Cells

26 pages, 2922 KiB

Open AccessArticle

Investigation and Distinction of Energy Metabolism in Proliferating Hepatocytes and Hepatocellular Carcinoma Cells

by Julia Nerusch, Gerda Schicht, Natalie Herzog, Jan-Heiner Küpper, Daniel Seehofer and Georg Damm

Cells 2025, 14(16), 1254; https://doi.org/10.3390/cells14161254 (registering DOI) - 14 Aug 2025

Abstract

Metabolic rewiring is a hallmark of both hepatic regeneration and malignant transformation, complicating the identification of cancer-specific traits. This study aimed to distinguish the metabolic profiles of proliferating hepatocytes and hepatocellular carcinoma (HCC) cells through integrated analyses of mRNA and protein expression, along [...] Read more.

Metabolic rewiring is a hallmark of both hepatic regeneration and malignant transformation, complicating the identification of cancer-specific traits. This study aimed to distinguish the metabolic profiles of proliferating hepatocytes and hepatocellular carcinoma (HCC) cells through integrated analyses of mRNA and protein expression, along with functional characterization. We compared non-malignant Upcyte^® hepatocytes (HepaFH3) cultured under proliferative and confluent conditions with primary human hepatocytes, primary human hepatoma cells, and hepatoma cell lines. Proliferating HepaFH3 cells exhibited features of metabolic reprogramming, including elevated glycolysis, increased HIF1A expression, and ketone body accumulation, while maintaining low c-MYC expression and reduced BDH1 levels, distinguishing them from malignant models. In contrast, HCC cells showed upregulation of HK2, c-MYC, and BDH1, reflecting a shift toward aggressive glycolytic and ketolytic metabolism. Functional assays supported the transcript and protein expression data, demonstrating increased glucose uptake, elevated lactate secretion, and reduced glycogen storage in both proliferating and malignant cells. These findings reveal that cancer-like metabolic changes also occur during hepatic regeneration, limiting the diagnostic utility of individual metabolic markers. HepaFH3 cells thus provide a physiologically relevant in vitro model to study regeneration-associated metabolic adaptation and may offer insights that contribute to distinguishing regenerative from malignant processes. Our findings highlight the potential of integrated metabolic profiling in differentiating proliferation from tumorigenesis. Full article

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29 pages, 3651 KiB

Open AccessArticle

Donor Variability and Seeding Density Shape NK-Cell Proliferation and Surface Receptor Expression: Insights from an Integrated Phenotypic and Genetic Analysis

by Neele Kusch, Jonathan Storm, Antonia Macioszek, Cornelius Knabbe, Barbara Kaltschmidt and Christian Kaltschmidt

Cells 2025, 14(16), 1252; https://doi.org/10.3390/cells14161252 (registering DOI) - 14 Aug 2025

Abstract

Natural killer (NK) cells are promising candidates for adoptive immunotherapy, but their clinical application requires standardized expansion protocols that yield functional cells in sufficient numbers. This study examined how initial seeding density and donor-intrinsic variability affect NK cell proliferation and receptor phenotype during [...] Read more.

Natural killer (NK) cells are promising candidates for adoptive immunotherapy, but their clinical application requires standardized expansion protocols that yield functional cells in sufficient numbers. This study examined how initial seeding density and donor-intrinsic variability affect NK cell proliferation and receptor phenotype during in vitro expansion in a G-Rex^® 24-well plate under IL-2 stimulation. NK cells from healthy donors were analyzed longitudinally by flow cytometry, and targeted SNP sequencing of selected receptor genes (IL2RA, IL2RB, FCGR3A, NCR1, KLRK1, and ICAM-1) was performed to assess potential genetic contributions. A seeding density of 2.0 × 10⁶ cells/cm² promoted high expansion rates and favorable expression of activating receptors including CD16a, NKp46, and NKG2D. Nonetheless, marked inter-donor differences were observed. Some donors exhibited impaired proliferation and aberrant receptor expression, possibly associated with high-priority SNPs and distinct haplotype structures. Others showed robust proliferation despite the absence of identifiable genetic drivers, suggesting the involvement of variants in other genes or non-genetic mechanisms such as epigenetic priming or adaptive NK-cell differentiation. These results highlight the influence of both culture conditions and donor-intrinsic factors on NK-cell expansion outcomes. Integrating phenotypic and genetic analyses may improve the reproducibility and personalization of NK-cell-based manufacturing protocols for therapeutic use. Full article

(This article belongs to the Special Issue Natural Killer (NK) Cells in Immunity: Limitations and Potential)

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11 pages, 1868 KiB

Open AccessArticle

A Single Subcutaneous Dose of BMD-1141 Stimulates Hair Regrowth Comparable to Daily, High-Dose Ruxolitinib in a Mouse Model of Alopecia Areata

by Robert Gensure, Brett King, Stephen Sikkink, Andrei Mardaryev and Robyn Goforth

Cells 2025, 14(16), 1251; https://doi.org/10.3390/cells14161251 (registering DOI) - 14 Aug 2025

Abstract

Alopecia areata (AA) is an autoimmune disorder of hair loss resulting from a T-cell mediated attack on hair follicles. Three Janus kinase (JAK) inhibitors have been approved for the treatment of moderate-to-severe alopecia areata; however, safety concerns for immunosuppressive therapy have limited their [...] Read more.

Alopecia areata (AA) is an autoimmune disorder of hair loss resulting from a T-cell mediated attack on hair follicles. Three Janus kinase (JAK) inhibitors have been approved for the treatment of moderate-to-severe alopecia areata; however, safety concerns for immunosuppressive therapy have limited their use. We previously demonstrated that BMD-1141, consisting of parathyroid hormone (PTH) fused to a collagen-binding domain (CBD) (PTH-CBD) improved hair retention, increased anagen hair follicles counts, and reduced hair follicle dystrophy in C3H/HeJ-engrafted mice). We now compare the effects of a single subcutaneous injection of BMD-1141 with the daily, high-dose, oral administration of the JAK inhibitor ruxolitinib on anagen hair follicle counts and hair regrowth in C3H/HeJ-engrafted mice. BMD-1141-treated mice exhibited a significant increase in anagen hair follicle counts (p < 0.05) and enhanced hair regrowth compared to ruxolitinib-treated mice after 8 weeks. Hair follicles from the BMD-1141-treated mice showed increased beta-catenin, consistent with a mechanism of stimulating the anagen transition of hair follicles, and did not increase immune cell infiltration. Thus, a single subcutaneous dose of BMD-1141 stimulated hair regrowth comparable to daily ruxolitinib, apparently by stimulating the hair cycle, rather than inhibiting the autoimmune response. Full article

(This article belongs to the Special Issue Molecular Signalings in Hair Regeneration)

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12 pages, 3454 KiB

Open AccessArticle

DIRAS3 Inhibits Ovarian Cancer Cell Growth by Blocking the Fibronectin-Mediated Integrin β1/FAK/AKT Signaling Pathway

by Jing Guo, Janice M. Santiago-O’Farrill, Vivian Orellana, Rumeysa Ozyurt, Hailing Yang, Marc Pina, Gamze Bildik, Weiqun Mao, Robert C. Bast and Zhen Lu

Cells 2025, 14(16), 1250; https://doi.org/10.3390/cells14161250 - 13 Aug 2025

Abstract

Autophagy is a crucial cellular process responsible for sustaining homeostasis through the degradation and recycling of proteins and organelles, providing energy during amino acid starvation and hypoxia. In cancer, autophagy can either inhibit tumor growth or support cancer cell survival. Our previous studies [...] Read more.

Autophagy is a crucial cellular process responsible for sustaining homeostasis through the degradation and recycling of proteins and organelles, providing energy during amino acid starvation and hypoxia. In cancer, autophagy can either inhibit tumor growth or support cancer cell survival. Our previous studies have shown that re-expression of the tumor suppressor gene DIRAS3 inhibits growth of ovarian cancer cells, promotes autophagic cell death in vitro, and induces tumor dormancy in vivo. Growth factors and extracellular matrix (ECM) components can, however, inhibit DIRAS3-induced autophagic cell death. This study explores whether fibronectin (FN) can counteract the growth inhibition induced by DIRAS3 in ovarian cancer cells. FN was found to inhibit DIRAS3-induced autophagy and to partially rescue ovarian cancer cells from DIRAS3-induced cell death while reducing DIRAS3-induced inhibition of p-FAK and p-AKT. Inhibiting FAK with defactinib in ovarian cancer cells enhanced DIRAS3-induced autophagy and cell death. Re-expression of DIRAS3 and treatment with defactinib produced tumor regression in xenograft models. Our findings suggest that ECM components in the tumor microenvironment like FN enhance the activities of β1 integrin, FAK, and AKT to inhibit DIRAS3-induced autophagic cell death, thereby promoting ovarian cancer cell survival. Full article

(This article belongs to the Section Autophagy)

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17 pages, 14998 KiB

Open AccessPerspective

A Biophysics of Epigenetic Rejuvenation

by Prim B. Singh

Cells 2025, 14(16), 1249; https://doi.org/10.3390/cells14161249 - 13 Aug 2025

Abstract

We present a synthesis based on epigenetics, machine learning and polymer physics from which emerges new relationships between the thermodynamic Flory–Huggins parameter (χ), epigenetic age (eAge) and Shannon entropy. Using a framework for the estimation of χ in the nuclear environment we show [...] Read more.

We present a synthesis based on epigenetics, machine learning and polymer physics from which emerges new relationships between the thermodynamic Flory–Huggins parameter (χ), epigenetic age (eAge) and Shannon entropy. Using a framework for the estimation of χ in the nuclear environment we show that

χ \propto {e A g e}^{- 1}

and

χ \propto {S h a n n o n E n t r o p y}^{- 1}

. As cells age, epigenetic drift results in “smoothing out” of the epigenetic landscape reducing the magnitude of χ. Epigenetic rejuvenation reverses epigenetic drift and restores χ to levels found in young cells with concomitant reduction in both eAge and Shannon entropy. Full article

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21 pages, 1389 KiB

Open AccessReview

Hypoimmunogenic Human iPSCs for Repair and Regeneration in the CNS

by Haiwei Zhang, Hongxia Zhou, Xugang Xia, Qilin Cao and Ying Liu

Cells 2025, 14(16), 1248; https://doi.org/10.3390/cells14161248 - 13 Aug 2025

Abstract

Human induced pluripotent stem cells (iPSCs) can be genetically engineered to evade host immune recognition, rendering them hypoimmunogenic and suitable as “universal donor” cells for allogeneic transplantation. Such modifications enable the development of off-the-shelf iPSC-derived therapeutics that are immediately available for clinical use [...] Read more.

Human induced pluripotent stem cells (iPSCs) can be genetically engineered to evade host immune recognition, rendering them hypoimmunogenic and suitable as “universal donor” cells for allogeneic transplantation. Such modifications enable the development of off-the-shelf iPSC-derived therapeutics that are immediately available for clinical use without the need for patient-specific derivation or immunosuppression. This review focuses on recent developments in strategies for generating hypoimmunogenic human iPSCs, with particular emphasis on their applications in central nervous system (CNS) cell therapy and repair. We assess immunomodulatory factors based on their immune functions and potential roles in CNS development and disease, with the goal of identifying strategies to use these factors either individually, in combination, or alongside gene editing to reduce immune rejection without compromising neurogenesis or tissue repair. Full article

(This article belongs to the Special Issue Stem Cells and Beyond: Innovations in Tissue Repair and Regeneration)

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23 pages, 8052 KiB

Open AccessArticle

The Capability to Undergo ACSL4-Mediated Ferroptosis Is Acquired During Brown-like Adipogenesis and Affected by Hypoxia

by Markus Mandl, Elisabeth Heuboeck, Peter Benedikt, Florian Huber, Olga Mamunchak, Sonja Grossmann, Michaela Kotnik, Esma Hamzic-Jahic, Charnkamal Singh Bhogal, Anna-Maria Lipp, Edeltraud Raml, Werner Zwerschke, Martin Wabitsch, Jakob Voelkl, Andreas Zierer and David Bernhard

Cells 2025, 14(16), 1247; https://doi.org/10.3390/cells14161247 - 13 Aug 2025

Abstract

Adipose tissue enlargement in obesity leads to hypoxia, which may promote premature aging. This study aimed to understand the hypoxic response in 3D cultures of SGBS cells, a model for brown-like adipose tissue expressing uncoupling protein 1 (UCP1). Single-nucleus RNA sequencing of SGBS [...] Read more.

Adipose tissue enlargement in obesity leads to hypoxia, which may promote premature aging. This study aimed to understand the hypoxic response in 3D cultures of SGBS cells, a model for brown-like adipose tissue expressing uncoupling protein 1 (UCP1). Single-nucleus RNA sequencing of SGBS organoids revealed a heterogeneous composition and sub-population-specific responses to hypoxia. The analysis identified a cluster of transcriptional repression, indicating dying cells, and implied a role of ferroptosis in this model. Further experiments with SGBS cells and white adipose tissue-derived stem/progenitor cells showed that Acyl-CoA synthetase long-chain family member 4 (ACSL4), a key enzyme in ferroptosis, is expressed only in the presence of browning factors. Hypoxia downregulated ACSL4 protein in SGBS organoids but induced an inflammaging phenotype. Analysis of brown-like epicardial adipose tissue from cardiac surgery patients revealed a significant positive correlation of ACSL4 mRNA with UCP1 and hypoxia-inducible pro-inflammatory markers, while ACSL4 protein appeared to be inversely correlated. In conclusion, this study demonstrates that adipocytes’ capability to undergo ACSL4-mediated ferroptosis is linked to brown-like adipogenesis, suggesting an opportunity to modulate ferroptotic signaling in adipose tissue. The dual role of hypoxia by inhibiting ACSL4 but promoting inflammaging indicates a relationship between ferroptosis and aging that warrants further investigation. Full article

(This article belongs to the Special Issue Gene Expression in Adipocytes During Obesity: Understanding Challenges and Future Prospects)

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24 pages, 4894 KiB

Open AccessArticle

Single-Cell mRNA Analysis for the Identification of Molecular Pathways of IRF1 in HER2+ Breast Cancer

by Laura Vilardo, Paride Pelucchi, Antonia Brindisi, Edoardo Abeni, Eleonora Piscitelli, Ettore Mosca, Giovanni Bertalot, Mira Palizban, Theodoros Karnavas, Angelos D. Gritzapis, Ioannis Misitzis, Martin Götte, Ileana Zucchi and Rolland Reinbold

Cells 2025, 14(16), 1246; https://doi.org/10.3390/cells14161246 - 13 Aug 2025

Abstract

Clonally established tumor cell lines often do not recapitulate the behavior of cells in tumors. The sequencing of a whole tumor tissue may not uncover transcriptome profiles induced by the interactions of all different cell types within a tumor. Interferons for instance have [...] Read more.

Clonally established tumor cell lines often do not recapitulate the behavior of cells in tumors. The sequencing of a whole tumor tissue may not uncover transcriptome profiles induced by the interactions of all different cell types within a tumor. Interferons for instance have a vast number of binding sites in their target genes. Access to the DNA binding sites is determined by the epigenomic state of each different cell type within a tumor mass. To understand how genes such as interferons appear to have both tumor-promoting and tumor-inhibiting functions, single-cell transcript analysis was performed in the breast cancer tissue of HER2+ (epidermal growth factor receptor 2) patients. We identified that potential antagonistic oncogenic activities of cells can be due to diverse expression patterns of genes with pleiotropic functions. Molecular pathways both known and novel were identified and were similar with those previously identified for patients with rheumatoid arthritis. Our study demonstrates the efficacy in using single-cell transcript analysis to gain insight into genes with apparent contradictory or paradoxical roles in oncogenesis. Full article

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16 pages, 2238 KiB

Open AccessArticle

Gene Expression Pattern Associated with Cytoskeletal Remodeling in Lipid-Loaded Human Vascular Smooth Muscle Cells: Crosstalk Between C3 Complement and the Focal Adhesion Protein Paxillin

by Maisa Garcia-Arguinzonis, Rafael Escate, Roberta Lugano, Esther Peña, Maria Borrell-Pages, Lina Badimon and Teresa Padro

Cells 2025, 14(16), 1245; https://doi.org/10.3390/cells14161245 - 12 Aug 2025

Abstract

Mechanical and contractile forces in the vascular wall regulate smooth muscle cell migration. We previously demonstrated the presence of C3 complement products in atherosclerotic lesions of human aortas and showed that that C3-derived fragments promote key cellular processes, such as actin cytoskeleton organization [...] Read more.

Mechanical and contractile forces in the vascular wall regulate smooth muscle cell migration. We previously demonstrated the presence of C3 complement products in atherosclerotic lesions of human aortas and showed that that C3-derived fragments promote key cellular processes, such as actin cytoskeleton organization and cell migration, in lipid-loaded human vascular smooth muscle cells (hVSMCs). In the present study, we aimed to investigate gene expression profiles related to cytoskeletal remodeling and cell adhesion in migrating hVSMCs with a particular focus on modulatory effect of the C3 complement pathway on these processes. We analyzed gene expression in migrating and non-migrating hVSMCs using real-time PCR and in silico network analysis. Additionally, we investigated cytoskeletal remodeling through Western blotting and confocal microscopy. PCR profiling revealed 30 genes with significantly altered expression in migrating hVSMCs compared to non-migrating control cells. In silico analysis identified six of these genes—PXN, AKT1, RHOA, VCL, CTNNB1, and FN1—as being associated with cytoskeletal remodeling and focal adhesion, with PXN occupying a central position in the interaction network. PXN expression was reduced at both the transcript and protein levels and showed altered subcellular localization in migrating lipid-loaded hVSMCs. Protein–protein interaction analysis using STRING predicted an association between PXN and the integrin complex αMβ2 (comprising ITGAM (CD11b) and ITGB2 (CD18)), which functions as receptors for the iC3b complement fragment. Confocal imaging of cell adhesion structures revealed that lipid-loaded hVSMCs stimulated with iC3b displayed a more diffuse PXN distribution and significantly increased PXN–F-actin colocalization in active cytoplasmic regions compared to lipid-loaded control cells. PXN–F-actin colocalization increased from 1.26% to 19.68%. Subcellular fractionation further confirmed enhanced PXN enrichment in the membrane fraction, with no significant changes observed in the cytosolic or cytoskeletal compartments. In conclusion, iC3b-mediated molecular signaling in lipid-loaded hVSMCs alters PXN distribution and enhances cytoskeletal remodeling, revealing novel molecular interactions in vascular remodeling and the progression of atherosclerotic lesions. Full article

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45 pages, 2976 KiB

Open AccessReview

Lipoxins as Modulators of Diseases

by Uzma Saqib, Monika Pandey, Anjali Vyas, Preeti Patidar, Sumati Hajela, Asgar Ali, Meenakshi Tiwari, Sutripta Sarkar, Neelam Yadav, Shivani Patel, Deepali Shukla, Grace N. Lienemann, Fletcher A. White, Herney Andrés García-Perdomo, Mirza Saqib Baig, Ganesh V. Halade, Krishnan Hajela, Sadhana Sharma and Alexander G. Obukhov

Cells 2025, 14(16), 1244; https://doi.org/10.3390/cells14161244 - 12 Aug 2025

Abstract

Lipoxins were discovered 40 years ago, and since then, their beneficial roles for human health have been confirmed in numerous studies. These small molecules belong to the eicosanoid class of compounds, which are generated metabolically by lipoxygenases. Lipoxins are released during various diseases [...] Read more.

Lipoxins were discovered 40 years ago, and since then, their beneficial roles for human health have been confirmed in numerous studies. These small molecules belong to the eicosanoid class of compounds, which are generated metabolically by lipoxygenases. Lipoxins are released during various diseases and conditions, including but not limited to systemic inflammation, infection, asthma, cancer, diabetes, and cardiovascular disorders. Recently, several synthetic lipoxin analogs have been developed that also exhibit potent anti-inflammatory properties. In this review, we discuss the inflammation-resolving roles of lipoxins in various major diseases. Further, we summarize the latest reports on the use of synthetic lipoxins as potential therapeutic agents and discuss the role of aspirin-dependent lipoxin production in alleviating various diseases, including cancer. Full article

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16 pages, 2323 KiB

Open AccessArticle

DNA and Histone Modifications Identify a Putative Controlling Element (CE) on the X Chromosome of Sciara coprophila

by Olga V. Posukh, Victor V. Shloma, Polina A. Skrypnik, Daniil A. Maksimov, Polina A. Antoshina, Daria A. Kalashnikova, Artem Nurislamov, Varvara A. Lukyanchikova, Nikita Torgunakov, Nariman R. Battulin, Veniamin S. Fishman, Yuri V. Vyatkin, Arina A. Smelova, Stanislav E. Romanov, Petr P. Laktionov, Daulet Valishayev, Stepan N. Belyakin and Prim B. Singh

Cells 2025, 14(16), 1243; https://doi.org/10.3390/cells14161243 - 12 Aug 2025

Abstract

The term chromosomal imprinting was introduced to denote the parent-of-origin-dependent behavior of chromosomes in the fungus gnat originally named Sciara coprophila (current taxonomic name is Bradysia coprophila). Such behavior is observed in Sciara coprophila embryos, where paternal X chromosomes (X_p) [...] Read more.

The term chromosomal imprinting was introduced to denote the parent-of-origin-dependent behavior of chromosomes in the fungus gnat originally named Sciara coprophila (current taxonomic name is Bradysia coprophila). Such behavior is observed in Sciara coprophila embryos, where paternal X chromosomes (X_p) are specifically eliminated during the 7th–8th cleavage divisions. Elimination is regulated by a controlling element (CE) that has been mapped to heterochromomere II (H2) within the sub-telomeric short arm of polytene X chromosomes. Here, using a combination of a new Sciara genome assembly, along with ChIP-Seq and MeDIP analyses, we show that a 1.2 Mb region within the CE locus has a repressive epigenetic signature that is characterised by enrichments of H3K9me3, H4K20me3 and 5′-methyl cytosine (5meC). These data provide evidence for a model where the H3K9me3/HP1/H4K20me3 pathway operates to assemble a heterochromatin-like complex at the CE that renders it silent on X_p chromosomes that are not eliminated. In this regard, our findings support the idea that the H3K9me3/HP1/H4K20me3 pathway represents the most evolutionarily conserved mechanism linked to chromosomal imprinting in animals. Full article

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29 pages, 912 KiB

Open AccessReview

Chimeric Antigen Receptor T Cell Immunotherapy for Autoimmune Rheumatic Disorders: Where Are We Now?

by Panagiota Anyfanti, Paschalis Evangelidis, Nikolaos Kotsiou, Anna Papakonstantinou, Ioannis Eftychidis, Ioanna Sakellari, Theodoros Dimitroulas and Eleni Gavriilaki

Cells 2025, 14(16), 1242; https://doi.org/10.3390/cells14161242 - 12 Aug 2025

Abstract

Chimeric antigen receptor (CAR) T cell immunotherapy has changed the landscape of B cell hematological malignancies’ management, while it has recently shown promising results in the treatment of refractory autoimmune rheumatic disorders (ARDs). Targeting B cell antigens such as CD19 and BCMA, CAR-T [...] Read more.

Chimeric antigen receptor (CAR) T cell immunotherapy has changed the landscape of B cell hematological malignancies’ management, while it has recently shown promising results in the treatment of refractory autoimmune rheumatic disorders (ARDs). Targeting B cell antigens such as CD19 and BCMA, CAR-T cell therapy can induce sustained remission by the elimination of autoreactive B cell populations resistant to the standard of care treatment options. Clinical data from case reports and small case series demonstrate profound clinical responses in ARDs, including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), idiopathic inflammatory myopathies (IIMs), rheumatoid arthritis (RA), antiphospholipid syndrome (APS), and primary Sjögren’s syndrome (pSS). Treatment outcomes include reduced disease activity, normalization of serologic markers, improved organ function, and drug-free remission, even after B cell reconstitution. Additionally, toxicities, primarily limited to mild cytokine release syndrome (CRS), were generally manageable with supportive care. Encouraging preliminary results have led to the development of several ongoing clinical trials investigating CAR-T cell therapy across multiple ARDs and patient populations, including pediatric patients. This review summarizes the current clinical experience and provides a comprehensive overview of ongoing clinical trials exploring CAR-T cell immunotherapy for ARDs. Full article

(This article belongs to the Special Issue Genetic and Cellular Basis of Autoimmune Diseases)

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Graphical abstract

14 pages, 1292 KiB

Open AccessArticle

The Adipose Tissue-Derived Secretome (ADS) in Obesity Uniquely Regulates the Na-Glucose Transporter SGLT1 in Intestinal Epithelial Cells

by Vivian Naa Amua Wellington and Soudamani Singh

Cells 2025, 14(16), 1241; https://doi.org/10.3390/cells14161241 - 12 Aug 2025

Abstract

Obesity is a complex chronic inflammatory condition that results from excess fat accumulation. It increases the risk of developing numerous co-morbidities such as Type 2 diabetes mellitus, cardiovascular disease, hypertension, and stroke. The adipose tissue is itself a vital endocrine organ that secretes [...] Read more.

Obesity is a complex chronic inflammatory condition that results from excess fat accumulation. It increases the risk of developing numerous co-morbidities such as Type 2 diabetes mellitus, cardiovascular disease, hypertension, and stroke. The adipose tissue is itself a vital endocrine organ that secretes numerous adipokines, cytokines, and exosomes, which are collectively known as the adipose-derived secretome (ADS). This ADS has been shown to influence and modulate many physiological processes. During obesity, the composition of ADS is altered, which may contribute to the development of obesity-associated diseases. Type-2 diabetes mellitus is one of the most common complications of obesity due to alterations in glucose homeostasis. Glucose absorption occurs via Na-glucose co-transport via SGLT1 at the brush border membrane (BBM) of small intestinal villus cells. This process of transepithelial glucose uptake is the primary method of glucose absorption from diet. However, how ADS mediates the function of SGLT1 is not yet known. This study aims to determine the mechanism of regulation of SGLT1 by ADS in intestinal epithelial cells. We show that ADS from OZR (but not LZR) stimulates SGLT1 in IEC-18 cells. OZR-ADS treatment diminished Na/K-ATPase activity in IEC-18 cells. Kinetic studies indicated that the mechanism of stimulation for SGLT1 during OZR-ADS treatment was secondary to an increase in the affinity (1/K_m) of the co-transporter for glucose without a change in co-transporter number. Western blot studies revealed that SGLT1 protein expression was unaltered in the two groups, confirming our kinetic studies. Immunoprecipitation demonstrated that an increase in the affinity of the SGLT1 protein was mediated by altered phosphorylation. In conclusion, during obesity, the adipose tissue secretome stimulates SGLT1 in intestinal epithelial cells, leading to an increase in affinity for glucose. The affinity change is due to alterations in SGLT1 phosphorylation. Together, these results may provide important insight into the mechanisms underlying altered glucose homeostasis in obesity and how this may lead to the development of Type 2 diabetes mellitus. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Browning of Adipocytes in Metabolic Health and Disease)

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15 pages, 6702 KiB

Open AccessArticle

CREB5 Promotes the Proliferation of Neural Stem/Progenitor Cells in the Rat Subventricular Zone via the Regulation of NFIX Expression

by Tao Yu, Hanyue Zhang, Chuang Zhang, Guorui Ma, Tu Shen, Yan Luan and Zhichao Zhang

Cells 2025, 14(16), 1240; https://doi.org/10.3390/cells14161240 - 12 Aug 2025

Abstract

Neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of the central nervous system (CNS) are critical for tissue repair following injury or disease. These cells retain the capacity to proliferate, migrate, and differentiate into neurons, astrocytes, and oligodendrocytes, making them a promising [...] Read more.

Neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of the central nervous system (CNS) are critical for tissue repair following injury or disease. These cells retain the capacity to proliferate, migrate, and differentiate into neurons, astrocytes, and oligodendrocytes, making them a promising therapeutic target for neurodegenerative disorders and traumatic injuries. However, the molecular mechanisms regulating their proliferation remain incompletely understood. This study investigates the role of cAMP responsive element-binding protein 5 (CREB5) in the proliferation of rat SVZ-derived NSPCs and elucidates its regulatory mechanism. Using RNA interference, we demonstrated that CREB5 knockdown significantly reduced cell viability, neurosphere formation capacity, and the number of proliferating cells (BrdU- and Ki-67-positive cells) both in vitro and in vivo. In contrast, CREB5 overexpression played opposing roles in cell proliferation. Additionally, alteration of CREB5 expression did not affect apoptosis, as assessed by TUNEL staining, indicating a specific role in proliferation rather than in cell death. Mechanistically, we identified Nuclear Factor One X (NFIX) as a transcriptional target of CREB5. CREB5 binds to the AP-1 site in the NFIX promoter, enhancing its expression. CREB5 knockdown inhibited NFIX expression, while CREB5 overexpression exerted the opposite function. ChIP and luciferase reporter assays further confirmed that CREB5 directly regulates NFIX promoter activity. More importantly, alteration of NFIX expression could reverse the effect of CREB5 on NSPC proliferation. These findings highlight CREB5 as a key regulator of NSPC proliferation through its interaction with NFIX, providing a potential therapeutic target for stem cell-based treatments of CNS disorders. Full article

(This article belongs to the Special Issue Advances in the Regulation of Proteins and Genes for Stem Cells)

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27 pages, 5035 KiB

Open AccessArticle

Mycn Is Essential for Pubertal Mammary Gland Development and Promotes the Activation of Bcl11b-Maintained Quiescent Stem Cells

by Zuobao Lin, Chunhui Wang, Huiru Bai, Yue Zhang, Meizhen Lin, Xiaoqin Liu, Tian’en Hu and Yuan Meng

Cells 2025, 14(16), 1239; https://doi.org/10.3390/cells14161239 - 12 Aug 2025

Abstract

This investigation examines the function of the mouse Mycn gene in regulating and activating quiescent mammary stem cells, which are vital for mammary gland development. The mammary gland, consisting of luminal and basal cells, progresses through complex developmental stages from embryonic development through [...] Read more.

This investigation examines the function of the mouse Mycn gene in regulating and activating quiescent mammary stem cells, which are vital for mammary gland development. The mammary gland, consisting of luminal and basal cells, progresses through complex developmental stages from embryonic development through puberty, adulthood, pregnancy, lactation, and involution. Quiescent stem cells, existing in a reversible non-proliferative state, are essential for gland maintenance, yet their activation mechanisms remain poorly understood. Mycn, a member of the Myc/MYC oncogene family, is recognized for its roles in embryonic development and cancer, notably aggressive neuroblastoma and triple-negative breast cancer. Through single-cell RNA sequencing (scRNA-seq), CRISPR knockout, and overexpression experiments, this study demonstrates that Mycn is highly enriched in the terminal end buds (TEBs) of the pubertal mammary gland, particularly in basal cells, and is critical for ductal development. Both deletion and overexpression of Mycn diminish the stemness and regenerative capacity of mammary stem cells. Mycn enhances cell proliferation while downregulating quiescent stem cell markers and regulators, including Bcl11b and Tspan8, affecting stem cell maintenance and differentiation. This research clarifies the regulatory role of Bcl11b in controlling Tspan8 expression and demonstrates that Mycn indirectly targets both under normal conditions. Maintaining appropriate levels of Mycn expression is essential for normal development and cancer prevention. These insights contribute to understanding diseases and aggressive cancers, including triple-negative breast cancer (TNBC), and suggest potential therapeutic approaches. Full article

(This article belongs to the Special Issue Regulation of Cell Division)

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16 pages, 2967 KiB

Open AccessArticle

Adeno-Associated Viral Gene Delivery of Wild-Type Human Tau Induces Progressive Hyperphosphorylation and Neuronal Cell Death in the Hippocampi of Middle-Aged Rats

by Ryan C. Gorzek, Aurelie Joly-Amado, Natalia Hurst-Calle, Graham L. Gabrielson, Maxine Miller, Sue Osting, Kevin R. Nash and Corinna Burger

Cells 2025, 14(16), 1238; https://doi.org/10.3390/cells14161238 - 11 Aug 2025

Abstract

Tau aggregation and the subsequent formation of neurofibrillary tangles are hallmarks of Alzheimer’s disease (AD) and other dementias. While accumulation of tau aggregates is believed to contribute to cell death and neurodegeneration, tau aggregation and hyperphosphorylation are also correlated with cognitive impairment in [...] Read more.

Tau aggregation and the subsequent formation of neurofibrillary tangles are hallmarks of Alzheimer’s disease (AD) and other dementias. While accumulation of tau aggregates is believed to contribute to cell death and neurodegeneration, tau aggregation and hyperphosphorylation are also correlated with cognitive impairment in AD. To understand the role of tau in neurodegeneration, we used adeno-associated virus serotype 9 (AAV9) to express human wild-type 4-repeat, 0-N-terminus tau isoform (AAV-htau) in the Cornu ammonis area 1 (CA1) region of the dorsal hippocampus of adult 6-month-old Fischer 344 rats. AAV expressing green fluorescent protein (AAV-GFP) or uninjected rats were used as controls. To characterize early phenotypes, we investigated pathological changes at 3, 8, and 12 weeks post-injection of AAV-htau. Our results show that at 3 weeks post-injection, there was already robust expression of human tau in the CA1 region of animals injected with AAV-htau compared to those injected with AAV-GFP or the uninjected controls. At 12 weeks post-injection, area CA1 showed a statistically significant reduction in cell number and a thinner neuronal layer all throughout the anterior dorsal hippocampus, as well as redistribution to the somatodendritic areas of CA1. We also found hyperphosphorylation of tau at all three timepoints. In spite of this pathology, we did not find any hippocampal-dependent cognitive impairment in rats overexpressing human tau. These results provide evidence of AAV-htau as a progressive model of tauopathy pathology to study changes in phosphorylation status and neuronal cell death that might precede cognitive impairment. Full article

(This article belongs to the Special Issue Experimental Systems to Model Aging Processes)

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22 pages, 1700 KiB

Open AccessReview

Microbial Metabolites and Cardiovascular Dysfunction: A New Era of Diagnostics and Therapy

by Jitendra Kumar

Cells 2025, 14(16), 1237; https://doi.org/10.3390/cells14161237 - 11 Aug 2025

Abstract

Cardiovascular diseases (CVDs) pose a significant threat to human life and mortality worldwide, encompassing a variety of conditions that affect the heart and blood vessels. These diseases are influenced by both genetic and environmental factors, which play a critical role in their development. [...] Read more.

Cardiovascular diseases (CVDs) pose a significant threat to human life and mortality worldwide, encompassing a variety of conditions that affect the heart and blood vessels. These diseases are influenced by both genetic and environmental factors, which play a critical role in their development. Recent research has highlighted the importance of gut microbes—the diverse community of bacteria in the gastrointestinal tract—that function as a “super organ” within the human body. These microbes have a remarkable impact on metabolic pathways and are increasingly recognized for their role in serious conditions like CVDs. They contribute to metabolic regulation, provide essential nutrients and vitamins, and help protect against diseases. Various internal and external factors influence the dynamic relationship between the human host and gut microbiota, thereby regulating overall metabolism. This review explores the complex connection between gut microbiota and microbial metabolites—such as short-chain fatty acids (SCFAs), bile acids (BAs), and trimethylamine N-oxide (TMAO)—and their potential influence on the development and progression of CVDs. We also examine the interaction between dietary interventions and gut microbes in the context of conditions including atherosclerosis, obesity, type 2 diabetes, heart failure, hypertension, atrial fibrillation, and myocardial infarction. Gaining a deeper understanding of the gut microbiota’s role in maintaining physiological balance creates exciting possibilities for identifying novel diagnostic biomarkers and therapeutic targets for treating CVDs. This knowledge offers hope for early disease prediction, improved clinical management, and innovative treatments. Full article

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15 pages, 2451 KiB

Open AccessArticle

Systematic Comparison of Temperature Effects on Antibody Performance via Automated Image Analysis: A Key for Primary Ciliary Dyskinesia Diagnostic

by Hanna Przystalowska-Maciola, Malgorzata Dabrowska, Ewa Zietkiewicz and Bukowy-Bieryllo Zuzanna

Cells 2025, 14(16), 1236; https://doi.org/10.3390/cells14161236 - 11 Aug 2025

Abstract

Immunofluorescence (IF) microscopy of ciliated epithelium is gaining increased popularity as a pre-genetic diagnostic method in primary ciliary dyskinesia (PCD). Ensuring reliable IF-based diagnostics in PCD requires robust standardization of staining methods and antibody performance. We applied whole slide scanning and automated image [...] Read more.

Immunofluorescence (IF) microscopy of ciliated epithelium is gaining increased popularity as a pre-genetic diagnostic method in primary ciliary dyskinesia (PCD). Ensuring reliable IF-based diagnostics in PCD requires robust standardization of staining methods and antibody performance. We applied whole slide scanning and automated image analysis to systematically evaluate the influence of various sample storage conditions on the specificity of IF staining. We tested eight polyclonal antibodies targeting diverse axonemal protein epitopes, routinely used for PCD diagnostics, under seven different temperature and time combinations. The storage conditions simulated handling of epithelial brushing on glass slides: after material collection at the clinic, during transport, or after reception at the diagnostic laboratory. Our study revealed that proper slide storage conditions are essential for the reliable PCD diagnosis via IF staining. We suggest continuous storage at −80 °C or −20 °C for slides prepared at the diagnostic laboratory, and storage at −20 °C or 4 °C for slides prepared remotely and shipped. Moreover, the IF sensitivity to slide storage conditions differs among antibodies targeting various ciliary elements, with molecular ruler proteins being particularly sensitive to prolonged storage at room temperature. We emphasize the inclusion of additional control slides to mitigate the inter-individual differences and the crucial correlation of IF results with comprehensive patient clinical history for enhanced diagnostic reliability. Full article

(This article belongs to the Special Issue Imaging Methods in Cell Biology)

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20 pages, 6384 KiB

Open AccessArticle

Identification of Epigenetic Regulatory Networks of Gene Methylation–miRNA–Transcription Factor Feed-Forward Loops in Basal-like Breast Cancer

by Larissa M. Okano, Alexandre L. K. de Azevedo, Tamyres M. Carvalho, Jean Resende, Jessica M. Magno, Bonald C. Figueiredo, Tathiane M. Malta, Mauro A. A. Castro and Luciane R. Cavalli

Cells 2025, 14(16), 1235; https://doi.org/10.3390/cells14161235 - 10 Aug 2025

Abstract

Basal-like breast cancer (BLBC) is associated with poor prognosis, high recurrence rates, and limited therapeutic options, largely due to its molecular heterogeneity and complexity, which include epigenetic alterations. This study investigated epigenetic regulatory networks in BLBC by analyzing DNA methylation in distal cis-regulatory [...] Read more.

Basal-like breast cancer (BLBC) is associated with poor prognosis, high recurrence rates, and limited therapeutic options, largely due to its molecular heterogeneity and complexity, which include epigenetic alterations. This study investigated epigenetic regulatory networks in BLBC by analyzing DNA methylation in distal cis-regulatory regions and its impact on genes, transcription factors (TFs), and microRNAs (miRNAs) expression. Data from TCGA were processed using the ELMER and DESeq2 tools to identify differentially methylated regions and differentially expressed genes, TFs, and miRNAs. The FANMOD algorithm was used to identify the regulatory interactions uncovering the feed-forward loops (FFLs). The analysis identified 110 TF-mediated FFLs, 43 miRNA-mediated FFLs, and five composite FFLs, involving 18 hypermethylated and 32 hypomethylated genes, eight upregulated and nine downregulated TFs, and 21 upregulated and seven downregulated miRNAs. The TF-mediated FFLs major regulators involved the AR, EBF1, FOS, FOXM1, and TEAD4 TFs, while key miRNAs were miR-3662, miR-429, and miR-4434. Enriched pathways involved cAMP, ErbB, FoxO, p53, TGF-beta, Rap1, and Ras signaling. Differences in hallmark gene set categories reflected distinct methylation and miRNA expression profiles. Overall, this integrative analysis mapped the intricate epigenetic landscape of BLBC, emphasizing the role of FFLs as regulatory motifs that integrate DNA methylation, TFs, and miRNAs in orchestrating disease’s development and progression and offering potential targets for future diagnostic and therapeutic strategies. Full article

(This article belongs to the Special Issue Epigenetic Regulatory Networks in Cancer: Understanding the Mechanisms of Disease Progression)

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