Transendothelial migration (TEM), is a complex, multistep process impacted by diseases like autoimmune disorders and cancer. Deciphering aspects of the process enhances our understanding and possibilities for disease treatments. The extent to which this potential can be leveraged is often limited due to conventional assays neither accurately mimicking specific in vivo conditions like sheer stress nor visualizing the whole transmigration cascade, hence missing distinct mechanisms. Flow-based adhesion assays overcome these limitations and allow the use of various endothelial cells from different tissues with distinct properties. So far, a broader and translational assay application is hampered by potential operator-based bias/lack of standardization, as well as poor scalability due to time-consuming manual analysis. In this study, we successfully combined this assay with AI-based analysis including subsequent classification of cell-transmigration phases by a Keras/TensorFlow-based deep learning model. Trained on healthy-donor and pancreatic cancer patient-derived T cells, the model achieved a high accuracy of 91.6 % in identifying/categorizing cell transmigration, surpassing the currently accepted 80 %-threshold, therefore qualifying as a fast, standardized AI-based live-cell imaging tool. Additionally, its architecture grants highly convenient reconfiguration for various disease-model investigations. Hence, by combining an affordable and simplistic, yet potent live-cell-imaging technique with a comprehensive AI-approach, we have established a powerful tool which allows for integrating TEM-assays into various disease models.

Melanocyte stem cells (McSCs) are a crucial melanocyte reservoir within the hair follicle niche. This review provides an overview of the processes for McSC quiescence and activation. Because McSCs closely interact with hair follicle stem cells, we have focused on this interaction. Given the high prevalence of hair graying, the McSC system serves as a model for cellular aging. Here, we highlight current research on the mechanisms of hair graying.

Iron metabolism is increasingly recognized as a key player in the development and progression of various cancers. Iron is required for vital cellular processes such as energy production; however, it can also interact with reactive oxygen species to cause cellular toxicity. Consequently, a host of proteins coordinate iron homeostasis, and ferritin stands out as a promising therapeutic target due to its pivotal role in buffering cellular iron levels. This review explores the relevance of ferritin in brain cancers, shedding light on how it influences the biology of both tumor cells and cancer stem cells (CSCs), a population of tumor cells that is notable in their resistance to conventional treatment strategies. Ferritin plays a critical role in protecting against oxidative stress and boosting resistance to ferroptosis, a form of cell death often evaded by CSCs. Development of cutting-edge strategies designed to target ferritin, including ferritinophagy-inducing compounds and novel redox-based therapies that can capitalize on the iron dependency of CSCs is discussed in context. We propose that the iron addiction of brain cancer cells provides a specific susceptibility, whereby removing their iron buffering mechanism via targeting of ferritin can result in favorable treatment outcomes, including the induction of iron-dependent cell death. Future studies on the modulation of ferritin offer a ground-breaking therapeutic strategy to undermine CSC-driven tumor growth, overcome resistance to conventional therapies, and ultimately improve treatment outcomes for patients battling brain cancers.

Dental tissues development involves two distinct cell lineages: mesenchymal cells (derived from the cranial neural crest) and epithelial cells (derived from oral ectoderm and pharyngeal epithelium). Emerging evidence highlights the remarkable functional heterogeneity of cranial neural crest-derived dental mesenchymal stem cells (DMSCs), exhibiting pluripotency, self-renewal, and differentiation capacities. This heterogeneity enables a single DMSC population to generate specialized subpopulations with unique roles in teeth and periodontal tissues formation. Significant progress has been made in characterizing six major types of DMSCs and two populations of closely related cells: Tooth germ progenitor cells (TGPCs) and dental follicle stem cells (DFSCs), critical during early morphogenesis; Stem cells from human exfoliated deciduous teeth (SHEDs) and apical papilla stem cells (SCAPs), pivotal for root development; Dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), gingival mesenchymal stem cells (GMSCs) and alveolar bone mesenchymal stem cells (ABMSCs), essential for maintaining and regenerating mature dental tissues. A key breakthrough has unveiled the development and hierarchy of DMSCs by applying new techniques like single-cell RNA sequencing (scRNA-seq). To integrate insights into the development of teeth and periodontal tissues, this review synthesizes current knowledge on both developmental heterogeneity and subpopulation heterogeneity within DMSCs and related cells. These insights not only advance fundamental understanding of the developmental mechanisms of teeth and periodontal tissues, but also establish a promising framework for achieving more efficient tissue regeneration and repair engineering.

With population aging, chemotherapy-induced thrombocytopenia (CIT) is a severe complication in elderly cancer patients, yet effective preventive and therapeutic strategies remain limited. Here, we demonstrate that dietary restriction (DR) significantly mitigates 5-fluorouracil (5-FU)-induced thrombocytopenia and promotes platelet recovery in both young and aged mice. Mechanistically, DR improves mitochondrial homeostasis in hematopoietic stem and progenitor cells and enhances their hematopoietic reconstitution capacity. This preconditioning facilitates mitochondrial activation after chemotherapy, thereby promoting megakaryocytic lineage recovery. Pharmacological mitochondrial activation in ad libitum-fed mice mimics the protective effects of DR, whereas mitochondrial inhibition in DR-treated mice markedly attenuates these benefits. Clinically, cancer patients with lower pre-chemotherapy body mass index ([BMI] 18.5-22.95 kg/m2) showed a lower incidence of CIT following 5-FU treatment than those with higher BMI. Together, we show that short-term DR significantly mitigates CIT and that targeting mitochondria may represent a novel therapeutic strategy for CIT in elderly cancer patients.

Neurons derived from human pluripotent stem cells (hPSCs) are valuable models for studying brain development and developing therapies for brain disorders. Evaluating hPSC-derived neurons requires assessing their electrical activity, which can be achieved using multi-electrode arrays (MEAs) for extracellular recordings. Because each electrode channel generally detects activity from multiple neurons, resolving the activity of single neurons requires a process called spike sorting. However, currently available methods were not developed for analyzing data from hPSC-derived neurons and require complex workflows and time-consuming manual intervention. Here, we introduce a semi-automated MEA spike sorting software (SAMS) designed specifically for low-density MEA recordings of cultured neurons. SAMS outperforms commercially available automated spike sorting algorithms in terms of accuracy and greatly reduces computational and human processing time. By providing an accessible, efficient, and integrated platform for spike sorting, SAMS enhances the resolution and utility of MEA in disease modeling and drug development using hPSC-derived neurons.

Accumulating evidence shows that excess cholesterol and glucose uptake stimulates the expansion of hematopoietic stem/progenitor cells and myeloid progenitors, resulting in increased production of inflammatory cells and atherosclerotic progression. However, the role of other metabolites in plaque progression remains unclear. Hereby, we observed elevated α-ketoglutarate levels in granulocyte-monocyte progenitors (GMPs) of Ldlr-/- mice on a high-fat diet (HFD), determined by targeted metabolomics. On top of HFD, α-ketoglutarate administration further increased GMP proportion, myeloid cell production, and plaque progression in Ldlr-/- mice. The regulation of α-ketoglutarate in atherosclerosis required the expression of its receptor, oxoglutarate receptor 1 (OXGR1), in bone marrow cells (BMCs), as transplantation of OXGR1-/- BMCs attenuated plaque progression compared to transplantation of OXGR1+/+ BMCs in HFD-fed Ldlr-/- recipients. Using targeted metabolomics, single-cell RNA sequencing and validation experiments, we demonstrated that the α-ketoglutarate/OXGR1 axis upregulated the expression of purine nucleoside phosphorylase (PNP) in GMPs, which promoted de novo purine biosynthesis and reduced the levels of nicotinamide mononucleotide and nicotinamide adenine dinucleotide (NAD), thereby disturbing mitochondrial homeostasis and increasing the production of myeloid cells. Furthermore, proteomics data revealed that PNP treatment regulated the redox status by increasing the expression of NAD kinase (NADK), thereby accelerating NAD consumption. Additionally, PNP promoted the transcriptional activation of NF-κB via ubiquitin, enhancing ROS production and inflammation in lineage-/low cells. Spearman's correlation analysis revealed a positive association between isocitrate and low-density lipoprotein cholesterol levels in human plasma. Overall, HFD potentiated α-ketoglutarate, contributing to atherosclerosis.

NOTCH3 variants cause CADASIL (cerebral autosomal dominant arteriopathy and subcortical infarcts and leukoencephalopathy), the most common monogenetic form of small vessel disease (SVD) and vascular dementia (VaD). The molecular mechanisms driving CADASIL pathogenesis remain poorly understood, and no specific treatments are currently available. NOTCH3 is mainly expressed in vascular smooth muscle cells (VSMCs) that arise from different embryonic origins. Using human induced pluripotent stem cell (iPSC) models, we generated origin-specific VSMCs and found that cerebral, but not peripheral, VSMC mimics are selectively vulnerable to NOTCH3 variants. CADASIL iPSC-derived brain-specific VSMCs acquired a synthetic phenotype, accompanied with extensive extracellular matrix accumulation and impaired cell adhesion leading to anoikis. Furthermore, an endothelial-independent nitric oxide signaling was substantially impaired in CADASIL iPSC-derived VSMCs. Phosphodiesterase-5 inhibition successfully reversed the functional abnormality and survival of mutant VSMCs. Our findings uncovered mechanistic insights and suggest a viable therapeutic strategy for NOTCH3-associated SVD/VaD, reinforcing the value of patient-specific iPSCs for disease modeling and potential drug discovery.

It has been demonstrated that both hUCMSC-exo and Cyasterone exhibit protective effects against steroid-induced osteonecrosis of the femoral head (SIONFH). Additionally, studies have shown that CTSD N-glycosylation influences BMSC apoptosis. Based on these findings, we aim to investigate the mechanism of hUCMSCs-exo@Cyasterone in the Dex-induced BMSCs model of SIONFH, focusing on its regulatory role in CTSD N-glycosylation during apoptosis.

Developmental potency-the ability of cells to differentiate into specialized identities-is progressively lost during development. This phenomenon, known as lineage restriction, is poorly understood. Here, we show that a cell's developmental potency is associated with which genes retain transcriptional potency, and that a molecular relay progressively strips the genome of transcriptional potency during differentiation. Mechanistically, genes chromatinized into nucleosomes can intrinsically lack transcriptional potency, remaining silent even when their transcriptional activators are present. Naive pluripotent stem cells uniquely possess the capacity to restore genome-wide transcriptional potency, but such capacity is decommissioned when cells transition into the primed pluripotent state. Thereafter, placeholder factors (PFs) in stem cells counteract chromatinization to preserve transcriptional potency of specific silent genes, thereby maintaining developmental potency for downstream lineages requiring these genes. As differentiation proceeds, PFs disappear. The genes they protect can then permanently lose transcriptional potency via chromatinization, leading to irreversible lineage restriction.

Toxoplasma gondii is a widespread parasite that impacts both human and animal health. Increasing use of organoid model systems has made previously challenging aspects of the T. gondii lifecycle more accessible. The media for these organoid systems are highly complex with many growth factors and pathway inhibitors that promote stem cell retention or differentiation of the cells. We noticed changes in T. gondii growth and development in our intestinal organoid system and wanted to determine if this was driven by cell type or media components. We found that low concentrations of SB202190 (a p38 MAPK inhibitor) and A83-01 (an ALK 4/5/7 receptor inhibitor) are each sufficient to alter T. gondii growth even in fibroblast cells. Further investigation with our qPCR panel of T. gondii stage markers revealed that these compounds promote bradyzoite cyst development and prime parasites for pre-sexual and sexual stage gene expression. As these complex organoid systems become more common in microbiology research, this study highlights the role of organoid media components in controlling pathogen growth and development.

Glioblastoma (GBM) is a highly aggressive, therapy-resistant brain tumor with inevitable recurrence despite maximal multimodal treatment. Increasing evidence suggests that this intractability arises from coordinated cellular programs rather than a single dominant pathway. Central to these programs are glioma stem-like cells (GSCs), which sustain self-renewal, phenotypic plasticity, and resistance to genotoxic and metabolic stress, and yet the molecular basis of their long-term tumor-propagating capacity remains incompletely understood. Here, we synthesize recent advances to propose an integrated conceptual framework-the Triadic Nexus-in which GSC stemness, telomere maintenance mechanisms, and metabolic reprogramming function as a self-reinforcing regulatory system. We review how telomerase reactivation versus alternative lengthening of telomeres (ALT) differentially shape genomic stability, immune signaling, and metabolic states and how metabolic plasticity feeds back to regulate stemness and telomere-associated stress responses. Drawing on single-cell, spatial, and multi-omics studies, we highlight how these interdependent axes collectively sustain therapy resistance and tumor recurrence. Finally, we discuss the translational implications of the Triadic Nexus, emphasizing rational combinatorial therapeutic strategies and biomarker-guided patient stratification based on telomere and metabolic signatures. By unifying stemness, telomere biology, and metabolism into a mechanistically testable model, this review provides a systems-level framework for understanding GBM persistence and guiding next-generation therapeutic interventions.

Both PRMT5 and EphA2 proteins are overexpressed and play a crucial role in multiple cancers, and have been used as targets to develop new anticancer drugs. However, the function and significance of the PRMT5-EphA2 interaction are unclear. Here, we report that PRMT5 bound to EphA2, catalyzed the dimethylation of EphA2 at arginine 816, and then stabilized EphA2 via inhibiting Cbl-mediated EphA2 ubiquitination and degradation in nasopharyngeal carcinoma (NPC) cells. Functionally, PRMT5 promoted in vitro and in vivo NPC stem cell properties by methylating and stabilizing EphA2. Based on the interacting regions of PRMT5 and EphA2 proteins, we developed a 20 amino acid-long PRMT5-derived peptide, P20, which disrupted the connection of PRMT5 with EphA2, degraded EphA2, and suppressed NPC stem cell properties in vitro and in mice. Moreover, the expression levels of PRMT5 and EphA2 in the NPC tissues were significantly higher than those in the normal nasopharyngeal mucosal tissues, and both proteins for predicting the patient's prognosis are superior to individual proteins. Our findings suggest that PRMT5 methylates and stabilizes EphA2 to promote NPC stem cell properties, and the PRMT5-derived peptide P20 can serve as a novel strategy for targeting EphA2 degradation and inhibiting NPC stem cell properties.

Corneal endothelial failure can cause blindness, with transplantation as the only treatment. Due to donor shortages, establishing robust methods for generating corneal endothelial-like cells (CECs) from induced pluripotent stem cells (iPSCs) is critical. Differentiation protocols included iPSC-to-CEC induction with or without neural crest cell differentiation. CECs directly differentiated from iPSCs demonstrated robust expression of CEC-specific markers and a hexagonal morphology. The wash-out protocol is a novel, efficient, noncytotoxic method for removing undifferentiated iPSCs and obtaining CEC populations with high purity. Single-cell sequencing data showed that iPSC-CECs with wash-out were similar to human primary CECs. In vivo transplantation of iPSC-CECs into a corneal endothelial dysfunction (CED) rabbit model demonstrated their safety and therapeutic efficacy, with improved corneal transparency. Notable recovery of corneal clarity in the CED model, without graft rejection, highlights the in vitro and in vivo potential of iPSC-CECs as a powerful source for clinical therapy in patients with CED. This work establishes an effective stem cell-based platform for producing corneal endothelium-like cells with clinical-grade quality, offering a scalable and regenerative alternative to conventional transplantation.

Pancreatic cancer is highly refractory and aggressive, with cancer stem cells (CSCs) being primarily responsible for its metastasis and chemoresistance. Deregulated cellular bioenergetics is a hallmark of cancer cells. However, the influence of bioenergetics on the maintenance of pancreatic CSC stemness and its underlying mechanisms have not been fully elucidated. In this study, pancreatic CSCs, isolated either by sorting ALDH+ subpopulation or enriching serially passaged tumorspheres from pancreatic cancer cells and PDX model, exhibited active mitochondrial complex I activity and increased oxidative phosphorylation. Complex I maintains stemness and tumorigenicity through its core subunit, NDUFS1. NDUFS1-mediated pancreatic CSC stemness is reinforced by high expression of CD147, which promotes pSTAT3Tyr705-mediated NDUFS1 transcription. To promote stemness, CD147-NDUFS1 initiates SIRT1-DNMT1 metaboloepigenetic signaling, decreasing promoter hypomethylation and increasing the mRNA expression of the stem cell transcript factor PAX2. Moreover, NDUFS1 and CD147 expressions were highly correlated in pancreatic cancer tissues, and their co-expression was significantly associated with poor patient survival. Taken together, our study provides evidence that mitochondrial complex I functions as a key player in CSC stemness maintenance through NDUFS1-mediated retrograde metaboloepigenetic signaling. Blocking a key regulator of mitonuclear communication by targeting CD147 may be a novel therapy for pancreatic cancer.

We have recently hypothesized that the hematogenous metastatic cancer cell of solid tumors is a hybrid between a primary cancer cell and a memory/trained macrophage (doi: 10.3389/fonc.2024.1412296). The hybrid cell respectively acquires mutator phenotype and overgrowth/hyperplasia property from the primary cancer cell and migratability/metastability from the memory/trained macrophage. We name this hypothesis Cancer Cell-Memory Macrophage Hybrid Theory. Since the publication of the article, a number of questions related to this Theory have been raised by colleagues in the oncology community, including intratumoral microbes and microbiomes/microbiotas, oncolytic viruses and bacteria, human papilloma virus vaccines, anti-cancer effects of γδ T-cells, and immune checkpoint inhibitors. The current article is prepared to address these issues. Additional to resolving questions like "Why metastatic cancer cells enter dormancy and can recur via stem-like self-renewal?", the Cancer Cell-Memory Macrophage Hybrid Theory distinguishes itself from other carcinogenesis and metastasis hypotheses/theories by offering answers to many puzzling clinical features including metastasis of seemingly malignant parasitic cells within the human body, intracellular microbes (including viruses, bacteria, fungi, and parasites) within cancer cells, paradoxal effects (recurrence vs. regression) of microbes on cancer, contradictory immune effects of human papilloma virus vaccines between young and adult/senior females, and immune context-dependent effects (stimulatory and inhibitory) of T-lymphocytes on cancer cells. The Theory also predicts that quantitatively and functionally dampening innate macrophages that have hybridized with cancer cells (i.e., cancer cell-memory macrophage hybrids), should be explored as a fundamental anti-cancer strategy. The Theory further forecasts how to prepare an organotropic/tumoritropic Coley's toxin-like anti-cancer microbe, which could potentially circumvent direct injection of microbial preparations into a tumor. A testable experiment that uses zebrafish larva models can potentially either validate or falsify the Theory.

Breast cancer metastasis remains the leading cause of mortality and frequently targets the bone. Breast cancer cells release soluble factors and extracellular vesicles that disrupt bone marrow (BM)/bone homeostasis, promoting osteoclastogenesis and the accumulation of senescent cells. In line with updated cancer hallmarks, senescent mesenchymal stem/ stromal cells (MSCs), osteoblasts, and osteocytes contribute to remodeling of the BM microenvironment, thereby favoring pre-metastatic niche (PMN) formation and subsequent bone metastasis. We previously demonstrated that untreated stage III-B breast cancer patients (BCPs) exhibit increased oxidative stress and elevated reactive oxygen species (ROS) levels, accompanied by senescent and functionally impaired BM-MSCs-key regulators of BM/bone homeostasis. In the present study, we sought to identify the molecular targets affected by oxidative stress that drive MSC senescence in these patients.

Focal post-traumatic cartilage lesions frequently progress to early osteoarthritis, highlighting the limited regenerative capacity of adult articular cartilage. Compared to native tissue, conventional surgical interventions often produce fibrocartilage with inferior biomechanical properties, representing a persistent therapeutic challenge. This review assessed preclinical studies exploring cartilage repair strategies using autologous mesenchymal stem cells (MSCs) in animal models. MSCs therapies demonstrated superior cartilage regeneration, matrix organization, and integration into the surrounding tissue compared to the control groups. The most efficient source was found to be bone marrow - derived mesenchymal stem cells (BM-MSCs) combined with biodegradable scaffolds, suggesting their potential in tissue engineering applications. Despite methodological heterogeneity across studies - including variations in stem cells sources, implant types, and deliver strategies - cumulative evidence strongly supports the regenerative potential of autologous MSCs for cartilage repair. Current research identifies key knowledge gaps, including the absence of standardized experimental protocols and limited insight into the mechanisms of tissue remodeling and maturation. Collectively, these gaps limit direct clinical translation, highlighting the need for further, standardized studies in large animal models with long-term follow-up (>2 years) to assess integration, functional maturation, and the full regenerative potential of the repair tissue.