhanced vascular ingrowth in the CAM model. In the organ‐on‐chip system, naiveDPSCs formed perfusable vasculature with HUVECs and differentiated into perivascular cell types. Most notably, endoDPSCs alone successfully generated vascularised tissue with both CD31(+) and αSMA(+) cells present in GelMA hydrogels after prolonged stimulation. Conclusion: Long‐term biobanked DPSCs preserve their angiogenic potential and, following extended endothelial induction, can independently generate vascularised tissue in 3D in vitro culture models. This is the first report demonstrating the comprehensive pro‐angiogenic characterisation and the feasibility of using biobanked DPSCs for vascularised tissue engineering, highlighting their strong clinical applicability for future regenerative therapies.

IRX3 is linked to predisposition to obesity through the FTO locus and is upregulated during early adipogenesis in risk-allele carriers, shifting adipocyte fate toward fat storage. However, how this elevated IRX3 expression influences later developmental stages remains unclear. Here we show that IRX3 regulates adipocyte fate by modulating epigenetic reprogramming. ChIP-sequencing in preadipocytes identifies over 300 IRX3 binding sites, predominantly at promoters of genes involved in SUMOylation and chromatin remodeling. IRX3 knockout alters expression of SUMO pathway genes, increases global SUMOylation, and inhibits PPARγ activity and adipogenesis. Pharmacological SUMOylation inhibition rescues these effects. IRX3 KO also reduces SUMO occupancy at Wnt-related genes, enhancing Wnt signaling and promoting osteogenic fate in 3D cultures. This fate switch is partially reversible by SUMOylation inhibition. We identify IRX3 as a key transcriptional regulator of epigenetic programs, acting upstream of SUMOylation to maintain mesenchymal identity and support adipogenesis while suppressing osteogenesis in mouse embryonic fibroblasts.

Periodontal and craniofacial regeneration presents significant challenges owing to the complex tissue architecture, inadequate vascularization, and diminished stem cell populations within damaged tissues. Traditionally, autologous bone grafts or alternative bone substitute materials have been employed to address these conditions; however, these approaches are constrained by donor site morbidity, limited availability, and suboptimal regenerative efficacy. The advancement of mesenchymal stem/stromal cell (MSC) biology has accelerated the development of cell‐based therapies in modern dentistry, which now focuses on biologically driven approaches to regenerate tissues. MSC‐based therapies currently under investigation, both preclinically and clinically, show promise for improving tissue integration and healing processes of both soft and hard tissues, attributable to their multipotent nature, immunomodulatory properties, and paracrine signaling capabilities. Nevertheless, obstacles persist, including inconsistent standardization, limited scalability, regulatory hurdles, a paucity of controlled studies, and restricted biomaterial options. This review evaluates MSC‐based treatments for periodontal and craniofacial reconstruction by discussing recent research findings and existing obstacles. This review also examines future prospects, such as advanced biofabrication methods, including 3D printing and bioprinting, which have the potential to improve personalized cell therapy for periodontal and craniofacial regeneration.