Most cellular and physiological activities are temporally organized, with many following robust circadian oscillations over the 24-h day. Mineralized dental tissues formation during odontogenesis provide an especially tractable model to study such temporal control, as they display clear daily patterns of cell behavior and matrix accretion. In this pilot study, the temporal dimension of odontogenesis was examined using two complementary circadian knockout (KO) mouse models that exhibit opposing chronotypes; Cryptochrome1 (Cry1) KO, characterized by a 1-h shortened circadian period, and Cryptochrome2 (Cry2) KO, characterized by a 1-h lengthened period. Our data revealed distinct chrono/genotype-phenotype coupling across the dental tissues examined. Cry2 targeting resulted in more pronounced changes with significantly augmented dentin/cementum deposition accompanied by constricted pulp chambers. In enamel, targeting the Cry genes resulted in less noticeable effects with an accelerated early enamel matrix deposition rate observed in Cry2 mutants, while enamel mineralization was slightly slowed down, whereas targeting Cry1 resulted in an opposite pattern, slowed down secretion, and precocious enamel mineralization. Collectively, our results strongly suggest that there is a correlation between circadian period length and dental matrix proteins deposition, whereby longer periods favor elevated secretion rates while shorter periods result in a slightly reduced output. On the other hand, no similar effects were noted in mineral content, as mature enamel, bone, and dentin mineral densities did not differ significantly across the two KO models. Overall, this work identifies the cryptochrome 2 gene as a potent regulator of dentinogenesis and mandibular bone remodeling with a limited impact on amelogenesis. It also highlights intrinsic biological time as a fundamental axis along which the architecture of mineralized craniofacial tissues is patterned.

Autophagy, an evolutionarily conserved and tightly controlled process in eukaryotic cells, allows them to respond to stress by selectively eliminating dysfunctional or unwanted materials to promote metabolic flexibility and maintain homeostasis. While autophagy is orchestrated by different autophagy (ATG)-related genes, whose regulation varies considerably according to tissue type and developmental stage. In this review, we investigate how regardless of the different players involved in autophagy, ATG5 emerges as a unique, highly conserved, critical molecule that acts as a central rheostat to control the stem cell fate, metabolic adaptability, and govern the immune signature pattern in cells. The journey of ATG5 modulation from physiological developmental variation to pathological scenario brings out the translational impact of ATG5. On the one hand, ATG5 promotes exit from the pluripotent state by c-Myc degradation during differentiation of specific lineages (involved in neurogenesis, adipogenesis, and hematopoiesis), while on the other hand, it has a critical involvement in metabolic circuitry via rewiring autophagy through modulation of lipophagy, mitophagy, and acetyl-CoA epigenetics. Taken together, this work summarizes new findings that centrally place ATG5 as a driver that engineers a coordinated crosstalk between the metabolic state and cell fate decisions and immune responses. These insights position ATG5 as a critical and therapeutic target for developmental disorders, cancer, and immune-metabolic diseases.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are emerging as potent cell-free mediators of tissue repair, whose composition and function can be tuned by the cellular microenvironment. Although inflammatory cues modulate mesenchymal stromal cell (MSC) behavior, how defined preconditioning strategies program extracellular vesicle (EV) functional outputs remains incompletely understood. Here, we systematically evaluated how priming bone marrow-derived MSCs with interferon-gamma/tumor necrosis factor-alpha (I/T) or lipopolysaccharide (LPS) generates EV populations with distinct immunomodulatory and regenerative properties. Using a murine full-thickness wound model, we performed an integrative analysis of biodistribution, immune response, and extracellular matrix (ECM) remodeling, complemented by single-cell, transcriptomic, and proteomic profiling. All EV populations were retained at the wound site following subcutaneous delivery and supported wound contraction; however, they drove distinct, treatment-specific repair trajectories. I/T-EVs promoted a coordinated regenerative response characterized by balanced macrophage (MΦ) activation, controlled immune modulation, and efficient resolution of inflammation, resulting in organized ECM remodeling. In contrast, LPS-EVs induced a more pro-inflammatory response, accelerating wound contraction and promoting compensatory matrix stiffening with reduced structural coordination. Control EVs primarily facilitated early immune resolution with limited induction of regenerative remodeling pathways. Proteomic profiling of EVs identified enrichment of proteins associated with insulin-like growth factor signaling, MΦ recruitment, and ECM remodeling, consistent with in vivo protein expression patterns and linking EV cargo to functional outcomes. These findings demonstrate that preconditioning does not uniformly enhance EV efficacy but instead selectively programs distinct MSC-EV functional states, establishing EV preconditioning as a tunable strategy for engineering cell-free therapeutics with predictable and context-specific therapeutic outcomes.

The cancer stem cell (CSC) paradigm has evolved from a rigid hierarchical model to a systems-level perspective in which stemness is a reversible and context-dependent phenotype. Evidence from lineage tracing and single-cell/spatial multiomics indicates that tumor cells occupy continuously evolving phenotypic states governed by complex gene regulatory networks. Within this landscape, CSCs can be interpreted as metastable attractors maintained through coupled signaling, epigenetic, metabolic, transcriptional, and microenvironmental interactions. Tumor heterogeneity and therapeutic resistance emerge through phenotypic reprogramming, regulatory network rewiring, and niche-dependent stabilization under environmental and therapeutic stress. This reframes resistance as an emergent property of tumor ecosystems, underscoring the limitations of targeting static CSC populations or single pathways. Therefore, durable therapeutic control will require network-oriented interventions capable of reshaping attractor topology and disrupting stemness-supportive microenvironments.

Focal segmental glomerulosclerosis (FSGS) is a major cause of nephrotic syndrome and end-stage kidney disease (ESKD). Many cases are attributable to pathogenic variants in podocyte-related genes, such as inverted formin 2 (INF2). However, no specific treatment exists for hereditary FSGS, and experimental platforms that faithfully model podocyte injury remain limited. Therefore, in this study, we developed an injury model by generating induced pluripotent stem cells (iPSCs) from a patient with INF2-associated FSGS and differentiating them into kidney organoids. Podocytes were validated by immunofluorescence staining for podocyte-specific markers. We induced podocyte injury in this model using puromycin aminonucleoside (PAN) in a dose-dependent manner. Injury severity was quantified by measuring podocalyxin fluorescence intensity. Cyclosporine A (CsA) or voclosporin (VCS) was administered as a 1-h pretreatment before PAN exposure to evaluate their podocyte-protective effects. The kidney organoids exhibited well-defined podocyte marker expression, confirming successful differentiation. PAN exposure caused a significant and concentration-dependent reduction in podocalyxin fluorescence, indicating robust induction of podocyte injury in organoids harboring the INF2 variant. Pretreatment with CsA or VCS significantly attenuated PAN-induced podocyte injury and preserved podocyte marker expression. CsA and VCS reduced podocyte injury to similar extents. In conclusion, we established a patient-specific kidney organoid model of INF2-associated FSGS that reliably recapitulated podocyte vulnerability to toxic injury. This platform demonstrated that calcineurin inhibitors, including the novel agent VCS, exert direct podocyte-protective effects in a genetic FSGS background and provide a practical system for mechanistic studies and therapeutic screening.

Aneuploidy in cultured embryonic stem (ES) cells severely limits research and therapeutic potential. To begin to understand when chromosomal imbalances arise, primary ES cell lines were derived in feeder-free, fully defined culture media from 44% of two-cell CF1 embryos (5 lines), 37% of CF1 multi-drug resistance (Mdr1/Abcb1) mutant embryos (19 lines), and 2 parthenogenetic stem (mPS) lines from Mdr1 mutant eggs for array comparative genomic hybridization analysis. By passage 2, trivial somatic chromosome euploidy was detected in all wild-type lines and in 9 (64%) of the 14 mutant cell lines. Five Mdr1 mutant ES cell lines and both mPS cell lines exhibited mosaic gain of chromosome 8 in at least 30% of cells, a common finding in mouse ES cells. X chromosomes exhibited more aneuploidy than somatic chromosomes. Five of the Mdr1 mutant lines were male, while the nine female ES cell lines and both mPS lines exhibited almost complete loss of one X chromosome. Two ES lines analyzed at passage 3 exhibited partial gains and losses in chromosomes 15 and 19, suggesting that aneuploidy in murine ES cells begins by passage 3 in standard culture conditions. The somatic cell euploidy in early passage wild-type CF1 ES lines, in contrast to the early accumulation of chromosome 8 triploidy in the Mdr1 mutant lines, suggests that component(s) of the culture system not well tolerated by ES cells with the Mdr1 mutation may be responsible for triploid chromosome 8 in multipassaged mouse ES cells. The ease of deriving euploid mouse stem cell lines in fully defined culture conditions from individual females encourages the use of primary ES cell lines for a variety of purposes. Adjusting culture components to correct early chromosome 8 accumulation in primary Mdr1 mutant lines may reveal pathways critical to maintaining euploidy in cultured ES cells.

Hematopoietic stem cell (HSC) transplantation is an established therapy for malignant and nonmalignant hematologic disorders; however, clinical application remains constrained by limited graft availability and challenges in maintaining stemness during ex vivo manipulation, as well as transplant-related complications. Accordingly, alternative strategies to generate hematopoietic-competent cells from accessible stem cell sources are being actively explored. This study investigated whether OCT3/4 overexpression enhances the hematopoietic trans-differentiation potential of dental tissue-derived mesenchymal stem cells (DMSCs) and whether the resulting HSC-like cells exert therapeutic effects in a cyclophosphamide-induced myelosuppressed mouse model following intra-femoral delivery. OCT3/4 was introduced into DMSCs using the Neon transfection system, and OCT3/4-overexpressing DMSCs (DMSCs[OCT3/4+]) were subsequently exposed to hematopoietic cytokines to induce an HSC-like phenotype. Cytokine-treated DMSCs[OCT3/4+] exhibited a rounded morphology, increased expression of HSC-associated surface markers (CD34 and CD45), and upregulated hematopoietic transcription factors, including GATA2, C/EBPα, RUNX1, and SCL. The derived HSC-like cells (D-HSCs) were transplanted into the femoral bone marrow cavity of myelosuppressed mice, and therapeutic outcomes were assessed by complete blood counts and histological analyses. D-HSC transplantation was associated with recovery of bone marrow cellularity and partial restoration of spleen and thymus cellularity and size, accompanied by improvement in body weight and peripheral blood parameters compared with myelosuppressed controls. Collectively, these findings indicate that OCT3/4-enhanced DMSCs can be directed toward an HSC-like state under hematopoietic cues and that the resulting cells may support hematopoietic and immune recovery in myelosuppressed hosts, supporting their potential as an alternative, autologous cell source for hematopoietic regeneration.

Hematopoietic stem cells (HSCs) are multipotent stem cells capable of differentiating into all types of blood and immune progeny and endowed with self-renewal capacity to enable lifelong hematopoiesis. Based on these unique characteristics, HSCs are utilized as a cell source for cell and gene therapies. During its dawning period, HSC-based therapies faced significant challenges due to inefficient gene transfer and insertional leukemogenesis. However, technological advances, such as the use of HIV-derived lentiviral vectors and cellular promoters, have established HSC gene therapy as a powerful treatment modality for patients with congenital monogenic diseases, leading to approved therapies commercially available in Europe and the United States. HSC-based therapies are now being explored for broader indications, including cancer, autoimmune, and infectious diseases. Innovative concepts achievable with HSCs-such as delivering therapeutic proteins to hard-to-reach tissues, in vivo delivery of antibodies and immune cells, and molecular shielding-have been proposed, offering new therapeutic approaches. Moreover, technological innovations in related fields, including more precise gene expression control and reduced-toxicity bone marrow conditioning, are expanding the range of applications. HSC-based cell and gene therapies are therefore evolving into a therapeutic modality applicable beyond monogenic diseases to a broader range of indications, to provide therapeutic value to patients with intractable diseases.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease lacking effective therapies. Mesenchymal stem cells (MSCs) show therapeutic potential; however, their efficacy is often limited. Aggregates of human umbilical cord-derived MSCs (hUC-MSCs) were generated using AggreWell™ 400 plates. The paracrine and mechanotransduction profiles of three-dimensional (3D)-MSCs were assessed by reverse transcription quantitative PCR and western blot. MASH was induced in C57BL/6J mice via a 32-week high-fat, high-fructose, and high-cholesterol (HFFC) diet, followed by tail vein injection of PBS, two-dimensional (2D)-MSCs, or 3D-MSCs. Therapeutic efficacy was evaluated via histological staining, immunohistochemistry, and serum biochemistry. RNA sequencing was performed to elucidate underlying molecular mechanisms. Under 3D culture conditions, hUC-MSCs formed relatively uniform aggregates with preserved MSC phenotypic characteristics after recovery. 3D aggregate culture enhanced the paracrine, anti-inflammatory, mechanosensitive phenotype, pro-survival, and matrix-remodeling properties of hUC-MSCs, evidenced by increased expression of hepatocyte growth factor, tumor necrosis factor-stimulated gene 6, cyclooxygenase-2, prostaglandin E synthase, B-cell lymphoma-2, and mechanotransduction-related genes, accompanied by elevated matrix metalloproteinase-2 (MMP2) and MMP9 expression in vitro. In vivo fluorescence imaging showed no significant difference in early hepatic retention between intravenously infused single-cell hUC-MSCs and 3D aggregates. In the MASH model, 3D-MSCs more effectively reduced lipid accumulation, collagen deposition, α-smooth muscle actin, hepatomegaly, and serum total cholesterol levels compared with 2D-MSCs. Moreover, 3D-MSCs enhanced the expression of anti-inflammatory interleukin-10, while suppressing inducible nitric oxide synthase. 3D-MSC therapy induced broader transcriptional remodeling than conventional MSCs, with enrichment in extracellular matrix organization, focal adhesion, and the phosphatidylinositol 3-kinase-protein kinase B signaling pathway. Consistently, western blot analysis showed that 3D-MSCs more effectively reduced hepatic p-PI3K and p-AKT levels, indicating stronger inhibition of PI3K/AKT pathway activation. 3D aggregate culture enhances the therapeutic efficacy of hUC-MSCs against MASH, supporting its translational potential for MSC-based liver therapy.

In pancreatic cancer, increased collagen I impairs the efficacy of gemcitabine; however, the role of gemcitabine itself in collagen I accumulation remains unclear. This study aims to explore the mechanism of gemcitabine-induced fibrosis and provide new insights to enhance its therapeutic efficacy. We analyzed COL1A1 expression in pancreatic cancer patient tumor tissues and found that gemcitabine treatment upregulated COL1A1 expression. Subsequently, cancer-associated fibroblasts (CAFs) were modeled by inducing human adipose-derived mesenchymal stem cells with tumor-derived exosomes. Using the autophagy inhibitor chloroquine (CQ) and the protein kinase B (AKT) activator SC79, we demonstrated that gemcitabine downregulated P62 expression and upregulated LC3BII, Beclin-1 expression, inducing autophagy in CAFs via decreasing AKT phosphorylation, which further led to collagen I accumulation. In addition, gemcitabine combined with CQ enhanced cell death in both CAFs and tumor cells, while inhibiting tumor cell proliferation and migration. In animal models, this combination therapy reduced gemcitabine-induced autophagy and collagen I deposition, contributing to delayed tumor growth. Collectively, gemcitabine upregulates collagen I by inducing CAF autophagy via reducing AKT phosphorylation. Targeting CAF autophagy can reduce collagen deposition, offering a promising strategy to improve the therapeutic efficacy of gemcitabine in pancreatic cancer.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into several cell types that hold great promise for therapeutic applications. However, the maintenance of proliferative and stemness capacity following in vitro expansion remains a significant challenge. Triiodothyronine (T3) plays a crucial role in embryogenesis and fetal development, yet the knowledge of its effects on MSCs' survival and function is limited. Here, we investigate the impact of T3 treatment in bone marrow (BM)-MSCs and adipose tissue (AT)-MSCs isolated from C57BL/6J mice, to assess stemness preservation, proliferation, and gene expression during in vitro expansion. To this end, MSCs were treated with T3 at various concentrations for 24 and 48 h, and thyroid hormone-responsive and stemness-related genes expression, proliferation, clonogenic potential, and surface marker profiles were analyzed using reverse transcript quantitative PCR, Cell Counting Kit-8, colony-forming unit-fibroblast assays, and flow cytometry. Our results show that T3 exposure did not affect variability or clonogenic potential of BM-MSCs and AT-MSCs, and the expression of T3-responsive genes is activated by distinct time- and dose-dependent responses to T3 in AT-MSCs and BM-MSCs. However, in BM-MSCs, a transient increase in pluripotent markers was observed. Conversely, AT-MSCs exhibited sustained increases in Nano-g, Sca-1, and Ssea-1, particularly at 10 and 100 nM. Collectively, we observed that T3 exposure during in vitro expansion enhanced stemness features in MSCs. This finding was more prominent in AT-MSCs compared with BM-MSCs. The data suggest that T3 exposure during AT-MSCs expansion could be a valuable tool to increase the yield and stemness of these MSCs, facilitating their therapeutic use.

Human embryonic stem cell (hESC) can be differentiated into definitive endoderm (DE) through multiple branching lineage choices. Although different DE differentiation methods have been established, there are still several limitations, such as the yield of heterogeneous cell populations containing undifferentiated or non-DE cells. Therefore, this study aimed to suppress the alternate fates at branch points and establish a robust and highly efficient differentiation protocol for hESC-derived hepatocytes (hESC-Heps). We developed a two-step DE induction protocol. First, hESCs were treated with a GSK-3α/β inhibitor and an mTOR inhibitor combined with TGF-β activation to generate an anterior primitive streak (progenitor to endoderm). Subsequently, a BMP inhibitor combined with TGF-β activation was used to abolish the mesoderm lineage. The resulting DE cells were further differentiated into hESC-Heps to evaluate their functionality. By regulating the branching lineage choices, we established an efficient two-step method that yielded up to 96% DE cells with minimal expression of pluripotency and mesodermal markers. Notably, this method reduced the dosage of Activin A, which makes it cost-effective for future applications. The derived hESC-Heps exhibited mature hepatocyte characteristics, including glycogen storage, indocyanine green uptake, and cytochrome P450 activity. Additionally, these cells demonstrated robust liver-specific functions such as sensitive innate immune responses and permissiveness to hepatitis B virus infection. In summary, we developed a novel and cost-effective method that achieves high-purity DE by precisely modulating cell fate decisions in the early stages. The derived hESC-Heps can serve as a model for further studies, such as host-virus interaction and hepatotoxicity testing.

Regenerative medicine for stroke patients has been attracting attention. However, the effects of rehabilitation after the cell transplantation have not been fully elucidated. The purpose of the present study was to investigate whether intensive gait-focused rehabilitation using a robotic orthosis after regenerative medicine improved gait function and induced plastic changes in cortical networks. The present study was conducted in a retrospective cohort study. We selected seven chronic stroke patients, those who had undergone adipose-derived mesenchymal stem cells (MSC) transplantation therapy after the onset of stroke and had been receiving adequate subsequent gait rehabilitation with a robot for more than 2 months. During hospitalization, each patient received at least 2 h of rehabilitation, including robotic-assisted gait training more than five times per week. As the assessments, gait performance and M1 seed-based resting state-functional connectivity (rs-FC) obtained by a magnetoencephalography were compared before and after hospitalization. After rehabilitation, cadence and spatial gait symmetry ratio were significantly improved, and a significant negative correlation was found between the changes in the gait symmetry ratio and the time from transplant to rehabilitation. Seed-based rs-FC in the beta band between the lesioned M1 and multiple brain regions (e.g., both frontal areas, ipsilateral postcentral gyrus) was significantly decreased after the rehabilitation. Significant negative correlations were also observed between the changes in the gait symmetry ratio and the changes in lesioned M1 seed-based rs-FC in the paracentral gyrus and regions associated with the default mode network. It was revealed that intensive gait-focused rehabilitation using a robotic orthosis improved gait function and induced plastic changes in the cortical networks. The improvements were significantly correlated with the timing of the start of rehabilitation after MSC transplantation.

Since our previous studies have indicated retinol promotes self-renewal of embryonic stem cells in vitro culture, we speculate that retinol may be directly involved in regulating adult stem cell self-renewal or developmental function in vivo. Vitamin A or retinoic acid (RA) solution was first injected into the abdominal cavities of mice, and then self-renewal and development marker gene expressions were investigated. The in vivo effects of retinol and RA on RA receptor expressions were further examined. The results showed that retinol not only significantly promotes self-renewal of neural stem cells in vivo but also induces orientational development of neural stem cells in vivo and significantly downregulates the expression of some RA receptor gene expression in the brain. This study provides experimental and theoretical bases for elucidating the regulation mechanism of retinol-mediated cell development in vivo, especially in brain, and the development of therapeutic drugs for neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Multiple sclerosis, and Huntington's disease.

Muse cells are endogenous pluripotent-like stem cells identified as stage-specific embryonic antigen-3 (SSEA-3)-positive subpopulations in the bone marrow, peripheral blood, and connective tissues of various organs. Clinical trials conducted by intravenous injection of donor-Muse cells, without the use of immunosuppressive drugs, have demonstrated safety and efficacy across multiple diseases. Since the epitope recognized by the anti-SSEA-3 antibody is a glycolipid, rather than a protein produced by a genetic code, the antibody may detect Muse cells across different species. Muse cells possess unique properties, including the ability to survive under stressful conditions, spontaneously turn into different cell types from all three primary layers of the body, and repair tissues in living organisms. They have been isolated from several mammalian species. However, their presence and characteristics in companion animals, such as canine and feline, remain unexplored, despite the growing demand for treatments that regenerate tissues in veterinary medicine. Adipose-derived stem cells (ADSCs) were established from adipose tissue taken during routine veterinary procedures. SSEA-3-positive cells were isolated using fluorescence-activated cell sorting. SSEA-3-positive cells were found in both canine (0.93 ± 0.16%) and feline (2.9 ± 0.15%) ADSCs, similar to human rates. Gene expression analysis revealed that SSEA-3-positive cells exhibited significantly higher levels of the pluripotency markers Oct3/4 and NANOG compared with SSEA-3-negative ADSCs. In suspension culture, SSEA-3-positive cells formed ES cell-like M-clusters. These cells could differentiate into endodermal (SOX17, AFP), mesodermal (GATA2, DESMIN, SMA), and ectodermal (NESTIN, NF) marker-positive cells, as measured by quantitative polymerase chain reaction and immunocytochemistry. These results show that canine and feline ADSCs contain SSEA-3-positive cells. These cells express pluripotency markers and can differentiate into endodermal, mesodermal, and ectodermal lineages. Their properties match those of Muse cells in humans and other mammals. This study offers basic evidence for isolating Muse cells from pets and demonstrates their potential for use in veterinary regenerative therapies.