Cells

16 pages, 5538 KiB

Open AccessArticle

Establishing Minimum Criteria for Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) Cultured in Human Platelet Lysate (hPL)-Contained Media as Cell Therapy Candidates: Characterization and Predictive Analysis of Secretome Effects

by Ji-Young Yoon, Bình Do Quang, Ji-Sun Shin, Jong-Bin Kim, Jun Hee Lee, Hae-Won Kim and Jung-Hwan Lee

Cells 2025, 14(4), 316; https://doi.org/10.3390/cells14040316 - 19 Feb 2025

Viewed by 205

Abstract

SHEDs have demonstrated significant potential in cell therapy due to their superior proliferation rate, self-renewal and differentiation capacity (particularly neurogenesis attributed to their neural crest origin), and the less invasive procedure required for tissue collection compared to other stem cells. However, there is [...] Read more.

SHEDs have demonstrated significant potential in cell therapy due to their superior proliferation rate, self-renewal and differentiation capacity (particularly neurogenesis attributed to their neural crest origin), and the less invasive procedure required for tissue collection compared to other stem cells. However, there is no established criterion to verify the minimum qualification to select one from numerous candidates, especially for SHEDs’ cultured FBS-free medium for clinic application. For that, we performed a characteristic analysis containing the growth rate, colony-forming unit (CFU) number, average colony size, and migration capacity with hPL-cultured SHEDs from 21 different donors, and we suggest the result as a minimum standard to filter out unqualified candidates. In addition, in the secretome analysis to predict the paracrine effect, it was found that upregulated proteins compared to the control were related to angiogenesis, immune response, and BMP signaling, and this was found to have a strong correlation only with protein concentration. This study presents a minimum standard for selecting cell therapy candidates and suggests the protein concentration of a conditioned medium as a cost-effective tool to expect the paracrine effect of SHEDs. Full article

(This article belongs to the Special Issue Human Dental Pulp Stem Cells: Isolation, Cultivation and Applications)

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Figure 1
Proliferation and self-renewal capacity analysis of SHEDs derived from 21 different donors. (A,B) Doubling times (A) and growth rates (B) were calculated based on cell counts at 24 and 72 h post 50,000 cell seeding. (C,D) Results from colony formation unit (CFU) assay show colony numbers (C) and average colony sizes (D) measured 10 days after seeding 500 cells. The light gray area and red bar in panels (A–D) indicate the average ± standard deviation (s.d.), with values above or below the average categorized as Good (red) or Poor (blue). (E,F) Representative images of CFU assays are shown at low magnification ((E), full 100 mm dish scan) and high magnification (F). Colonies were stained with crystal violet for visualization. Scale bar: 500 μm. (G) Correlation plots with linear regression showing the relationship between colony sizes and growth rates (left) or doubling times (right). The shaded blue area represents the 95% confidence interval (CI) for the regression line. Pearson’s correlation coefficient (r) values and two-tailed p-value are indicated (p). (A–D) Data are presented as mean ± s.d. (n = 3), with statistical significance determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 1 Cont.
Proliferation and self-renewal capacity analysis of SHEDs derived from 21 different donors. (A,B) Doubling times (A) and growth rates (B) were calculated based on cell counts at 24 and 72 h post 50,000 cell seeding. (C,D) Results from colony formation unit (CFU) assay show colony numbers (C) and average colony sizes (D) measured 10 days after seeding 500 cells. The light gray area and red bar in panels (A–D) indicate the average ± standard deviation (s.d.), with values above or below the average categorized as Good (red) or Poor (blue). (E,F) Representative images of CFU assays are shown at low magnification ((E), full 100 mm dish scan) and high magnification (F). Colonies were stained with crystal violet for visualization. Scale bar: 500 μm. (G) Correlation plots with linear regression showing the relationship between colony sizes and growth rates (left) or doubling times (right). The shaded blue area represents the 95% confidence interval (CI) for the regression line. Pearson’s correlation coefficient (r) values and two-tailed p-value are indicated (p). (A–D) Data are presented as mean ± s.d. (n = 3), with statistical significance determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">Figure 2
Migration capacity analysis of SHEDs derived from 21 different donors. (A,B) SHED (40,000 cells per well) were seeded in the upper chamber of a transwell insert and allowed to migrate for 24 h. (A) Quantification of migrated cell numbers based on DAPI-stained images. The red bar and the light gray area indicate the average ± standard deviation (s.d.). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test against the average. Data are presented as mean ± s.d. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (B) Representative images of migrated cells stained with crystal violet (upper). Scale bar: 100 μm. (C) Correlation plots with linear regression showing the relationship between colony size and migrated cell numbers. The shaded blue area represents the 95% confidence interval (CI). Pearson’s correlation coefficient (r = 0.46) and p-value (p = 0.03) were used to evaluate statistical significance. Full article ">Figure 3
Secretome analysis using conditioned media from SHEDs derived from 21 different donors. Differentially secreted proteins relative to blank (FC > 2.5, normalized value (log2) > 2) were analyzed. (A) Heatmap of secretome. (B) PCA analysis: #15, #17, #18, and #19 show distinct characteristics compared to others (yellow circle). (C) Biological process gene ontology from David analysis, with particularly interesting biological processes marked with different colored lines. (D) Heatmap of secretome from 21 different donors (FC > 2.5, normalized value (log2) > 2) assigned to angiogenesis, immune response, and BMP signaling pathway (as representative differentiation). FC: Fold change. Full article ">Figure 4
Total protein amounts from conditioned media can predict general secretome factors. (A) Schematic summary of secretome factor calculation. Fold changes of proteins upregulated over 2.5-fold compared to blank medium were summed to calculate the secretome factor. (B–F) Correlation plots with linear regression showing the relationship between secretome factor and various stem cell parameters including growth rate, CFU number, average colony size, migrated cell numbers, and total secreted proteins amounts. The blue shaded areas represent the 95% confidence interval of the regression line, illustrating the uncertainty in the correlation. Pearson’s correlation coefficient (r) was used to evaluate relationships, and statistical significance was assessed with two-tailed p-values. (G) Pearson’s correlation matrix showing relationships among the secretome factor, total protein amounts, and stem cell parameters. Only the total protein amount showed significant correlation with the secretome factor (*, p < 0.0001). (H) Pearson’s correlation matrix depicting associations between secretome factors and stem cell parameters. Total protein amount showed significant positive correlation with angiogenesis factors, immune response factors, and BMP signaling factor (*, p < 0.0001). Full article ">Figure 5
Schematic image of the current study. We conducted assays of proliferation, self-renewal, and migration on SHEDs obtained from 21 different donors. Additionally, we found that the secretome concentration related to angiogenesis, immunomodulation, and differentiation could be predicted by protein concentration. Based on these results, we propose criteria for the minimum qualification as a candidate for cell therapy. Cell candidates passing these criteria can undergo further studies, including preclinical studies, clinical research, and clinical trials, for eventual clinical application. Full article ">

14 pages, 3188 KiB

Open AccessArticle

Role of Hemocytes in the Aging of Drosophila Male Germline

by Virginia Varga, Janka Szinyákovics, Anikó Bebes, Fanni Szikszai and Tibor Kovács

Cells 2025, 14(4), 315; https://doi.org/10.3390/cells14040315 - 19 Feb 2025

Viewed by 109

Abstract

Stem cells are essential for the proper functioning of tissues, replacing damaged, senescent cells to ensure tissue regeneration. However, as age advances, the number of these stem cells can change, and their self-renewal abilities can become impaired, leading to disruption of homeostasis, loss [...] Read more.

Stem cells are essential for the proper functioning of tissues, replacing damaged, senescent cells to ensure tissue regeneration. However, as age advances, the number of these stem cells can change, and their self-renewal abilities can become impaired, leading to disruption of homeostasis, loss of regenerative capacity, and, ultimately, deterioration of tissue function. In Drosophila testis, in addition to the germline and somatic cells involved in spermatogenesis, there are immune cells (hemocytes) with macrophage function. In our study, we aimed to investigate the role of hemocytes in maintaining germline stem cells throughout their lifespan. Our results show that in the absence of plasmatocytes and crystal immune cells, the number of germline stem cells (GSCs) and apoptotic germline cells also increases significantly during senescence, which may have detrimental effects on the differentiation processes of germline cells. The size of the hub increases in aged male testes. It is therefore conceivable that changes in the hub may induce dysfunction of differentiation processes. The fertility of aged immunodeficient animals is decreased. Furthermore, we show that the expression of the JAK/STAT signaling pathway, which is essential for the maintenance of the stem cell niche, is impaired in the lack of hemocytes. We found an increased expression of Socs36e, an inhibitor of JAK-STAT, which correlates with decreased JAK-STAT activity. Overexpression of Socs36e in the apical part of the germline led to a phenotype similar to the immunodeficient aged germline, where an increased GSC number and hub size were also observed. However, spermatogenesis was also disturbed in this case. Our study shows that hemocytes are required to regulate the number of GSCs. This regulation could be mediated through the JAK-STAT signaling pathway. These results may help to provide a more complex insight into the relationships between immune cells and stem cells. Full article

(This article belongs to the Special Issue Microenvironmental Factors and Functional Modulation in Mesenchymal Stem Cells 2.0)

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Hemocytes are necessary to regulate the number of GSCs in the male germline during lifespan. (A,A′,B,B′) Hemolectin (Hml) and croquemort (Crq) hemocyte-specific Gal4s were used to express the UAS-GFPnls transgene. We found a decrease in the number of hemocytes in the apical part of the germline of aged male animals. (C) By immunohistochemistry, we labeled the germline with anti-VASA (green) and hub cells with anti-Faciclin III (red). The VASA-positive cells adjacent to the hub were considered GSCs according to the literature (dashed line). We induced immune cell apoptosis by hemocyte-specific overexpression of reaper (rpr) proapoptotic factor. (C′) The number of GSCs was significantly increased in immunodeficient 30- and 50-day samples. (C″) Due to the alteration in GSC number, we also measured the change in the size of hubs in testes from control and rpr-overexpressing animals at various ages. We also studied the size of the hub in testes at different ages by measuring the circumference of the hubs at their largest diameter (red dashed lines). Immunodeficiency significantly increased the hub diameter in older samples compared to age-matched controls. It is worth noting that hub size also increases in control samples during lifespan. The significance level was indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 2
Drosophila male fertility declines during the lifespan due to the absence of immune cells. (A) Apoptotic cells were labeled with TUNEL assay in control and immunodeficient testes of different ages. (A′) Germline cells have larger nuclei. These TUNEL-positive cells are quantified. Our results show increased apoptosis at the apical tip of old immunodeficient male germlines. (B,B′) We compared the fertility of control and immunodeficient young and old males. Males were crossed one at a time with wild-type Oregon virgin females. In young animals, no differences were found between the two investigated genotypes. However, at age 50 days, the fertility of immunodeficient males was reduced, with significantly fewer offspring than age-matched controls. (C) Anti-Hts/1B1 labeling (green) was used to examine the number of fusome structures in the testes of 50-day-old control and immune-depleted animals. (C′) Fewer fusomes were observed in immune-depleted testes. We used Hoechst for nuclei staining. The significance level was indicated as follows: *** p < 0.001. Full article ">Figure 3
The lack of immune cells leads to increased expression of socs36e and inhibition of JAK-STAT. (A,A′) Quantitative real-time PCR was used to investigate the mRNA levels of two well-known target genes of JAK-STAT (chinmo and Scocs36e) in young (10 days old) and old (50 days old) control and immune cell-deficient animals. The mRNA was isolated from the testes of the animals. Immune depletion was induced by overexpression of rpr in Hml-Gal4 positive cells. Both chinmo and socs36e expression were downregulated in aged control animals. However, increased socs36e mRNA levels were detected in 50-day immunodeficient animals. (B) The activity of the JAK-STAT signaling pathway was investigated by using 10xStat92e-GFP (green) reporter. Stat92e is a transcription factor of the JAK-STAT signaling pathway, and the expression of the regulated transgene can monitor its activity. (B′) In immunodeficient cells, the expression of 10xStat92e-GFP reporter was significantly decreased. (C) Anti-Hts/1B1 labeling (green) was used to examine the amount of fusosome structures in the testes in 50-day-old animals. Nanos-Gal4 was used to induce Socs36e overexpression in germline stem cells (GSCs) and gonialblast cells. (C′) Elevated expression of Socs36e reduced the amount of fusosome structures. (D) We examined the effect of Socs36e overexpression on both the number of GSCs and the size of the hub. Similar results were obtained in immunodeficient animals, (D′) significant increase in GSC number and (D″) hub size was observed. Anti-Fasciclin III (red) was used to label hub, and anti-VASA (green) antibody labels were used to label germline cells. In fluorescence images, nuclei were labeled using Hoechst staining. The significance level was indicated as follows: * p < 0.05; *** p < 0.001. Full article ">

19 pages, 6384 KiB

Open AccessArticle

A Critical Role of Intracellular PD-L1 in Promoting Ovarian Cancer Progression

by Rui Huang, Brad Nakamura, Rosemary Senguttuvan, Yi-Jia Li, Antons Martincuks, Rania Bakkar, Mihae Song, David K. Ann, Lorna Rodriguez-Rodriguez and Hua Yu

Cells 2025, 14(4), 314; https://doi.org/10.3390/cells14040314 - 19 Feb 2025

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Abstract

Disrupting the interaction between tumor-cell surface PD-L1 and T cell membrane PD-1 can elicit durable clinical responses. However, only about 10% of ovarian cancer patients respond to PD-1/PD-L1 blockade. Here, we show that PD-L1 expression in ovarian cancer-patient tumors is predominantly intracellular. Notably, [...] Read more.

Disrupting the interaction between tumor-cell surface PD-L1 and T cell membrane PD-1 can elicit durable clinical responses. However, only about 10% of ovarian cancer patients respond to PD-1/PD-L1 blockade. Here, we show that PD-L1 expression in ovarian cancer-patient tumors is predominantly intracellular. Notably, PARP inhibitor treatment highly increased intracellular PD-L1 accumulation in both ovarian cancer-patient tumor samples and cell lines. We investigated whether intracellular PD-L1 might play a critical role in ovarian cancer progression. Mutating the PD-L1 acetylation site in PEO1 and ID8^Brca1−/− ovarian cancer cells significantly decreased PD-L1 levels and impaired colony formation, which was accompanied by cell cycle G2/M arrest and apoptosis induction. PEO1 and ID8^Brca1−/− tumors with PD-L1 acetylation site mutation also exhibited significantly reduced growth in mice. Furthermore, targeting intracellular PD-L1 with a cell-penetrating antibody effectively decreased ovarian tumor-cell intracellular PD-L1 level and induced tumor-cell growth arrest and apoptosis, as well as enhanced DNA damage and STING activation, both in vitro and in vivo. In conclusion, we have shown the critical role of intracellular PD-L1 in ovarian cancer progression. Full article

(This article belongs to the Special Issue Ovarian Cancer and Endometriosis)

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17 pages, 10990 KiB

Open AccessArticle

Rotavirus Spreads in a Spatially Controlled Manner

by Gianna V. Passarelli, Patricio Doldan, Camila Metz-Zumaran, Yagmur Keser, Steeve Boulant and Megan L. Stanifer

Cells 2025, 14(4), 313; https://doi.org/10.3390/cells14040313 - 19 Feb 2025

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Abstract

Rotavirus is an enteric virus that leads to 200,000 deaths worldwide every year. The live-cell imaging evaluating rotavirus infection of MA104 cells revealed that rotavirus replication and spread occurs in a spatially controlled manner. Specifically, following initial rotavirus infection, the infected cells die, [...] Read more.

Rotavirus is an enteric virus that leads to 200,000 deaths worldwide every year. The live-cell imaging evaluating rotavirus infection of MA104 cells revealed that rotavirus replication and spread occurs in a spatially controlled manner. Specifically, following initial rotavirus infection, the infected cells die, and the second round of infection occurs in the restricted area surrounding the initially infected cell. Interestingly, we found that the time required to establish the secondary infection is shorter compared to the time required for the initial infection. To determine if this increase in the kinetic of secondary infection was due to the early release of viruses or priming of the cells that are infected during the secondary infection, we used a combination of live-cell microscopy, trypsin neutralization assays, and the pharmacological inhibition of calcium signaling. Together, our results show that the second round of infection required rotavirus to be released and accessible to extracellular proteases. In addition, we found that the calcium wave induced upon rotavirus infection was critical for initial infection but did not play a role in the establishment of a secondary infection. Finally, we uncovered that high viral titers released from the initial infection were sufficient to accelerate the rate of the secondary infection. Full article

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21 pages, 15816 KiB

Open AccessReview

Exploratory Review and In Silico Insights into circRNA and RNA-Binding Protein Roles in γ-Globin to β-Globin Switching

by Alawi Habara

Cells 2025, 14(4), 312; https://doi.org/10.3390/cells14040312 - 19 Feb 2025

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Abstract

β-globin gene cluster regulation involves complex mechanisms to ensure proper expression and function in RBCs. During development, switching occurs as γ-globin is replaced by β-globin. Key regulators, like BCL11A and ZBTB7A, repress γ-globin expression to facilitate this transition with other factors, like KLF1, [...] Read more.

β-globin gene cluster regulation involves complex mechanisms to ensure proper expression and function in RBCs. During development, switching occurs as γ-globin is replaced by β-globin. Key regulators, like BCL11A and ZBTB7A, repress γ-globin expression to facilitate this transition with other factors, like KLF1, LSD1, and PGC-1α; these regulators ensure an orchestrated transition from γ- to β-globin during development. While these mechanisms have been extensively studied, circRNAs have recently emerged as key contributors to gene regulation, but their role in β-globin gene cluster regulation remains largely unexplored. Although discovered in the 1970s, circRNAs have only recently been recognized for their functional roles, particularly in interactions with RNA-binding proteins. Understanding how circRNAs contribute to switching from γ- to β-globin could lead to new therapeutic strategies for hemoglobinopathies, such as sickle cell disease and β-thalassemia. This review uses the circAtlas 3.0 database to explore circRNA expressions in genes related to switching from γ- to β-globin expression, focusing on blood, bone marrow, liver, and spleen. It emphasizes the exploration of the potential interactions between circRNAs and RNA-binding proteins involved in β-globin gene cluster regulatory mechanisms, further enhancing our understanding of β-globin gene cluster expression. Full article

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12 pages, 1895 KiB

Open AccessArticle

Comparison Between Signal Transduction Pathway Activity in Blood Cells of Sepsis Patients and Laboratory Models

by Wilbert Bouwman, Reinier Raymakers, Tom van der Poll and Anja van de Stolpe

Cells 2025, 14(4), 311; https://doi.org/10.3390/cells14040311 - 19 Feb 2025

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Abstract

Sepsis represents a serious disease burden that lacks effective treatment. Drug development for sepsis requires laboratory models that adequately represent sepsis patients. Simultaneous Transcriptome-based Activity Profiling of Signal Transduction Pathway (STAP-STP) technology quantitatively infers STP activity from mRNA levels of target genes of [...] Read more.

Sepsis represents a serious disease burden that lacks effective treatment. Drug development for sepsis requires laboratory models that adequately represent sepsis patients. Simultaneous Transcriptome-based Activity Profiling of Signal Transduction Pathway (STAP-STP) technology quantitatively infers STP activity from mRNA levels of target genes of the STP-associated transcription factor. Here, we used STAP-STP technology to compare STP activities between sepsis patients and lipopolysaccharide (LPS)-based models. Activity scores of Androgen Receptor (AR), TGFβ, NFκB, JAK-STAT1/2, and JAK-STAT3 STPs were calculated based on publicly available transcriptome data. Peripheral blood mononuclear cells (PBMCs) from patients with Gram-negative sepsis, nor PBMCs stimulated with LPS in vitro, showed altered STP activity. Increased NFκB, JAK-STAT1/2, and JAK-STAT3 STP activity was found in whole blood stimulated with LPS in vitro, and in whole blood obtained after intravenous injection of LPS in humans in vivo; AR and TGFβ STP activity only increased in the in vivo LPS model. These results resembled previously reported STP activity in whole blood of sepsis patients. We provide the first comparison of STP activity between patients with sepsis and laboratory model systems. Results are of use for the refinement of sepsis model systems for rational drug development. Full article

(This article belongs to the Section Cell Signaling)

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Figure 1
Pathway activity scores of PBMCs from sepsis patients and after stimulation with LPS in vitro. (A) GSE9960 [<a href="#B28-cells-14-00311" class="html-bibr">28</a>]: PBMCs isolated from healthy and sepsis patients infected with Gram-positive, Gram-negative, or mixed Gram-positive and Gram-negative bacteria, or an unknown pathogen. (B) GSE46914 [<a href="#B30-cells-14-00311" class="html-bibr">30</a>]: PBMCs isolated from healthy donors: unstimulated (medium), stimulated once with LPS (LPS unprimed) or twice with LPS (LPS primed). Boxplot: Depicted are median and interquartile range (IQR, 25–75% percentile). A two-sided Mann–Whitney t-test was used to compare PAS across groups within the dataset. p-values (Bonferroni corrected) are indicated in the figures as ** p < 0.01 or ns (not significant). Pathway activity score (PAS) on Y-axis in log2odds. Full article ">Figure 1 Cont.
Pathway activity scores of PBMCs from sepsis patients and after stimulation with LPS in vitro. (A) GSE9960 [<a href="#B28-cells-14-00311" class="html-bibr">28</a>]: PBMCs isolated from healthy and sepsis patients infected with Gram-positive, Gram-negative, or mixed Gram-positive and Gram-negative bacteria, or an unknown pathogen. (B) GSE46914 [<a href="#B30-cells-14-00311" class="html-bibr">30</a>]: PBMCs isolated from healthy donors: unstimulated (medium), stimulated once with LPS (LPS unprimed) or twice with LPS (LPS primed). Boxplot: Depicted are median and interquartile range (IQR, 25–75% percentile). A two-sided Mann–Whitney t-test was used to compare PAS across groups within the dataset. p-values (Bonferroni corrected) are indicated in the figures as ** p < 0.01 or ns (not significant). Pathway activity score (PAS) on Y-axis in log2odds. Full article ">Figure 2
Pathway activity scores of whole blood from in vitro and in vivo endotoxemia LPS-based models. (A) GSE20114 [<a href="#B29-cells-14-00311" class="html-bibr">29</a>]: Whole blood in vitro LPS-based model. Samples were stimulated for 4 h with LPS or vehicle. (B) GSE3284 [<a href="#B31-cells-14-00311" class="html-bibr">31</a>]: Whole blood in vivo LPS endotoxemia model. Healthy humans received LPS. Whole blood was collected before LPS administration and 2 and 6 h thereafter. (A) Pathway activity score (PAS) of each individual patient is connected by a line and tested with a paired two-sided method. (B) A linear mixed-effects model was used to compare PAS across groups, and p-value annotations were added to the plot. p-values are indicated in the figures as ** p < 0.01, *** p < 0.001, **** p < 0.0001, or ns (not significant). PAS on Y-axis in log2odds. Full article ">Figure 2 Cont.
Pathway activity scores of whole blood from in vitro and in vivo endotoxemia LPS-based models. (A) GSE20114 [<a href="#B29-cells-14-00311" class="html-bibr">29</a>]: Whole blood in vitro LPS-based model. Samples were stimulated for 4 h with LPS or vehicle. (B) GSE3284 [<a href="#B31-cells-14-00311" class="html-bibr">31</a>]: Whole blood in vivo LPS endotoxemia model. Healthy humans received LPS. Whole blood was collected before LPS administration and 2 and 6 h thereafter. (A) Pathway activity score (PAS) of each individual patient is connected by a line and tested with a paired two-sided method. (B) A linear mixed-effects model was used to compare PAS across groups, and p-value annotations were added to the plot. p-values are indicated in the figures as ** p < 0.01, *** p < 0.001, **** p < 0.0001, or ns (not significant). PAS on Y-axis in log2odds. Full article ">

28 pages, 7761 KiB

Open AccessArticle

Therapeutic Targeting of the Galectin-1/miR-22-3p Axis Regulates Cell Cycle and EMT Depending on the Molecular Subtype of Breast Cancer

by Ju Yeon Kim, Jun Ho Lee, Eun Jung Jung, Young Sim Son, Hee Jin Park, Jae Myung Kim, Taejin Park, Sang-Ho Jeong, Jinkwon Lee, Tae Han Kim, Seon Min Lee and Jeong Doo Heo

Cells 2025, 14(4), 310; https://doi.org/10.3390/cells14040310 - 19 Feb 2025

Viewed by 197

Abstract

Breast cancer is a highly heterogeneous disease; hence, it is crucial to understand its biology and identify new targets for the development of effective treatments. Galectin-1 is known to play an oncogenic role in breast cancer progression. It is known that oncogenic factors [...] Read more.

Breast cancer is a highly heterogeneous disease; hence, it is crucial to understand its biology and identify new targets for the development of effective treatments. Galectin-1 is known to play an oncogenic role in breast cancer progression. It is known that oncogenic factors can influence cancer progression through interactions with miRNAs. The purpose of this study is to identify the clinical significance and biological role of galectin-1 and miR-22-3p in cancer progression according to the molecular subtype of breast cancer. We analyzed the expression of galectin-1 and miR-22-3p using cancer tissues and the correlation with clinical pathological characteristics. In addition, we investigated the regulation of the cell cycle and EMT processes of cancer progression through the galectin-1/miR-22-3p axis using cell lines of different breast cancer subtypes. miR-22-3p negatively regulates galectin-1 expression and the two molecules have opposite patterns of oncogenic and tumor-suppressive functions, respectively; furthermore, these two molecules are associated with metastasis-free survival. Cell experiments showed that miR-22-3p overexpression and galectin-1 knockdown inhibited the proliferation and invasion of breast cancer cells. Galectin-1 regulates different cancer progression pathways depending on the molecular subtype. In hormone receptor-positive breast cancer cells, galectin-1 knockdown mainly inhibited cell cycle-related substances and induced G0/G1 arrest, whereas in triple-negative breast cancer cells, it suppressed molecules related to the epithelial–mesenchymal transition pathway. In conclusion, the miR-22-3p/galectin-1 axis regulates different cancer metastasis mechanisms depending on the specific molecular subtype of breast cancer, and miR-22-3p/galectin-1 axis modulation may be a novel target for molecular subtype-specific personalized treatment. Full article

(This article belongs to the Special Issue Molecular Mechanism and Therapeutic Opportunities of Breast Cancer)

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17 pages, 1432 KiB

Open AccessReview

The CD39/CD73/Adenosine and NAD/CD38/CD203a/CD73 Axis in Cutaneous T-Cell Lymphomas

by Liyun Lin, Gabriele Roccuzzo, Yuliya Yakymiv, Sara Marchisio, Erika Ortolan, Ada Funaro, Rebecca Senetta, Valentina Pala, Martine Bagot, Adèle de Masson, Maxime Battistella, Emmanuella Guenova, Simone Ribero and Pietro Quaglino

Cells 2025, 14(4), 309; https://doi.org/10.3390/cells14040309 - 19 Feb 2025

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Abstract

Cutaneous T-cell lymphoma (CTCL), characterized by malignant T-cell proliferation primarily in the skin, includes subtypes such as mycosis fungoides (MF) and Sézary syndrome (SS). The tumor microenvironment (TME) is central to their pathogenesis, with flow cytometry and histology being the gold standards for [...] Read more.

Cutaneous T-cell lymphoma (CTCL), characterized by malignant T-cell proliferation primarily in the skin, includes subtypes such as mycosis fungoides (MF) and Sézary syndrome (SS). The tumor microenvironment (TME) is central to their pathogenesis, with flow cytometry and histology being the gold standards for detecting malignant T cells within the TME. Alongside emerging molecular markers, particularly clonality analysis, these tools are indispensable for accurate diagnosis and treatment planning. Of note, adenosine signaling within the TME has been shown to suppress immune responses, affecting various cell types. The expression of CD39, CD73, and CD38, enzymes involved in adenosine production, can be elevated in MF and SS, contributing to immune suppression. Conversely, the expression of CD26, part of the adenosine deaminase/CD26 complex, that degrades adenosine, is often lost by circulating tumoral cells. Flow cytometry has demonstrated increased levels of CD39 and CD73 on Sézary cells, correlating with disease progression and prognosis, while CD38 shows a variable expression, with its prognostic significance remaining under investigation. Understanding these markers’ roles in the complexity of TME-mediated immune evasion mechanisms might enhance diagnostic precision and offer new therapeutic targets in CTCL. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Lymphomas)

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14 pages, 409 KiB

Open AccessReview

Ultraviolet Radiation-Induced Tolerogenic Dendritic Cells in Skin: Insights and Mechanisms

by Gelare Ghajar-Rahimi, Nabiha Yusuf and Hui Xu

Cells 2025, 14(4), 308; https://doi.org/10.3390/cells14040308 - 18 Feb 2025

Viewed by 167

Abstract

Ultraviolet (UV) radiation has profound effects on the immune system, including the induction of tolerogenic dendritic cells (DCs), which contribute to immune suppression and tolerance. This review explores the roles of conventional CD11c⁺ DCs, as well as cutaneous Langerhans cells and CD11b⁺ myeloid [...] Read more.

Ultraviolet (UV) radiation has profound effects on the immune system, including the induction of tolerogenic dendritic cells (DCs), which contribute to immune suppression and tolerance. This review explores the roles of conventional CD11c⁺ DCs, as well as cutaneous Langerhans cells and CD11b⁺ myeloid cells, in UV-induced immune modulation. Two key mechanisms underlying the immunosuppressive relationship between UV and DCs are discussed: the inactivation of DCs and the induction of tolerogenic DCs. DCs serve as a critical link between the innate and adaptive immune systems, serving as professional antigen-presenting cells. In this context, we explore how UV-induced DCs influence the activity of specific T cell subsets, including regulatory T lymphocytes and T helper cells, and shape immune outcomes. Finally, we highlight the implications of UV-induced tolerogenic DCs in select dermatologic pathologies, including cutaneous lupus, polymorphic light eruption, and skin cancer. Understanding the mechanisms by which UV radiation alters DC function offers insights into the complex interplay between environmental factors and immune regulation, providing potential avenues for preventive and therapeutic intervention in UV-induced skin diseases. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Immune Regulation)

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23 pages, 3781 KiB

Open AccessReview

The Emerging Role of the Histone H2AK13/15 Ubiquitination: Mechanisms of Writing, Reading, and Erasing in DNA Damage Repair and Disease

by Qi Shu, Yun Liu and Huasong Ai

Cells 2025, 14(4), 307; https://doi.org/10.3390/cells14040307 - 18 Feb 2025

Viewed by 154

Abstract

Histone modifications serve as molecular switches controlling critical cellular processes. The ubiquitination of histone H2A at lysines 13 and 15 (H2AK13/15ub) is a crucial epigenetic modification that coordinates DNA repair and genome stability during the DNA damage response (DDR). This epigenetic mark is [...] Read more.

Histone modifications serve as molecular switches controlling critical cellular processes. The ubiquitination of histone H2A at lysines 13 and 15 (H2AK13/15ub) is a crucial epigenetic modification that coordinates DNA repair and genome stability during the DNA damage response (DDR). This epigenetic mark is dynamically regulated by three functional protein groups: “writer” enzymes (e.g., E3 ubiquitin ligase RNF168 that catalyzes H2AK13/15ub formation), “reader” proteins (including 53BP1 and BRCA1-BARD1 that recognize the mark to guide DNA repair), and “eraser” deubiquitinases (such as USP3 and USP16 that remove the modification). Dysregulation of the precisely coordinated network of H2AK13/15ub is strongly associated with various diseases, including RIDDLE syndrome, neurodegenerative disorders, immune deficiencies, and breast cancer. This review systematically analyzes the dynamic regulation of H2AK13/15ub in DDR and explores its therapeutic potential for disease intervention. Full article

(This article belongs to the Section Cell Microenvironment)

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20 pages, 3756 KiB

Open AccessArticle

Prenatal Exposure to Metals Is Associated with Placental Decelerated Epigenetic Gestational Age in a Sex-Dependent Manner in Infants Born Extremely Preterm

by Katelyn K. Huff, Kyle R. Roell, Lauren A. Eaves, Thomas Michael O’Shea and Rebecca C. Fry

Cells 2025, 14(4), 306; https://doi.org/10.3390/cells14040306 - 18 Feb 2025

Viewed by 254

Abstract

Prenatal exposure to metals can influence fetal programming via DNA methylation and has been linked to adverse birth outcomes and long-term consequences. Epigenetic clocks estimate the biological age of a given tissue based on DNA methylation and are potential health biomarkers. This study [...] Read more.

Prenatal exposure to metals can influence fetal programming via DNA methylation and has been linked to adverse birth outcomes and long-term consequences. Epigenetic clocks estimate the biological age of a given tissue based on DNA methylation and are potential health biomarkers. This study leveraged the Extremely Low Gestational Age Newborn (ELGAN) study (n = 265) to evaluate associations between umbilical cord tissue concentrations of 11 metals as single exposures as well as mixtures in relation to (1) placental epigenetic gestational age acceleration (eGAA) and the (2) methylation status of the Robust Placental Clock (RPC) CpGs. Linear mixed effect regression models were stratified by infant sex. Both copper (Cu) and manganese (Mn) were significantly associated with a decelerated placental eGA of −0.98 (95% confidence interval (CI): −1.89, −0.07) and −0.90 weeks (95% CI: −1.78, −0.01), respectively, in male infants. Cu and Mn levels were also associated with methylation at RPC CpGs within genes related to processes including energy homeostasis and inflammatory response in placenta. Overall, these findings suggest that prenatal exposures to Cu and Mn impact placental eGAA in a sex-dependent manner in ELGANs, and future work could examine eGAA as a potential mechanism mediating in utero metal exposures and later life consequences. Full article

(This article belongs to the Special Issue Molecular Advances in Prenatal Exposure to Environmental Toxicants)

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17 pages, 5006 KiB

Open AccessReview

The Terminal Segment of the Seminiferous Tubule: The Current Discovery of Its Morphofunctional Importance in Mammals

by Vicente Seco-Rovira, Ester Beltrán-Frutos, Jesús Martínez-Hernández, Juan Francisco Madrid and Luis Miguel Pastor

Cells 2025, 14(4), 305; https://doi.org/10.3390/cells14040305 - 18 Feb 2025

Viewed by 164

Abstract

The morphophysiology of intratesticular sperm pathways in mammals, including humans, is poorly understood. The seminiferous tubule is continuous with the straight tubule; however, its final portion—the terminal segment (TS)—has a different tissue composition. This paper reviews the most important histological results from mammal [...] Read more.

The morphophysiology of intratesticular sperm pathways in mammals, including humans, is poorly understood. The seminiferous tubule is continuous with the straight tubule; however, its final portion—the terminal segment (TS)—has a different tissue composition. This paper reviews the most important histological results from mammal studies from the last decades of the 20th century, including the different nomenclatures given to the TS. The TS presents a loss of spermatogenesis and is lined mainly with modified Sertoli cells. There is no unanimity among authors when it comes to naming and defining TS. In the last ten years, studies on rats and mice have highlighted the importance of this testicular zone, proposing that there is a high proliferation of modified Sertoli cells with an undifferentiated cellular profile associated with stem spermatogonia. In hamsters, an immunohistochemical study showed the existence of heterogeneity between these cells, and the surrounding interstitium presents numerous Leydig cells that are ultrastructurally different from those of the rest of the testis rest. In conclusion, we have only just begun to understand the tissue biology of TS. Emerging research is very promising; it can potentially modify our current knowledge of testicular biology and be very useful in promoting the advancement of male fertility restoration therapies in andrology. Full article

(This article belongs to the Special Issue Advances in Spermatogenesis)

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Light microscopy. Terminal segment of the hamster seminiferous tubule. (A) Seminiferous tubule with normal spermatogenesis (NST), followed below by the first subpart (between the dotted lines) in which the epithelium progressively loses spermatogenesis; hematoxylin and eosin staining (H&E). (B) Subpart with only modified SCs and a final zone of modified SCs that form a plug or valve (between continuous and dotted lines); H&E. (C) Modified SCs (arrows) in the plug or valve; H&E. (D) Receptacle or the initial part of the straight tubule (IPST). The continuous line demarcates the end of the SC valve (SV) with modified SCs (black arrows). The IPST exhibits a flattened epithelium (arrows red). Valve (V); H&E. (E,F) Semithin sections stained with Toluidine blue. (E) Some spermatogonia (red arrow) and spermatocytes (yellow arrows) are found in the TS epithelium. SCs: black arrows. The peritubular interstitium shows several layers of myoid cells (red square). (F) Clearly shows the valve plug (V). Flatted cells of the IPST epithelium (red arrows) and IPST lumen (asterisks). Scale bar = 25 µm. From [<a href="#B47-cells-14-00305" class="html-bibr">47</a>,<a href="#B48-cells-14-00305" class="html-bibr">48</a>]. Full article ">Figure 2
Summary of the results obtained in the terminal segment epithelium of Syrian hamster seminiferous tubules. NST: Seminiferous tubule with normal spermatogenesis. IPST: Initial part of the straight tubule. ST: Medial and final segment of the straight tubule. Vim: Vimentin intermediate filaments. CK8/18: Cytokeratin 8/18 intermediate filaments. Full article ">Figure 3
Light immunohistochemistry detection of desmin, actin, and cytokeratin 8/18 in the hamster TS and ST. (A) Desmin-positive SCs are only located in the initial subpart of the TS (red asterisk), where the epithelium progressively loses spermatogenesis. In the initial part of the straight tubule (IPST) and seminiferous tubules, in which spermatogenesis is complete, SCs are desmin-negative. (B) Transversal section of the initial subpart of the TS (red asterisk). Arrows: desmin-positive SCs. (C) The cytoplasm of modified SCs in the valve (V) is highly positive for actin (red asterisk). (D) Immunoreactivity to cytokeratin 8/18 is found in the epithelium of the IPST and ST. The modified SCs of the valve (V) are cytokeratin 8/18-negative. Scale bar = 25 µm. From [<a href="#B47-cells-14-00305" class="html-bibr">47</a>,<a href="#B48-cells-14-00305" class="html-bibr">48</a>]. Full article ">Figure 4
(A,B) Cyclin D1 immunohistochemistry. Most modified Sertoli cells (SCs) of the TS are Cyclin D1-positive, as are some from the initial part of the straight tubule (IPST). In normal spermatogenesis (NST), positive SCs are not observed. (C) In this double-stained confocal microscopy image, D1-positive cells are also vimentin-positive. (D) Modified SCs of the TS are strongly positive for HSP47. (E) Low-magnification electronography of modified SCs (mSC) with transmission electron microscopy (TEM). Indentations are seen in some nuclei, whereas others are more spherical. (F) Electrolucid cells (ELC) are observed with TEM in the ST and RT. Insert: These cells show proliferation and are positive for proliferating cell nuclear antigen (PCNA) (red arrows). Scale bar: (A–C) = 50 µm; (D) = 25 µm; (F) (light microscopy) = 50 µm; (E,F) = 5 µm. From [<a href="#B47-cells-14-00305" class="html-bibr">47</a>,<a href="#B48-cells-14-00305" class="html-bibr">48</a>]. Full article ">Figure 5
(A1,A2) Light microscopy of semithin sections. Hamster intratesticular pathway interstitium. (A1) shows a large group of Leydig cells can be seen in this interstitium. (A2) shows them close to blood capillaries. RT: rete testis. V: valve. (B1,B2) Transmission electron microscopy. A group of Leydig cells can be seen. LC: Leydig cells. (B2) is at higher magnification and it is easy to identify large mitochondria (M) as abundant rough and smooth endoplasmic reticula in the cytoplasm. Scale bar: A1 = 50 µm; A2 = 25 µm; B1,B2 = 5 µm. (C) Number of Leydig cells by area (mm2) and volume (mm3) in zone A (TS) and zone B (remaining testis). Results are expressed as mean ± SD (n = 12 per group). Significantly different results (p < 0.05) are expressed as: *—relative to the Zone B group. From [<a href="#B62-cells-14-00305" class="html-bibr">62</a>]. Full article ">

25 pages, 1622 KiB

Open AccessFeature PaperReview

NEMO Family of Proteins as Polyubiquitin Receptors: Illustrating Non-Degradative Polyubiquitination’s Roles in Health and Disease

by Chuan-Jin Wu

Cells 2025, 14(4), 304; https://doi.org/10.3390/cells14040304 - 18 Feb 2025

Viewed by 192

Abstract

The IκB kinase (IKK) complex plays a central role in many signaling pathways that activate NF-κB, which turns on a battery of genes important for immune response, inflammation, and cancer development. Ubiquitination is one of the most prevalent post-translational modifications of proteins and [...] Read more.

The IκB kinase (IKK) complex plays a central role in many signaling pathways that activate NF-κB, which turns on a battery of genes important for immune response, inflammation, and cancer development. Ubiquitination is one of the most prevalent post-translational modifications of proteins and is best known for targeting substrates for proteasomal degradation. The investigations of NF-κB signaling pathway primed the unveiling of the non-degradative roles of protein ubiquitination. The NF-κB-essential modulator (NEMO) is the IKK regulatory subunit that is essential for IKK activation by diverse intrinsic and extrinsic stimuli. The studies centered on NEMO as a polyubiquitin-binding protein have remarkably advanced understandings of how NEMO transmits signals to NF-κB activation and have laid a foundation for determining the molecular events demonstrating non-degradative ubiquitination as a major driving element in IKK activation. Furthermore, these studies have largely solved the enigma that IKK can be activated by diverse pathways that employ distinct sets of intermediaries in transmitting signals. NEMO and NEMO-related proteins that include optineurin, ABIN1, ABIN2, ABIN3, and CEP55, as non-degradative ubiquitin chain receptors, play a key role in sensing and transmitting ubiquitin signals embodied in different topologies of polyubiquitin chains for a variety of cellular processes and body responses. Studies of these multifaceted proteins in ubiquitin sensing have promoted understanding about the functions of non-degradative ubiquitination in intracellular signaling, protein trafficking, proteostasis, immune response, DNA damage response, and cell cycle control. In this review, I will also discuss how dysfunction in the NEMO family of protein-mediated non-degradative ubiquitin signaling is associated with various diseases, including immune disorders, neurodegenerative diseases, and cancer, and how microbial virulence factors target NEMO to induce pathogenesis or manipulate host response. A profound understanding of the molecular bases for non-degradative ubiquitin signaling will be valuable for developing tailored approaches for therapeutic purposes. Full article

(This article belongs to the Special Issue Non-Traditional Roles of Protein Ubiquitination in Cellular Processes and Health)

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Schematic representation of the molecular structure and functional domains of the NEMO family of human proteins. The intervening domain (IVD, aa112–195) of NEMO mediates allosteric communication between the N- and C-terminal regions and conformational change that enhances IKKβ activation, following the interaction of NEMO with polyubiquitin. The N-terminal aa1–127 of optineurin is responsible for its interaction with TBK1. KBD: IKK-binding domain; HLX: helical domain; CC: coiled coil; LZ: leucine zipper; UBAN: ubiquitin binding in ABIN and NEMO; ZF: zinc finger; LIR: LC3 interaction region; EABR: ESCRT- and ALIX-binding region; AHD: ABIN homology domain; NBD: NEMO binding domain [<a href="#B8-cells-14-00304" class="html-bibr">8</a>,<a href="#B9-cells-14-00304" class="html-bibr">9</a>,<a href="#B10-cells-14-00304" class="html-bibr">10</a>,<a href="#B18-cells-14-00304" class="html-bibr">18</a>]. Full article ">Figure 2
The signaling cascades generalized for ubiquitin-mediated IKK activation in IL-1R/TLR and TNF pathways. K63-linked ubiquitin chains modified to IRAK1 by TRAF6 in the IL-1R/TLR pathway or RIP1 by cIAP1/2 in the TNF pathway bind to TAB2/3 subunits of the TAK1 kinase complex, and this binding promotes autophosphorylation of TAK1, which results in its activation. In the meantime, the K63-linked and/or linear ubiquitin chains resulting from the LUBAC catalyzation on IRAK1 or RIP1 also bind to NEMO, bringing the IKK complex into the vicinity of TAK1, thereby facilitating the phosphorylation and subsequent activation of IKKβ by TAK1. An arrow with a curved dashed line indicates “ubiquitinate”, and an arrow with a curved solid line indicates “phosphorylate”. The different ubiquitin chain linkages are color-coded. The depiction of the signaling model is adapted from the articles [<a href="#B21-cells-14-00304" class="html-bibr">21</a>,<a href="#B47-cells-14-00304" class="html-bibr">47</a>]. Full article ">Figure 3
Ubiquitin-mediated IKK activation and Bcl10 autophagy in the T cell signaling pathway. The binding of the TCR to the antigen peptide presented by MHC molecules (p-MHC) induces T cell activation and leads to the initiation of a T cell response. When TCR:pMHC binding occurs, the TCR complex is phosphorylated by Lck. Phosphorylated signaling motifs in the TCR complex recruit the ZAP70 kinase. The recruitment of ZAP70 and its phosphorylation by Lck causes its activation. ZAP70 then phosphorylates the adaptor protein LAT, which recruits additional signaling effectors that become activated, leading to PKCθ activation. The activated PKCθ phosphorylates CARMA1 in the CARMA1-Bcl10-MALT1 complex, inducing K63-linked or linear ubiquitination of Bcl10, which in turn binds to NEMO in the IKK complex. The formation of supercomplexes leads to liquid phase separation, which is presented as punctate cytosolic structures called POLKADOTS under a microscope [<a href="#B104-cells-14-00304" class="html-bibr">104</a>]. The liquid phase separation and ubiquitin-mediated NEMO conformational changes induce IKKβ activation that triggers IκB phosphorylation, ubiquitination, and proteasomal degradation, then NF-κB activation. Subsequently, the complexes encompassing ubiquitinated Bcl10 undergo autophagy that leads to Bcl10 lysosomal degradation and the inhibition of T cell signaling-induced NF-κB and MAPK activation. Full article ">

22 pages, 4264 KiB

Open AccessArticle

Seasonal Influences on Human Placental Transcriptomes Associated with Spontaneous Preterm Birth

by Khondoker M. Akram, Eleanor Dodd and Dilly O. C. Anumba

Cells 2025, 14(4), 303; https://doi.org/10.3390/cells14040303 - 18 Feb 2025

Viewed by 303

Abstract

Demographic studies have revealed a strong association between exposure to high ambient temperatures during pregnancy and increased risks of preterm birth (PTB). The mechanism underlying this association is unclear, but it is plausible that altered placental function may contribute to it. In this [...] Read more.

Demographic studies have revealed a strong association between exposure to high ambient temperatures during pregnancy and increased risks of preterm birth (PTB). The mechanism underlying this association is unclear, but it is plausible that altered placental function may contribute to it. In this study, we conducted differential gene expression analysis, gene set enrichment analysis (GSEA), and gene ontology (GO) analysis on bulk RNA-seq data from human placentas delivered at term and preterm during the warmer months compared to placentas delivered at term and preterm during the colder months in the UK. We detected 48 differentially expressed genes in preterm placentas delivered during the warmer months compared to preterm placentas delivered during the colder months, the majority of which were inflammatory cytokines and chemokines, including SERPINA1, IL1B, CCL3, CCL3L3, CCL4, CCL4L2, CCL20, and CXCL8. The GSEA positively enriched 17 signalling pathways, including the NF-κB, IL17, Toll-like receptor, and chemokine signalling pathways in preterm placentas delivered during warmer months. These results were not observed in the placentas delivered at term during the same times of the year. The GO analysis revealed several enhanced biological processes, including neutrophil, granulocyte, monocyte, and lymphocyte chemotaxis, as well as inflammatory and humoral immune responses in preterm placentas, but not in placentas delivered at term in the summer. We conclude that maternal exposure to warm environmental temperatures during pregnancy likely alters the placental transcriptomes towards inflammation and immune regulation, potentially leading to PTB. Full article

(This article belongs to the Special Issue Molecular Insight into the Pathogenesis of Spontaneous Preterm Birth)

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Environmental temperature exposure during pregnancy. The rain-cloud plots showing monthly mean temperature (A) and average maximum temperature (B) when the women delivered. The clouds show the data kernel density, red dots with connectors show the mean, box plots show the median with the IQR, and each dot represents an individual subject. n = 10 warm group and n = 15 cold group (A,B). p values were calculated by the Mann–Whitney U test. (C) Box blots with jitters showing the duration of exposure of 4 groups of pregnant women to warm or cold weather temperatures prior to delivery. Data are presented as median values with the IQR, and each dot represents an individual subject. p = 0.82, as determined by one-way ANOVA. Full article ">Figure 2
Differential gene expression analysis between warm and cold groups of placentas. (A) PCA plots showing group separation between warm and cold groups in preterm and term placentas. (B) Volcano plots showing DEGs in preterm-warm placentas compared to the preterm-cold group. (C) A heat map showing the expression of 48 significant DEGs in preterm-warm and preterm-cold placentas. (D) Signalling pathways in preterm-warm placentas positively enriched by GSEA. (E) The comparison of expression by a selected set of genes between term and preterm placentas. Full article ">Figure 3
Cell-type enrichment and gene ontology analysis. (A) A bar plot showing the positive enrichment of cell types in preterm-warm placentas identified by GSEA. (B) A chord plot showing links between individual DEGs and enriched cell types. (C) A bar plot showing biological processes and cell components significantly enriched with the upregulated DEGs in preterm-warm placentas. (D) A bar plot showing disease phenotype enrichment with upregulated DEGs in the preterm-warm placentas. Full article ">Figure 4
Differential gene expression and KEGG pathway analyses between preterm and term placentas. (A) A Venn diagram showing the overlap between significantly upregulated DEGs from the preterm-warm vs. preterm-cold and preterm-warm vs. term-warm group analyses. The common upregulated DEGs (FDR < 0.05) are given in the box. (B) KEGG pathway enrichment with upregulated DEGs from preterm-warm vs. term-warm placentas. (C) Venn diagram showing the overlap of significantly enriched KEGG signalling pathways in Preterm-warm vs. Preterm-cold and Preterm-warm vs. Term-warm group analyses. The common upregulated pathways are shown in box (FDR < 0.05). Full article ">Figure 5
Individual gene expression in preterm placentas. (A–L) Box plots with jitters showing expressions of a selected set of DEGs in the warm and cold groups of preterm placentas. Data are presented as the median with the IQR. Each dot represents individual subjects. p values were determined by the Wilcoxon signed-rank test. (M) STRING protein–protein interaction analysis between the HPGD and related proteins (STRING v12). Full article ">Figure 6
ELISA assay on placenta tissue. Box plots showing SERPINA1 protein concentrations in preterm-warm and preterm-cold placentas (A) and in term-warm and term-cold placentas (B). A protein concentration of 300 µg/mL was used as the loading concentration for each sample in the ELISA assay. Data are presented as median with interquartile range (IQR). Each dot represents an individual subject. The p value was determined by a two-tailed unpaired Student’s t-test. ns = not significant. Full article ">Figure 7
Gene interaction network analysis by Cytoscape plug-in GeneMANIA. Striped bigger nodes indicate DEGs upregulated in preterm-warm placentas. Colour codes inside each node indicate their biological function as stated in the Functions legend. Coloured edges and their connections with other genes indicate the nature of interactions. Full article ">

32 pages, 3557 KiB

Open AccessArticle

Secretome Analysis of Human and Rat Pancreatic Islets Co-Cultured with Adipose-Derived Stromal Cells Reveals a Signature with Enhanced Regenerative Capacities

by Erika Pinheiro-Machado, Bart J. de Haan, Marten A. Engelse and Alexandra M. Smink

Cells 2025, 14(4), 302; https://doi.org/10.3390/cells14040302 - 18 Feb 2025

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Abstract

Pancreatic islet transplantation (PIT) is a promising treatment for type 1 diabetes (T1D) but faces challenges pre- and post-transplantation. Co-transplantation with mesenchymal stromal cells (MSCs), known for their regenerative properties, has shown potential in improving PIT outcomes. This study examined the secretome of [...] Read more.

Pancreatic islet transplantation (PIT) is a promising treatment for type 1 diabetes (T1D) but faces challenges pre- and post-transplantation. Co-transplantation with mesenchymal stromal cells (MSCs), known for their regenerative properties, has shown potential in improving PIT outcomes. This study examined the secretome of islets cultured alone compared to the secretomes of islets co-cultured with adipose-derived stromal cells (ASCs), a subtype of MSCs, under transplantation-relevant stressors: normoxia, cytokines, high glucose, hypoxia, and combined hypoxia and high glucose. Islet co-culture with ASCs significantly altered the proteome, affecting pathways related to energy metabolism, angiogenesis, extracellular matrix organization, and immune modulation. Key signaling molecules (e.g., VEGF, PDGF, bFGF, Collagen I alpha 1, IL-1α, and IL-10) were differentially regulated depending on culture conditions and ASC presence. Functional assays demonstrated that the co-culture secretome could enhance angiogenesis, collagen deposition, and immune modulation, depending on the stress conditions. These findings highlight possible mechanisms through which ASCs may support islet survival and function, offering insights into overcoming PIT challenges. Moreover, this work contributes to identifying biomarkers of the post-transplantation microenvironment, advancing therapeutic strategies for T1D and regenerative medicine. Full article

(This article belongs to the Special Issue Adipose-Derived Stem Cells: Current Applications and Future Directions)

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13 pages, 1406 KiB

Open AccessArticle

Humanized FcεRI Expressed on Mouse Eosinophils Mediates IgE-Facilitated Eosinophil Antigen Presentation

by Haibin Wang, Jean-Pierre Kinet and Peter F. Weller

Cells 2025, 14(4), 301; https://doi.org/10.3390/cells14040301 - 18 Feb 2025

Viewed by 146

Abstract

High-affinity IgE receptors (FcεRI) are expressed on human blood eosinophils and may be upregulated on eosinophils at sites of allergic inflammation including atopic dermatitis and allergic asthma. FcεRI engagement, however, fails to elicit “effector” responses from eosinophils. Thus, a functional role for FcεRI [...] Read more.

High-affinity IgE receptors (FcεRI) are expressed on human blood eosinophils and may be upregulated on eosinophils at sites of allergic inflammation including atopic dermatitis and allergic asthma. FcεRI engagement, however, fails to elicit “effector” responses from eosinophils. Thus, a functional role for FcεRI on eosinophils has been uncertain. We evaluated the role of FcεRI in enhancing eosinophil antigen presentation in vivo by using humanized FcεRI α chain (hFcεRIα) transgenic mice. Eosinophils from hFcεRIα transgenic mice expressed humanized FcεRIα, with higher levels of eosinophils from the bronchoalveolar lavage of experimental asthma than those from polymyxin-elicited peritoneal lavage. The hFcεRIα-bearing eosinophils instilled intratracheally (i.t.) into recipient wild-type mice migrated from airways into paratracheal lymph nodes (pLNs) and spleens. Eosinophils, pretreated in vitro with nitrophenyl-ovalbumin ((NP)-OVA) and anti-NP human IgE complexes and instilled i.t., presented NP antigen via hFcεRIα to T cells more effectively than those pretreated with NP-OVA only, as assessed by pLN cell proliferation. IgE/FcεRIα-facilitated eosinophil antigen presentation resulted in increased IL-4 but not INF-γ production by pLN cells, with a bias towards Th2 cytokine production. Furthermore, cross-linking hFcεRIα on eosinophils increased eosinophil expressions of T cell costimulatory proteins CD40, CD80, and CD86. Humanized FcεRIα on murine eosinophils functions to enhance eosinophil antigen presentation capacities by mediating IgE-facilitated antigen presentation and upregulating expression of requisite T cell costimulatory proteins. Thus, a functional, non-“effector” role for FcεRI on eosinophils is revealed through identifying a means by which IgE may act on eosinophils to mediate their immunomodulatory, enhanced antigen presentation capabilities. Full article

(This article belongs to the Special Issue Eosinophils and Their Role in Allergy and Related Diseases)

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Graphical abstract

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Full article ">Figure 1
HFcεRIα expressing eosinophils take up Ag in the lungs and migrate to pLNs and spleens. (A) Eosinophils from peritoneal exudates or from BAL of OVA-sensitized and airways-challenged hFcεRIα mice expressed hFcεRI detected by anti-hFcεRI 15.1 mAb vs control IgG1 mAb. (B) In OVA-sensitized and airways-challenged mice, FcεRI-facilitated Texas Red-OVA Ag uptake by hFcεRIα eosinophils (red line) in comparison with WT eosinophils (filled) or eosinophils from control i.t. recipients of unlabeled OVA. (C) Eosinophils from hFcεRIα mice, stained with DiIC16(3) and instilled i.t into WT mice, migrated from the airways to pLNs and spleens. (D) Trafficking of DiIC16(3) and hFcεRIα double positive airways instilled eosinophils into pLNs and spleens, calculated by multiplying total tissue cells by percentage of DiIC16(3) and hFcεRIα double positive eosinophils vs numbers of instilled DiIC16(3) and hFcεRIα double positive eosinophils (SD, triplicates). Full article ">Figure 2
IgE-hFcεRI engagement facilitates eosinophil antigen presentation in vivo to enhance pLN T cell proliferation and IL-4 release and to increase eosinophil CD40, CD80, and CD86 expression. (A,B) hFcεRIα eosinophils, incubated with NP-OVA or NP-OVA and chimeric anti-NP human IgE, were injected i.t. into NP-OVA-immunized mice. Controls received i.t instillation of PBS or eosinophils treated with OVA, NP-OVA, or control human IgE. (A) To block hFcεRIα-mediated cIgE-facilitated NP-OVA presentation, anti-FcεRIα mAb (15.1) doses were used. Three days after eosinophil instillation, pLN cells were assayed for T cell proliferation (B) Three days after i.t. eosinophil transfer, pLN cells were cultured and assayed for IL-4 and INF-γ release. (C) FcεRI engagement increases expressions of CD40, CD80, and CD86 on airway eosinophils. On BAL eosinophils from OVA-sensitized and -challenged hFcεRIα mice, hFcεRI was cross-linked with either cIgE Ab and mouse anti-human IgE (left panel) or anti-hFcεRIα mAb (15.1) and anti-mouse IgG1 (right panel). Histograms show expressions of costimulatory proteins on resting eosinophils (middle histograms) and FcεRI cross-linked eosinophils (bold line right histograms) vs isotype control Ab (left histograms). (D) Western blotting shows that hFcεRIα cross-linking, elicited by 15.1 mAb + anti-mIgG1 and cIgE+ anti-hIgE, increases CD40, CD80, and CD86 proteins in eosinophils. Eosinophils were treated with PBS, control mIgG1 mAb, 15.1 mAb only, 15.1 mAb + anti-mIgG, or cIgE only, cIgE + mouse isotype IgG, and cIgE+ anti-hIgE. Full article ">

17 pages, 2028 KiB

Open AccessArticle

Dual Roles of Plasma miRNAs in Myocardial Injuries After Polytrauma: miR-122-5p and miR-885-5p Reflect Inflammatory Response, While miR-499a-5p and miR-194-5p Contribute to Cardiomyocyte Damage

by Jiaoyan Han, Liudmila Leppik, Larissa Sztulman, Roberta De Rosa, Victoria Pfeiffer, Lewin-Caspar Busse, Elena Kontaxi, Elisabeth Adam, Dirk Henrich, Ingo Marzi and Birte Weber

Cells 2025, 14(4), 300; https://doi.org/10.3390/cells14040300 - 18 Feb 2025

Viewed by 174

Abstract

Cardiac injury after severe trauma is associated with higher mortality in polytrauma patients. Recent evidence suggests that miRNAs play a key role in cardiac pathophysiology and could serve as potential markers of cardiac damage after polytrauma. To explore this hypothesis, plasma miRNA profiles [...] Read more.

Cardiac injury after severe trauma is associated with higher mortality in polytrauma patients. Recent evidence suggests that miRNAs play a key role in cardiac pathophysiology and could serve as potential markers of cardiac damage after polytrauma. To explore this hypothesis, plasma miRNA profiles from polytrauma patients (ISS ≥ 16) with and without cardiac injury, stratified by troponin T levels (TnT, > 50 pg/mL vs. < 12 pg/mL), were analysed using NGS and validated via RT-qPCR. Five miRNAs (miR-122-5p, miR-424-5p, miR-885-5p, miR-194-5p, and miR-499a-5p) were found to be significantly upregulated in polytrauma patients with elevated TnT levels. miR-122-5p was associated with markers of right ventricular dysfunction (TAPSE) and left ventricular hypertrophy (IVS/LVPW), while miR-885-5p correlated with left ventricular hypertrophy (IVS/LVPW) and diastolic dysfunction (E/E’ ratio). In vitro, miR-194-5p mimic and miR-499a-5p mimic exhibited more active roles in cardiomyocyte injury by increasing caspase-3/7 activity and/or enhancing caspase-1 activity. Notably, the miR-194-5p mimic significantly enhanced the cytotoxic effects of the polytrauma cocktail, while miR-499a-5p boosted effects of LPS/nigericin stimulation in cardiomyocytes. Our findings identify miR-122-5p and miR-885-5p as potential biomarkers reflecting the cardiomyocyte response to polytrauma-induced inflammation, while miR-499a-5p and miR-194-5p appear to play a direct role in myocardial injury after polytrauma. Full article

(This article belongs to the Special Issue Advances in Cardiomyocyte and Stem Cell Biology in Heart Disease)

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4 pages, 904 KiB

Open AccessCorrection

Correction: Singh et al. Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models. Cells 2024, 13, 1463

by Surya P. Singh, Gopal Pathuri, Adam S. Asch, Chinthalapally V. Rao and Venkateshwar Madka

Cells 2025, 14(4), 299; https://doi.org/10.3390/cells14040299 - 18 Feb 2025

Viewed by 102

Abstract

In the original publication [...] Full article

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Figure 2

Figure 2
Effect of STAT3 inhibitors on proliferation of BCa spheroids. Representative images of spheroids generated using J82 (A), NBT-II (B), and MB49 (C) BCa cell lines. All images were captured at a 10X magnification. Growth curves of spheroid sizes (D). Schematic diagram illustrating treatment of spheroids with STAT3 inhibitors (E). Intracellular ATP content in (I–K) spheroids measured by luminescence on day six (F–H). Treatment with STAT3 inhibitors decreased the BCa spheroids size; bar represents 200 µM diameter (I–K). Spheroids were stained with calcein AM for live cells (green) and EtBr for dead cells (red). Representative images of control vs. treated BCa spheroids (L,M). All experiments were performed in triplicate (n = 3). Values are expressed as the mean ± SEM. Significance is indicated by * p < 0.05, ** p < 0.001, and *** p < 0.0001. Full article ">Figure 2 Cont.
Effect of STAT3 inhibitors on proliferation of BCa spheroids. Representative images of spheroids generated using J82 (A), NBT-II (B), and MB49 (C) BCa cell lines. All images were captured at a 10X magnification. Growth curves of spheroid sizes (D). Schematic diagram illustrating treatment of spheroids with STAT3 inhibitors (E). Intracellular ATP content in (I–K) spheroids measured by luminescence on day six (F–H). Treatment with STAT3 inhibitors decreased the BCa spheroids size; bar represents 200 µM diameter (I–K). Spheroids were stained with calcein AM for live cells (green) and EtBr for dead cells (red). Representative images of control vs. treated BCa spheroids (L,M). All experiments were performed in triplicate (n = 3). Values are expressed as the mean ± SEM. Significance is indicated by * p < 0.05, ** p < 0.001, and *** p < 0.0001. Full article ">

23 pages, 2955 KiB

Open AccessArticle

Radiation Retinopathy: Microangiopathy-Inflammation-Neurodegeneration

by Anja-Maria Davids, Inga-Marie Pompös, Norbert Kociok, Jens Heufelder, Sergej Skosyrski, Nadine Reichhart, Antonia M. Joussen and Susanne A. Wolf

Cells 2025, 14(4), 298; https://doi.org/10.3390/cells14040298 - 18 Feb 2025

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Abstract

Purpose: Proton irradiation is used to treat choroidal melanoma of the eye. The impact on non-malignant retinal cells is currently understudied. Therefore, we here report a mouse model to investigate the impact of proton irradiation on the retina. Methods: We performed a proton [...] Read more.

Purpose: Proton irradiation is used to treat choroidal melanoma of the eye. The impact on non-malignant retinal cells is currently understudied. Therefore, we here report a mouse model to investigate the impact of proton irradiation on the retina. Methods: We performed a proton beam irradiation of 5–15 Cobalt-Gray-Equivalent (CGE) of the eyes of female C57Bl6/J (Cx3cr1^+/+), Cx3cr1^gfp/+ and Cx3cr1^gfp/gfp mice mimicking the clinical situation and evaluated the structure, function and cellular composition of the retina up to 24 weeks after irradiation. Results: Proton beam irradiation of the eye with 15 CGE leads to cataract formation after 24 weeks without affecting the gross anatomy of the retinal vasculature as shown by Fundus imaging in all genotypes respectively. However, 10 and 15 CGE, lead to a significant decrease in NG2 positive cell numbers and all three dosages induced an increase in GFAP immunoreactivity. At 24 weeks a dosage of 15 CGE resulted in functional impairment and a decrease of NG2 positive cells in both WT and Cx3cr1 animals. Iba1 cell immunoreactivity was increased in all genotypes. However, in the Cx3cr1 animals the invasion of Iba1 cells into the deep vascular layer was partially prevented. This was accompanied by a less severe functional impairment in the irradiated Cx3cr1^gfp/gfp vs. WT. Conclusions: Although the gross anatomy of the retina does not seem to be affected by proton beam irradiation, the cellular composition and retinal function changed significantly in both WT and Cx3cr1 mice reflecting the clinical situation. Moreover, cataract formation was one of the major long-term effects of irradiation. We conclude that the murine model (WT and Cx3cr1 genotype) can be used to investigate proton-beam associated side effects in vivo as well as to test prospective interventions. Moreover, the loss of Cx3cr1 seems to be partially protective. Full article

(This article belongs to the Special Issue Radiation-Induced Brain Injury: Molecular Mechanisms and Therapeutic Strategies)

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18 pages, 641 KiB

Open AccessArticle

Selected Elements of the Tumor Microenvironment (MMP-2, MMP-7, TIMP-2, CXCL-9, CXCL-10) in the Serum of Pediatric Patients with Acute Lymphoblastic Leukemia

by Aleksandra Kaczorowska, Natalia Miękus-Purwin, Anna Owczarzak, Anna Gabrych, Małgorzata Wojciechowska, Ninela Irga-Jaworska, Sylwia Małgorzewicz, Małgorzata Rąpała and Joanna Stefanowicz

Cells 2025, 14(4), 297; https://doi.org/10.3390/cells14040297 - 17 Feb 2025

Viewed by 172

Abstract

In recent years, researchers have been paying special attention to the tumor microenvironment (TME). One of the most important factors contributing to the development and progression of cancer is the destruction of elements of the extracellular matrix (ECM). The most important substances involved [...] Read more.

In recent years, researchers have been paying special attention to the tumor microenvironment (TME). One of the most important factors contributing to the development and progression of cancer is the destruction of elements of the extracellular matrix (ECM). The most important substances involved in regulating the extracellular matrix degradation process are extracellular matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In the process of cancer cell migration, chemokines secreted by target tissues, as well as the profile of chemokine receptors presented on cancer cells, play a key role. In the presented work, five components of the TME were selected: MMP-2, MMP-7, TIMP-2, CXCL-9, and CXCL-10. In the years 2018–2021, peripheral blood samples were collected before the start of treatment and then on day 33 of intensive treatment from 31 patients diagnosed with ALL. The results indicate that the levels of MMP-2, MMP-7, and TIMP-2 did not statistically significantly change before and during treatment of ALL patients. The decrease in CXCL-9 and CXCL-10 levels in the patients’ serum on the 33rd day of therapy turned out to be statistically significant. Our study indicates that serum is also a valuable material for the evaluation of these substances. Conclusions: CXCL-9 and CXCL-10 could be used as one of markers for monitoring the response to treatment and a potential marker of ALL recurrence in pediatric patients. The role of MMP-2, MMP-7, and TIMP-2 in the assessment of response to therapy in children with ALL has not been confirmed. Full article

(This article belongs to the Special Issue Role of Matrix in Cancers)

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13 pages, 2730 KiB

Open AccessCommunication

Generation of a Transgenic Mouse Model for Investigating Mitochondria in Sperm

by Hironmoy Sarkar, Suryaprakash R. Batta, Neerja Wadhwa, Subeer S. Majumdar and Bhola Shankar Pradhan

Cells 2025, 14(4), 296; https://doi.org/10.3390/cells14040296 - 17 Feb 2025

Viewed by 198

Abstract

Mitochondria play a crucial role in sperm development; however, the mechanisms regulating their function in sperm remain poorly understood. Developing a method to regulate the expression of a target gene within the mitochondria of sperm is a vital step in this area of [...] Read more.

Mitochondria play a crucial role in sperm development; however, the mechanisms regulating their function in sperm remain poorly understood. Developing a method to regulate the expression of a target gene within the mitochondria of sperm is a vital step in this area of research. In this study, we aimed to create a system for expressing a transgene in the mitochondria of sperm. As a proof of concept, we generated transgenic mice that express green fluorescent protein (GFP) fused with a mitochondrial localization signal (MLS) driven by the phosphoglycerate kinase 2 (PGK2) promoter, which facilitates the transgene expression in the sperm. Although the PGK2 promoter has previously shown to drive gene expression in spermatocytes and spermatids, the novelty of our approach lies in the combination of PGK2-driven MLS-GFP expression to study mitochondria in vivo. We established two founder lines of transgenic mice through pronuclear microinjection, and MLS-GFP expression was confirmed in the mitochondria of sperm cells using fluorescence microscopy and flow cytometry. Consequently, we provide a novel platform for investigating mitochondrial function in sperm, where GFP can be substituted with other genes of interest to examine their effects on mitochondria. This system specifically targets sperm mitochondria, offering an innovative approach for studying mitochondrial function in vivo. Full article

(This article belongs to the Special Issue Sperm Biology and Reproductive Health—Second Edition)

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Figure 1
Validation of MLS sequence in HEK293 cells. (A) A schematic diagram of the constructs used in this study. (B) HEK 293 cells were transfected with CMV_EGFP (control) and CMV_MLS-EGFP and co-stained with MitoTracker red dye. The images were taken 24 h after transfection. The left panel was expression for GFP, middle panel was for the MitoTracker of the same cells, and the right panel was the co-localization of GFP and MitoTracker red. Scale bar: 10 µm. Full article ">Figure 2
A schematic diagram of generation of PGK2-MLS-GFP transgenic mice after embryo transfer. The male and female mice were indicated in the figure. The PCR-positive progenies were marked in red. After the embryo transfer, mice No. 1, 2, and 8 were transgene positive (founder). The founder mice No. 1 and No. 8 with wild type mice, the mice No 1.4, 1.5, 1.6, 1.7, 8.5, 8.6, and 8.10 were detected to be PCR positive for transgene integration. Full article ">Figure 3
Generation of PGK2-MLS-GFP transgenic mice. Detection of transgene by PCR. PCR screening of progeny for generation of PGK2-MLS-GFP mice. (A) A band size of 258 bp represented the transgene positive mice. (B) PCR of the gene Ppia (120 bp) was used to determine the quality of the isolated gDNA from mice (loading control). Lad: 100 bp DNA ladder; Bl = blank containing only PCR master mix, P.1 to P.10 indicated the progeny numbers in the gel. Full article ">Figure 4
Detection of GFP-positive spermatozoa isolated from the epididymis of the PGK2-MLS-GFP transgenic mice by microscopy. Microscopic images of the sperm cells of wild-type mice (A) and transgenic mice (B). The GFP-positive spermatozoa was detected by microscopy in the live sperm cells of the transgenic mice. Scale bar: 20 µm. Full article ">Figure 5
Detection of GFP-positive sperm cells collected from the epididymis of the PGK2-MLS-GFP transgenic mice by flow cytometry. (A) Scatter plot of one of the representative sperm samples and the corresponding histogram of wild-type mouse No 1.7.3. (B) Scatter plot of one of the representative sperm samples and the corresponding histogram of transgenic mouse No 1.7.5. Full article ">

21 pages, 2160 KiB

Open AccessArticle

Phenolic Compounds from Haskap Berries Have Structure, Combination, and Cell Line-Dependent Impacts on the Longevity-Associated Deacetylase Sirtuin 1

by Morgan A. Fleming, Nicholas H. Low and Christopher H. Eskiw

Cells 2025, 14(4), 295; https://doi.org/10.3390/cells14040295 - 17 Feb 2025

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Abstract

It is well established that phenolic compounds from plant sources impact readouts of cell health such as reduced radical and reactive oxygen species. However, it is unclear if specific phenolic structures impact other cellular processes or proteins, such as the evolutionary conserved deacetylase [...] Read more.

It is well established that phenolic compounds from plant sources impact readouts of cell health such as reduced radical and reactive oxygen species. However, it is unclear if specific phenolic structures impact other cellular processes or proteins, such as the evolutionary conserved deacetylase Sirtuin 1 (SIRT1), and if phenolic combinations interact synergistically to do so. We observed that structurally diverse haskap berry phenolics (caffeic acid, cyanidin, kaempferol-3-O-glucoside, and gentisic acid) differentially impacted normal primary human fibroblast growth, which has been linked to SIRT1. These results were consistent with previous work from our lab indicating that haskap phenolic extracts/fractions impact human cell growth via SIRT1-dependent mechanisms. Therefore, we furthered the investigation into SIRT1 and phenolic structure and observed that the individual phenolics or their combinations had no observable impact on SIRT1 transcript abundance or cellular localization. We also observed that select phenolics decreased SIRT1 protein abundance and increased SIRT1 activity. The catechol-containing phenolics outperformed those that lack a catechol group, indicating potential structure-dependent impact(s). Potential synergy between the specific phenolics analyzed was observed in Western blot, and potential antagonism was identified in the SIRT1 activity assay. Results were concomitant with the presence of different phenolic structures, phenolic combinations, and cell type (sex and/or individual differences). These results highlight the possible significance of the catechol structure and indicate that phenolics have the potential to impact cell processes, which the authors hypothesize to be due to mechanisms that are independent of antioxidant activity. Full article

(This article belongs to the Section Cellular Aging)

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Figure 1
Haskap berries and the structures of the four phenolics selected for this work. The phenolics were supplemented to normal primary human fibroblasts individually to determine their function(s) and in equimolar combinations to examine synergy. Full article ">Figure 2
The phenolic treatments have structure-dependent impacts on 2DD and 07124B cell growth behavior. (A) Representative images of DMSO and CY-treated 2DD cells following a 72 h treatment period. Scale bar = 100 μm. (B) Quantified PDT assay results. Following the treatment period, cells were collected and counted affording the calculation of PDT that was normalized to the vehicle control (DMSO). Error bars represent the standard error of the mean (n = 3; p = * p < 0.1, ** p < 0.05, *** p < 0.01). (C) Cell viability assay results, the phenolic treatments maintained high levels of cell viability indicating that these compounds did not increase 2DD or 07124B cell death. Error bars represent the standard error of the mean (n = 3). Treatment abbreviations are as follows: vehicle control (DMSO), caffeic acid (CA), cyanidin (CY), kaempferol-3-O-glucoside (K3G), and gentisic acid (GA). Full article ">Figure 3
The phenolic treatments had no significant impact on SIRT1 transcript abundance but had structure-, interaction-, and/or concentration-dependent impacts on SIRT1 protein abundance. (A) Cells were treated followed by RNA extraction, cDNA synthesis, and RT-qPCR, a fold change of ±2 was considered significant. (B) Impacts of the phenolic treatments on SIRT1 protein abundance. Cells were treated followed by whole cell lysis, harvesting, and Western blot (n = 3), representative Western blot and quantified densitometry results are presented. Coomassie blue gels were used as a load control. (C) Impacts of the phenolic treatments on SIRT1 protein abundance in response to H2O2 (n = 3; p = * p < 0.1, ** p < 0.05, *** p < 0.01). Abbreviations are as follows: vehicle control (DMSO), caffeic acid (CA), cyanidin (CY), kaempferol-3-O-glucoside (K3G), gentisic acid (GA), molecular weight (MW), and hydrogen peroxide (H2O2). Full article ">Figure 4
The phenolic treatments had no observable impact on SIRT1 localization but had structure and cell line-dependent impacts on SIRT1 activity level. (A) Representative immunofluorescence images. Chromatin was counter-stained with DAPI and is represented by the blue color, SIRT1 is represented by the red color. Greyscale images were false colored in Photoshop and all treatments were imaged with consistent exposure times and treated equally in Photoshop to avoid artifact creation. The DAPI (blue) and SIRT1 (red) images were overlayed to create the Merge image, where a purple color suggests co-localization of chromatin and SIRT1. A total of 30–50 cells were imaged per treatment replicate (n = 2). (B) SIRT1 activity assay results. Cells were treated with the selected phenolics prior to harvesting in non-denaturing lysis buffer. Error bars represent the standard error of the mean and fluorescence values were normalized to DMSO (n = 2; p = ** p < 0.05, *** p < 0.01). Treatment abbreviations are as follows: vehicle control (DMSO), caffeic acid (CA), cyanidin (CY), kaempferol-3-O-glucoside (K3G), gentisic acid (GA), and hydrogen peroxide (H2O2). Full article ">

25 pages, 3378 KiB

Open AccessArticle

Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells

by Ana Laura S. A. Matos, Ashley J. Ovens, Emil Jakobsen, Diego Iglesias-Gato, Jacob M. Bech, Stine Friis, Lasse Kristoffer Bak, Gunvor I. Madsen, Jonathan S. Oakhill, Pietri Puustinen and José M. A. Moreira

Cells 2025, 14(4), 294; https://doi.org/10.3390/cells14040294 - 17 Feb 2025

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Abstract

Aspirin has consistently shown preventive effects in some solid cancers, notably colorectal cancer. However, the precise molecular mechanisms underlying this positive effect have remained elusive. In this study, we used an azoxymethane-induced mouse model of colon carcinogenesis to identify aspirin-associated molecular alterations that [...] Read more.

Aspirin has consistently shown preventive effects in some solid cancers, notably colorectal cancer. However, the precise molecular mechanisms underlying this positive effect have remained elusive. In this study, we used an azoxymethane-induced mouse model of colon carcinogenesis to identify aspirin-associated molecular alterations that could account for its cancer-preventive effect. Transcriptomic analysis of aspirin-treated mice showed a strong reduction in c-Myc protein levels and effects on the Myc-dependent transcriptional program in colonic cells. Proto-oncogene c-Myc cooperates with AMP-activated protein kinase (AMPK) to control cellular energetics. Here, we show that salicylate, the active metabolite of aspirin, reduces c-Myc protein expression levels through multiple mechanisms that are both AMPK dependent and independent. This effect is cell-type dependent and occurs at both the transcriptional and post-translational levels. Salicylate-induced AMPK activation leads to the phosphorylation of c-Myc at Thr400, as well as its destabilization and degradation. Our results reveal a complex, multilayered, negative effect of salicylate on c-Myc protein abundance and suggest that chronic depletion of c-Myc can counteract the neoplastic transformation of colorectal epithelium, underpinning the preventive effect of aspirin on colorectal cancer. Full article

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Figure 1
Aspirin has a preventive effect on mice with chemically induced CRC lesions. (A) Representative images of lesions in AOM-treated mice. Lesions were examined by colonoscopy. Tumors were counted, and each tumor scored from 1 to 5 based on the burden relative to colon circumference, according to [<a href="#B29-cells-14-00294" class="html-bibr">29</a>]. (B) Tumor number and (C) tumor score in untreated, AOM-treated, and AOM+aspirin-treated mice. Significance was evaluated with unpaired Student’s t-test. (D) immunohistochemical analysis of c-Myc in normal colonic tissue in AOM-treated (subpanel (a)) and AOM+aspirin-treated mice (subpanel (b)). The entire intestinal tract was collected using the Swiss-rolling technique (subpanel (c)), and lesions (subpanel (d)) were identified and analyzed, both in AOM-treated (subpanel (e)) and AOM+aspirin-treated mice (subpanel (f)). Colonic cells with expression of c-Myc are indicated by black arrows and cells with no expression by red arrows. Yellow arrow indicates immune cells. Original magnification was 40× for subpanels (a,b), 4× for subpanels (c,d), and 20× for subpanels (e,f). (E) Histological analysis of colorectal adenomas. Depicted are representative c-Myc stainings of HG adenomas (subpanels (a–c): c-Myc high, intermediate, and low expression, respectively). Original magnification was 40× for all subpanels. (F) Bar graph represents quantification of c-Myc-positive cells (n = 20 samples; * p < 0.05, Student’s t-test) in samples with high or low p-AMPKα T172 immunoreactivity. Full article ">Figure 2
AMPK activation causes loss of c-Myc. (A) Exposure of HCT116 colon cancer cells to 3 mM salicylate for 1, 4, 24, or 48 h (lanes 2 through 5) elicited AMPK activation, evaluated by phosphorylation of AMPKα at T172, followed by loss of c-Myc expression. Acetyl-CoA carboxylase (ACC) phosphorylation (S79) was used as a marker of functional AMPK activation. (B) Schematic illustration of the molecular mechanisms leading to the activation or inhibition of AMPK. Modulators of AMPK used in this study, negative as well as positive, and their expected mechanism of action are represented. (C) Decreased levels of c-Myc are associated with AMPK activation. HCT116 cells were grown in complete medium for 48 h in the absence (lane 1) or presence of salicylate (lane 2), the synthetic AMPK activator A76966 (lane 3), phenformin (lane 5), or grown in glucose-free medium for 8 h (lane 6). Cells were also treated with a combination of salicylate (3 mM) and compound C (dorsomorphin 100 nM) for 48 h (lane 4). (D) HCT116 cells were cultured in glucose-free medium for up to 5 h in the absence (lanes 1 through 6) or presence of compound C (lanes 7 through 12). Representative western blots and quantification graphs of triplicate experiments are shown. Data are presented as mean ± SEM, n = 3. Full article ">Figure 3
Salicylate affects multiple cell types and has diverse effects. (A) U2OS osteosarcoma cells and (B) MCF-7 breast cancer cells were grown in complete media for 48 h in the presence of salicylate (lane 2, (A,B), respectively), the synthetic activator A76966 (lane 3, (A,B), respectively), phenformin (lane 5, (A,B), respectively), or in glucose-free media for 8 h (lane 6, (A,B), respectively). Cells were also treated with a combination of salicylate (3 mM) and compound C (dorsomorphin 100 nM) for 48 h (lane 4, (A,B), respectively). In MCF-7 cells, the expression of c-Myc was significantly decreased by 4-OHT alone (lane 9) and even more by a combination of salicylate and 4-OHT (lane 10), compared with the negative control (lane 7). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in all cases as loading control for protein normalization. (C) Cell viability of HCT116 cells was determined by MTT assay after treatment with indicated concentrations of salicylate for 48 h. IC50 for salicylate was determined using Prism v.9 (GraphPad Software, San Diego, CA, USA) based on the changes in HCT116 cell viability. (D) Cell viability was determined after 48 h in the presence of 1 mM or 3 mM of salicylate, the synthetic activator A76966, compound C (dorsomorphin: 100 nM), or combinations of these drugs. Mean values ± SEMs are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s.: not significant. (E,F) Seahorse extracellular flux analysis of the oxygen consumption rate (OCR) shows respiratory changes in HCT-116 cells brought about by sodium salicylate treatment. (E) Seahorse injection series analysis with OCR data presented for one experiment. (F) Calculated parameters from 3 independent experiments include ATP-linked respiration, proton leak, basal respiration, and maximal respiratory capacity. Basal respiration significantly increased when cells were treated with sodium salicylate (1 or 3 mM). Treatment with sodium salicylate caused an increased proton leak (uncoupling) and a decreased percental amount of oxygen being used for ATP production. The maximal respiratory capacity was lowered when treated with sodium salicylate, which suggests a compromised mitochondrial function. Full article ">Figure 4
Dynamic interaction between AMPK and c-Myc regulates phosphorylation of c-Myc. Endogenous protein complexes were immunoprecipitated from HEK293 cell lysates with an AMPK1/2 antibody before and after (A) exposure to salicylate (3 mM for 3 h) or (B) glucose deprivation (20 min). Immunoprecipitated proteins were analyzed by immunoblotting, as indicated. Normal serum IgG served as a negative control. Immunoblots of the corresponding cell lysates are shown in the lower panel (input 5%). (C) Quantitation of three replicate experiments. Conditions were compared using Student’s t-test and the results are presented. *** p < 0.001; **** p < 0.0001. (D) Immunoblotting of purified c-Myc-HA-tagged protein used for the in vitro kinase reaction. Cells were mock-transfected (lane 1) or transfected with a control HA-tag vector (lane 2), or recombinant c-Myc-HA (lane 3), and proteins immunopurified using the HA tag. Immunoblotting with HA-antibodies (upper panel) and c-Myc antibodies (lower panel) confirmed the identity of purified proteins. (E,F) AMPK phosphorylates c-Myc in vitro. In vitro kinase reaction was performed with (E) HEK293-expressed c-Myc-HA purified protein (lane 1), recombinant full-length active human AMPK complex (α1/β1/γ1) (lane 2), or a combination of both (lane 3). Addition of compound C to AMPK complex prior to the kinase reaction abolished phosphorylation of substrates in reaction. Recombinant GSK3β (lane 5) was used as control of reaction specificity. (E) Bacterially expressed rc-Myc protein (lane 1), active human AMPK complex (α1/β1/γ1) (lane 2), or a combination of both (lanes 3 and 4, at 1x and 2x amounts of AMPK). The * symbol in the autoradiograph indicates the presence of a phosphorylated substrate compatible with c-Myc. (G) AMPK substrate consensus sequence (upper panel). Lower panel shows the AMPK motifs present on the c-Myc protein ranked by score. (H) Phosphorylated peptides identified by in-gel tryptic digestion and mass spectrometry of in vitro phosphorylation of recombinant c-Myc with AMPK-α1/β1/ϒ1 complex (+AMPK). A mock reaction lacking AMPK complex (−AMPK) was used as control. Phosphorylated residues are shown in red. Results from two independent experiments are shown. Full article ">Figure 5
AMPK activation with sodium salicylate mediates c-Myc ubiquitinylation and nuclear depletion. (A) Exposure of HCT116 colon cancer cells to 1 or 3 mM salicylate for 48 h (lanes 2 and 3, respectively) elicited the loss of c-Myc expression accompanied by CIP2A, but no change in the levels of S62 phosphorylated c-Myc. Cells were also treated with a combination of salicylate (1 or 3 mM) and compound C (dorsomorphin 100 nM) for 8 h (lanes 5 and 6, respectively). (B) Salicylate induced phosphorylation of PP2A Y307. Samples were analyzed with an anti-pY307-PP2Ac antibody, and the blots were reprobed with an anti-PP2Ac antibody. (C) Sodium salicylate induced AMPK-mediated ubiquitinylation of c-Myc. HEK293 cells were transiently transfected with c-Myc-HA (lanes 1 through 4) or a mock construct (HA-vector, lane 5). Cells were then either left untreated (lanes 1 and 5) or treated with compound C (lane 2), sodium salicylate (lane 3), or a combination of both (lane 4), for three hours. C-Myc-HA-tagged protein was then immunopurified and its ubiquitination levels analyzed with a ubiquitin antibody by western blot (upper panel, ubiquitin). Immunoblots of the corresponding cell lysates (ubiquitin antibody) and purified proteins (HA antibody) are shown (lower panel). Red boxes highlight regions of interest corresponding to mono- and poly-ubiquitinylated c-Myc. (D,E) western blot detection of c-Myc in HCT116 cells treated with (D) MG-132 (20 μM) and (E) CHX (10 µg/mL) and in the presence or absence of 3 mM salicylate for 12 h. (F) QRT-PCR analysis of salicylate effects on relative levels of MYC mRNA in HCT116 cancer cells. MYC and GAPDH mRNA were quantified using a LightCycler Real-Time PCR system. Changes in mRNA levels of MYC were normalized to mRNA levels of GAPDH, and relative fold changes in mRNA levels were calculated compared to their respective vehicle-treated controls. Graphic bars indicate relative fold changes in mRNA ± SEM (arbitrary units) in each treatment group for each cell line. (G–I): sodium salicylate causes nuclear depletion of c-Myc. (G) AMPKα and c-Myc are visualized by indirect immunofluorescence in formaldehyde-fixed HCT116 cells. Also, COS cells were transfected with the GFP-tagged AMPK complex, either (H) α1β1γ1 or (I) α2β1γ1. Under normal growth conditions (control), c-Myc showed strong nuclear expression. Upon exposure to sodium salicylate (+salicylate), c-Myc was absent from the nuclear compartment. Scale bars: 10 µm. (J) Quantification of the nuclear signal of c-Myc relative to DAPI in HCT116 cells, and COS cells transfected with α1β1γ1 or α2β1γ1. *** p < 0.001; **** p < 0.0001. Full article ">Figure 6
Model of potential mode of action of salicylate on c-Myc. Salicylate decreases the expression of MYC mRNA at the transcriptional level. In addition, salicylate activates AMPK both through directly binding to the ADaM site and changes in the mitochondrial membrane potential, which lead to an increased AMP:ATP ratio. Activated AMPK can directly bind to c-Myc and phosphorylate it at the bHLH-LZ region (S373 and T400), disrupting DNA binding. Additionally, phosphorylation of PP2A at Y307 and downregulation of CIP2A further compound the regulatory loop, decreasing c-Myc levels and affecting the c-Myc transcriptional program. Modulation of miR-34 defines another regulatory loop, further linking AMPK activation and c-Myc expression. Full article ">

19 pages, 654 KiB

Open AccessReview

Shaping Rare Granulomatous Diseases in the Lab: How New Models Are Changing the Game

by Jessica Ceccato, Giulia Gualtiero, Maria Piazza, Samuela Carraro, Helena Buso, Carla Felice, Marcello Rattazzi, Riccardo Scarpa, Fabrizio Vianello and Francesco Cinetto

Cells 2025, 14(4), 293; https://doi.org/10.3390/cells14040293 - 16 Feb 2025

Viewed by 313

Abstract

In vitro models serve as valuable tools for understanding the complex cellular and molecular interactions involved in granuloma formation, providing a controlled environment to explore the underlying mechanisms of their development and function. Various models have been developed to replicate granulomatous diseases, even [...] Read more.

In vitro models serve as valuable tools for understanding the complex cellular and molecular interactions involved in granuloma formation, providing a controlled environment to explore the underlying mechanisms of their development and function. Various models have been developed to replicate granulomatous diseases, even though they may lack the sophistication needed to fully capture the variability present in clinical spectra and environmental influences. Traditional cultures of PBMCs have been widely used to generate granuloma models, enabling the study of aggregation responses to various stimuli. However, growing cells on a two-dimensional (2D) plastic surface as a monolayer can lead to altered cellular responses and the modulation of signaling pathways, which may not accurately represent in vivo conditions. In response to these limitations, the past decade has seen significant advancements in the development of three-dimensional (3D) in vitro models, which more effectively mimic in vivo conditions and provide better insights into cell–cell and cell–microenvironment interactions. Meanwhile, the use of in vivo animal models in biomedical research must adhere to the principle of the three Rs (replacement, reduction, and refinement) while ensuring that the models faithfully replicate human-specific processes. This review summarizes and compares the main models developed to investigate granulomas, focusing on their contribution to advancing our understanding of granuloma biology. We also discuss the strengths and limitations of each model, offering insights into their biological relevance and practical applications. Full article

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30 pages, 824 KiB

Open AccessReview

Advancing Glioblastoma Research with Innovative Brain Organoid-Based Models

by Cátia D. Correia, Sofia M. Calado, Alexandra Matos, Filipa Esteves, Ana Luísa De Sousa-Coelho, Marco A. Campinho and Mónica T. Fernandes

Cells 2025, 14(4), 292; https://doi.org/10.3390/cells14040292 - 16 Feb 2025

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Abstract

Glioblastoma (GBM) is a relatively rare but highly aggressive form of brain cancer characterized by rapid growth, invasiveness, and resistance to standard therapies. Despite significant progress in understanding its molecular and cellular mechanisms, GBM remains one of the most challenging cancers to treat [...] Read more.

Glioblastoma (GBM) is a relatively rare but highly aggressive form of brain cancer characterized by rapid growth, invasiveness, and resistance to standard therapies. Despite significant progress in understanding its molecular and cellular mechanisms, GBM remains one of the most challenging cancers to treat due to its high heterogeneity and complex tumor microenvironment. To address these obstacles, researchers have employed a range of models, including in vitro cell cultures and in vivo animal models, but these often fail to replicate the complexity of GBM. As a result, there has been a growing focus on refining these models by incorporating human-origin cells, along with advanced genetic techniques and stem cell-based bioengineering approaches. In this context, a variety of GBM models based on brain organoids were developed and confirmed to be clinically relevant and are contributing to the advancement of GBM research at the preclinical level. This review explores the preparation and use of brain organoid-based models to deepen our understanding of GBM biology and to explore novel therapeutic approaches. These innovative models hold significant promise for improving our ability to study this deadly cancer and for advancing the development of more effective treatments. Full article

(This article belongs to the Special Issue The Current Applications and Potential of Stem Cell-Derived Organoids)

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Graphical abstract
Full article ">Figure 1
Schematic representation of cerebral organoid generation from human pluripotent stem cells (hPSCs). At day 0, hPSCs are plated in ultra-low attachment plates to form embryoid bodies (EBs). After six days, the EBs are transferred to low-adhesion 24-well plates to induce neural differentiation. Following five days, the developing neuroepithelial tissues are embedded in Matrigel droplets, promoting the expansion of neuroepithelial buds. After an initial stationary growth phase, the organoids are transferred to an orbital shaker or spinning bioreactor to support further maturation. This figure was created based on the Lancaster and Knoblich protocol [<a href="#B50-cells-14-00292" class="html-bibr">50</a>]. Full article ">Figure 2
Cerebral organoid (CO)-based GBM models generated through the genetic engineering of human pluripotent stem cells (hPSCs). These models can be developed in three general ways, depending on the stage of CO differentiation at which the genetic modification is introduced. (A) When a genetic modification is applied at a later stage, during CO maturation, the resulting model consists of brain-like tissue with focal cancerous growths that originate at the surface of the CO and progressively invade its interior. (B) When a genetic modification is introduced during the expansion of neuroepithelial buds, mainly neural stem cells transform, better recapitulating the cells of origin of GBM. This approach leads to the formation of multiple tumors with a limited set of microenvironmental cells. (C) When genetic modification is performed at the hPSC stage, the resulting tumor model exhibits a disorganized structure, consisting primarily of transformed cells with only a few microenvironmental cells at later time points. Circular arrows illustrate the differentiation process. Full article ">Figure 3
Cerebral organoid (CO)-based GBM models generated through the co-culture of GBM-derived cells with pluripotent stem cells (PSCs) or COs. These models can be developed in three general ways, depending on the timing and method of GBM-derived cell addition. (A) Glioblastoma stem-like cells (GSCs) can be mixed with hPSCs before differentiation into COs. (B) GSCs can be introduced as a single-cell suspension into preformed COs. (C) GSCs can be added as spheroids to COs. Circular arrows represent the differentiation process. Full article ">

16 pages, 7294 KiB

Open AccessArticle

Differential Regulation of Nav1.1 and SCN1A Disease Mutant Sodium Current Properties by Fibroblast Growth Factor Homologous Factors

by Ashley Frazee, Agnes Zybura and Theodore R. Cummins

Cells 2025, 14(4), 291; https://doi.org/10.3390/cells14040291 - 15 Feb 2025

Viewed by 334

Abstract

Fibroblast growth factor homologous factors (FHFs) regulate the activity of several different voltage-gated sodium channels (Na_vs). However, more work is needed to determine how specific FHF isoforms and variants affect the properties of different Na_v isoforms. In addition, it is [...] Read more.

Fibroblast growth factor homologous factors (FHFs) regulate the activity of several different voltage-gated sodium channels (Na_vs). However, more work is needed to determine how specific FHF isoforms and variants affect the properties of different Na_v isoforms. In addition, it is not known if FHFs can differentially modulate the properties of Na_v variants associated with disease. Here, we investigated the effects of FHF2A and FHF2B on Nav1.1 properties as well as on a familial hemiplegic migraine 3 (FHM3) causing mutation in this channel, F1774S. We found that FHF2A, but not 2B, induced prominent long-term inactivation (LTI) in the wild-type (WT) Nav1.1. Interestingly, FHF2A induced LTI in the F1774S FHM3 mutant channel to a greater extent than in the WT. Furthermore, persistent currents caused by the F1774S mutation were attenuated by the co-expression of FHF2A, leading to a possible rescue of the mutant channel phenotype. By contrast, the P1894L mutation, which is associated with epilepsy and mild intellectual disability, greatly attenuated the LTI induced by FHF2A. Overall, our data show for the first time that FHF2A might be a significant modulator of Nav1.1 that can differentially modulate the impact of Nav1.1 disease-associated mutations. Full article

(This article belongs to the Special Issue Ion Channels in Pain: Mechanisms and Therapeutics)

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20 pages, 344 KiB

Open AccessReview

Significance of Measurable Residual Disease in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia

by Margery Gang, Megan Othus and Roland B. Walter

Cells 2025, 14(4), 290; https://doi.org/10.3390/cells14040290 - 15 Feb 2025

Viewed by 428

Abstract

Allogeneic hematopoietic cell transplantation (HCT) remains an important curative-intent treatment for many patients with acute myeloid leukemia (AML), but AML recurrence after allografting is common. Many factors associated with relapse after allogeneic HCT have been identified over the years. Central among these is [...] Read more.

Allogeneic hematopoietic cell transplantation (HCT) remains an important curative-intent treatment for many patients with acute myeloid leukemia (AML), but AML recurrence after allografting is common. Many factors associated with relapse after allogeneic HCT have been identified over the years. Central among these is measurable (“minimal”) residual disease (MRD) as detected by multiparameter flow cytometry, quantitative polymerase chain reaction, and/or next-generation sequencing. Demonstration of a strong, independent prognostic role of pre- and early post-HCT MRD has raised hopes MRD could also serve as a predictive biomarker to inform treatment decision-making, with emerging data indicating the potential value to guide candidacy assessment for allografting as a post-remission treatment strategy, the selection of conditioning intensity, use of small molecule inhibitors as post-HCT maintenance therapy, and preemptive infusion of donor lymphocytes. Monitoring for leukemia recurrence after HCT and surrogacy for treatment response are other considerations for the clinical use of MRD data. In this review, we will outline the current landscape of MRD as a biomarker for patients with AML undergoing HCT and discuss areas of uncertainty and ongoing research. Full article

(This article belongs to the Special Issue State of the Art and Future Prospects in Stem Cell Transplantation)

14 pages, 2315 KiB

Open AccessArticle

Angiotensin-(1-7) Provides Potent Long-Term Neurorepair/Neuroregeneration in a Rodent White Matter Stroke Model: Nonarteritic Ischemic Optic Neuropathy (rNAION)

by Kwang Min Woo, Yan Guo, Zara Mehrabian, Thomas Walther, Neil R. Miller and Steven L. Bernstein

Cells 2025, 14(4), 289; https://doi.org/10.3390/cells14040289 - 15 Feb 2025

Viewed by 318

Abstract

Nonarteritic anterior ischemic optic neuropathy (NAION) is an ischemic lesion of the anterior optic nerve (ON), currently untreatable due to the length of time from symptom onset until treatment. We evaluated angiotensin-(1-7) (Ang-(1-7)): the MAS1-receptor ligand, as a possible NAION treatment using the [...] Read more.

Nonarteritic anterior ischemic optic neuropathy (NAION) is an ischemic lesion of the anterior optic nerve (ON), currently untreatable due to the length of time from symptom onset until treatment. We evaluated angiotensin-(1-7) (Ang-(1-7)): the MAS1-receptor ligand, as a possible NAION treatment using the rodent NAION model (rNAION). Long-Evans rats were unilaterally rNAION-induced. One-day post-induction, lesion severity was quantified via optic nerve head (ONH) edema using spectral domain optical coherence tomography. Animals meeting rNAION induction criteria were randomized into (1) Subcutaneous Ang-(1-7) infusion for 28 days and (2) Vehicle. Visual function was assessed using both visual acuity and flash visual evoked potentials (fVEP). Tissues were collected >30d and RGC neurons were quantified by stereology. ONs were histologically examined for inflammation. Ang-(1-7) improved post-rNAION visual function. Ang-(1-7)-treated animals showed improved visual acuity (ANCOVA: p = 0.0084) and improved fVEP amplitudes (ANCOVA: p = 0.0378) vs vehicle controls. The relative degree of improvement correlated with ONH edema severity. Treated animals showed trends towards increased RGC survival, and reduced optic nerve inflammatory cell infiltration. Ang-(1-7) is the first agent effective ≥1 day after rNAION induction. Ang-(1-7) type agonists may be useful in improving long-term function and neuronal survival in clinical NAION and other forms of white matter ischemia. Full article

(This article belongs to the Special Issue Novel and Experimental Strategies for the Treatment of Neurodegenerative Diseases)

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ONH edema results in 1d post-rNAION for both rat groups prior to treatment (both sexes). Mean ONH edema for each animal was based on results for the three largest contiguous diameters measured by SD-OCT and shown in microns (µM). Mean ONH diameter of 16 uninduced (contralateral eyes used for comparison). Animals were eliminated if ONH edema was ≤450 µm. Vehicle: N = 26; Treatment: N = 24. The relative spread of the edema is similar in both groups. There is a significant difference in ONH diameter between uninduced and induced eyes (**** p < 0.0001; two-tailed t-test). No significant (ns) difference was seen in the mean ONH diameter between the two randomized rNAION-induced (Vehicle and Treated) groups. Full article ">Figure 2
Ang-(1-7) improves visual acuity by OptoMotry after rNAION. (A) Regression analysis using analysis of covariance (ANCOVA). Regression lines for both vehicle- (n = 25) and Ang-(1-7)-treated (n = 21) are shown. ANCOVA reveals a significant difference in the intercepts between TXA-127 and vehicle-treated rats. (F(1, 40) = 7.68, p = 0.0084) (B) Estimation plot for OptoMotry for animals with moderate-severe edema. The difference between treated and vehicle yielded an overall improvement of 16% for Ang-(1-7)-treated animals, compared with vehicle-treated animals with moderate-severe ONH edema, defined as edema ≥600 μm (t(25) = 2.753, p = 0.0108). (C) Subgroup analysis of Ang-(1-7) treatment effects post-rNAION. Ang-(1-7) provides an increasingly robust improvement in animals with progressively more severe ONH edema. There was a 3.6% improvement in the mild group (450–599 µm), a 13% improvement in the moderate group (600–699 µm), and an 18% improvement in the severe group (700–850 µm). (* p < 0.05). Full article ">Figure 3
Ang-(1-7) administered 1 day after rNAION improves fVEP-based visual function as measured using waveform amplitudes: Analyses. (A) fVEP vs Edema regression analysis using ANCOVA. There is a significant difference in the intercepts between Ang-(1-7)- and vehicle-treated rats (F(1, 37) = 4.64, p = 0.0378). (B) Estimation plot for fVEP differences for a vehicle vs treated animals with moderate-severe ONH edema. There is a 20% overall improvement in the fVEP of treated animals with moderate-severe ONH edema, defined as edema ≥600 µm. (C) Stratified fVEP results for mild (450–599 µm), moderate (600–699 µm) and severe (700–850 µm) ONH edema. There was an 8.0% improvement in the mild group, a 21% improvement in the moderate group, and a 19% improvement in the severe group. Ang-(1-7) neuroregenerative/neuroreparative effects are strongest in animals with a moderate-severe amount of ONH edema. Full article ">Figure 4
RGC loss in vehicle- and Ang-(1-7)-treated animals. RGCs were quantified by stereology. There is a similar distribution of RGC loss in both samples. The mean value for RGC loss in vehicle-treated animals was 74.29 ± 5.39% (n = 17), whereas Ang-(1-7)-treated animals yielded a mean RGC loss of 63.09 ± 7.17% (n = 14). This difference is not significant (NS: nonsignificant; unpaired t-test: p = 0.219). Full article ">Figure 5
Ang-(1-7) effects on late cellular inflammation after rNAION. All tissues from animals 30d post-treatment. Only animals with similar levels of RGC loss were used for comparing vehicle- and Ang-(1-7)-treated responses. (A) Immunostaining of rat control ON (Green: Iba-1; Red: ED1/CD68). Ramified inactive microglia are distributed throughout the ON diameter, with little ED1 activity. (B) Quantification of inflammation: Comparison graphs. Vehicle-treated rNAION-induced ON has ~5 times the Iba-1 expression of naïve (10.7 ± 0.7% vs 2.2 ± 0.2% naïve). Iba-1 expression in Ang-(1-7)-treated animals is 8.5 ± 1.0% sem (n = 5/group). The difference between naïve and rNAION-induced animals treated with vehicle or Ang-(1-7) is highly significant (p = 0.003 for Ang-(1-7)-treated animals; the lower value). The difference between vehicle and Ang-(1-7) treatment is non-significant (Mann–Whitney two-tailed U test, p = 0.211). ED1/CD68 expression is shown in densitometric units; ED1 expression in naïve ONH is virtually null. The pattern of ED1/CD68 expression between vehicle and Ang-(1-7) treatment was similar to that seen with Iba-1 (N = 7/group; nonsignificant (NS), Mann–Whitney 2-tailed U test: p = 0.522). (** p < 0.01, ns = no significance) (C) Immunostaining of an rNAION-induced vehicle-treated animal. RGC loss at 30d = 88%. The majority of microglia/macrophages are hypertrophic/active, with strong upregulation of Iba-1 activity. ED1 activity is also elevated, confirming the activation of the majority of inflammatory cells. (D) Immunostaining of an rNAION-induced Ang-(1-7)-treated animal. RGC loss = 84%. The overall expression of both Iba-1 and ED1/CD68 is considerably lower than that seen in vehicle-treated rNAION-induced ON. Full article ">

27 pages, 1786 KiB

Open AccessReview

Targeting Spinal Interneurons for Respiratory Recovery After Spinal Cord Injury

by Maha Paracha, Allison N. Brezinski, Rhea Singh, Elizabeth Sinson and Kajana Satkunendrarajah

Cells 2025, 14(4), 288; https://doi.org/10.3390/cells14040288 - 15 Feb 2025

Viewed by 355

Abstract

Spinal interneurons (SpINs) are pivotal to the function of neural circuits, orchestrating motor, sensory, and autonomic functions in the healthy, intact central nervous system. These interneurons (INs) are heterogeneous, with diverse types contributing to various neural systems, including those that control respiratory function. [...] Read more.

Spinal interneurons (SpINs) are pivotal to the function of neural circuits, orchestrating motor, sensory, and autonomic functions in the healthy, intact central nervous system. These interneurons (INs) are heterogeneous, with diverse types contributing to various neural systems, including those that control respiratory function. Research in the last few decades has highlighted the complex involvement of SpINs in modulating motor control. SpINs also partake in motor plasticity by aiding in adapting and rewiring neural circuits in response to injury or disease. This plasticity is crucial in the context of spinal cord injury (SCI), where damage often leads to severe and long-term breathing deficits. Such deficits are a leading cause of morbidity and mortality in individuals with SCI, emphasizing the need for effective interventions. This review will focus on SpIN circuits involved in the modulation of breathing and explore current and emerging approaches that leverage SpINs as therapeutic targets to promote respiratory recovery following SCI. Full article

(This article belongs to the Special Issue Understanding Spinal Cord Injury and Repair: From Molecules to Neurocircuits and Multimodal Prostheses)

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24 pages, 7771 KiB

Open AccessArticle

Mirvetuximab Soravtansine Induces Potent Cytotoxicity and Bystander Effect in Cisplatin-Resistant Germ Cell Tumor Cells

by Lucia Kucerova, Adriana Fekiacova, Natalia Udvorkova, Pavlina Malcharkova, Viktoria Blahova, Silvia Jochova, Katarina Kalavska, Zuzana Cierna and Michal Mego

Cells 2025, 14(4), 287; https://doi.org/10.3390/cells14040287 - 15 Feb 2025

Viewed by 360

Abstract

Patients with treatment-refractory/relapsing germ cell tumors (GCTs) have a dismal prognosis due to a lack of any effective therapy. Moreover, the efficacy of newly approved targeted therapies remains unexplored for cisplatin-resistant GCTs. Previously, it was demonstrated that folate receptor α (FRα) is overexpressed [...] Read more.

Patients with treatment-refractory/relapsing germ cell tumors (GCTs) have a dismal prognosis due to a lack of any effective therapy. Moreover, the efficacy of newly approved targeted therapies remains unexplored for cisplatin-resistant GCTs. Previously, it was demonstrated that folate receptor α (FRα) is overexpressed in many tumor types and efficiently targeted by the antibody–drug conjugate (ADC) mirvetuximab soravtansine (MIRV) in cisplatin-resistant cancers. We hypothesized that FRα represents an attractive target for treating treatment-refractory GCTs. We determined the expression of the FOLR1 gene in a broad range of GCT cell lines and tumor xenografts. We tested the antitumor efficacy of MIRV on cisplatin-resistant GCT cells in vitro and explored the ability of MIRV treatment to induce a bystander effect in the direct coculture of FRα-high and FRα-low cells. We found that the FOLR1 gene has significantly higher expression in testicular GCTs (TGCTs) than in normal testicular tissue. FOLR1 is highly expressed in the TCam2, JEG3, JAR, and NOY1 cell lines and their respective cisplatin-resistant variants. MIRV treatment induced apoptosis and a potent antiproliferative effect in cisplatin-resistant GCT cells in adherent and 3D spheroid cultures in vitro. A significant decrease in FRα-low 2102EP_R_NL cells was observed in the presence of FRα-high NOY1_R_SK in the presence of 12.5 nM MIRV, showing a potent bystander effect in the direct coculture. Immunohistochemical analysis confirmed significantly higher Folr1 protein expression in patients with TGCTs postchemotherapy than in chemo-naïve patients, as well as in patients with an unfavorable prognosis. In this study, we present data suggesting that the FOLR1 gene is highly expressed in (T)GCT cells in vitro and in vivo, and anti-FRα-targeting therapies should be investigated as a treatment modality in a subset of patients with TGCTs. Moreover, MIRV induced significant antitumor and bystander effects, thus showing its potential in further preclinical exploration and drug repurposing for a salvage treatment regime in refractory (T)GCT disease. Full article

(This article belongs to the Special Issue Signaling Pathways and Mechanisms in Cancer Therapy Resistance)

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FOLR1 gene expression in TGCTs. (A) The FOLR1 gene is highly expressed and significantly upregulated in TGCTs (red) compared with healthy testicular tissue (gray), as well as in other tumor types, such as ovarian cancer (OV), uterine carcinosarcoma (UCS), and uterine corpus endometrial carcinoma (UCEC). (B) FOLR1 expression in the cancer cell lines according to the tissue of origin available from the Cancer Cell Line Encyclopedia (CCLE) public database. Asterisks indicate FOLR1 expression in the TGCT and ChC cell lines; each color illustrates different tissue lineages. (C) Detailed image depicting FOLR1 expression in (T)GCT cell lines 1156QE8, 1618K, 1777NRPMET, 833KE, NCCIT, TERA1, TERA2, NTERA2CLD1 (EC, dark gray); GCT27 and SUSA (TC, blue); and JAR, JEG3, and T3M3 (ChC, light brown) according to the CCLE. (D) FOLR2 expression is also significantly upregulated in TGCT tissue (red) compared with normal tissue (gray), but there is no significant difference in the expression of FOLR3 or IZUMO1R (FRδ). * p ≤ 0.05. Full article ">Figure 1 Cont.
FOLR1 gene expression in TGCTs. (A) The FOLR1 gene is highly expressed and significantly upregulated in TGCTs (red) compared with healthy testicular tissue (gray), as well as in other tumor types, such as ovarian cancer (OV), uterine carcinosarcoma (UCS), and uterine corpus endometrial carcinoma (UCEC). (B) FOLR1 expression in the cancer cell lines according to the tissue of origin available from the Cancer Cell Line Encyclopedia (CCLE) public database. Asterisks indicate FOLR1 expression in the TGCT and ChC cell lines; each color illustrates different tissue lineages. (C) Detailed image depicting FOLR1 expression in (T)GCT cell lines 1156QE8, 1618K, 1777NRPMET, 833KE, NCCIT, TERA1, TERA2, NTERA2CLD1 (EC, dark gray); GCT27 and SUSA (TC, blue); and JAR, JEG3, and T3M3 (ChC, light brown) according to the CCLE. (D) FOLR2 expression is also significantly upregulated in TGCT tissue (red) compared with normal tissue (gray), but there is no significant difference in the expression of FOLR3 or IZUMO1R (FRδ). * p ≤ 0.05. Full article ">Figure 2
FOLR1 gene expression in cisplatin-resistant GCT cell lines and xenografts. (A) Quantitative RT-PCR was used to determine the relative expression level of the FOLR1 gene in a panel of GCT cell lines. The panel includes established cell lines inherently resistant to CPT and pairs of sensitive and derived cisplatin-resistant variants of GCTs of all histological subtypes. C33 cervical adenocarcinoma cell lines and MCF10 mammary epithelial cells were used to confirm low/absent expression in these cells, as expected according to the CCLE. Normal human testicular fibroblast Hs1.Tes and human foreskin fibroblast HuFib were used to confirm very low FOLR1 gene expression in nonmalignant cells. The data are expressed as fold changes in expression, where the expression in SuSa (TC) cells was taken as a reference. (B) Quantitative RT-PCR was used to determine the relative expression level of the FOLR1 gene in xenografts derived from selected GCT cell lines in vivo. High FOLR1 gene expression was confirmed in xenografts derived from seminoma, choriocarcinoma, and yolk sac tumor parental cells and their resistant variants. Full article ">Figure 3
Effect of mirvetuximab soravtansine (MIRV) on cisplatin-resistant GCT cells. (A) TCam2_R_SK cisplatin-resistant seminoma cells were treated with increasing doses of MIRV in vitro. Viability was determined using a luminescent viability assay, illustrating the unique sensitivity of the seminoma cells to MIRV treatment at nanomolar concentrations. (B) Live-cell imaging assays confirmed a significant decrease in the cell number of NLR-JEG3_R_SK choriocarcinoma cells upon treatment with a 3.13nM dose of MIRV. (C) Induction of apoptosis via MIRV in cisplatin-resistant NT2_R_SK cells. NT2_R_SK (EC) cells were treated with 500 nM of MIRV or 2.5 µM of CPT in vitro. Apoptosis induction was kinetically monitored using a green fluorescent signal from Incucyte® Caspase-3/7 Dye for apoptosis in a live-cell imaging system. A significant increase in green signal and apoptosis induction was observed starting 36 h post-treatment (left panel). Relative confluence in the same experiment exhibited a significant decrease starting 48 h post-treatment, corresponding to apoptosis induction and the antiproliferative effect of MIRV (right panel). No significant changes in fluorescence or relative confluence were observed upon treatment with 2.5 µM of CPT corresponding to the cisplatin resistance of the target NT2_R_SK cells. (D) MIRV induced a decrease in cell impedance, as a readout of cytolysis was determined in a kinetic-based impedance assay. JAR_R_SK (ChC) cells were plated onto CytoView plates, and after 24 h, various concentrations of MIRV were added. Dose–response and a decrease in impedance were monitored for 96 h. MIRV at a concentration of 1.56 nM induces complete cell detachment in vitro. Full article ">Figure 4
Antiproliferative effect of MIRV in adherent and spheroid cultures. (A) GCT cells were treated with MIRV, and viability was determined using a luminescent assay. GCT cell lines are aligned according to their sensitivity to the MIRV treatment in vitro, showing the highest sensitivity of TCam2_R_SK (SE), JEG3_R_SK (ChC), JAR_R_SK (ChC), and NOY1_R_SK (oYST). Cisplatin-resistant MDA-MB-231 triple-negative breast cancer (TNBC) cells were included to compare the cytotoxic effect (FOLR1 expression, 2.55 in CCLE). (B) GCT cells were grown as spheroids in 3D culture conditions and treated with increasing concentrations of MIRV. Subsequently, the spheroid viability was determined using an endpoint 3D luminescent assay. JEG3_R_SK (ChC), JAR_R_SK (ChC), and NT2_R_SK (EC) cells retained their sensitivity to MIRV treatment in 3D culture conditions. Mean viability values are shown for each treatment condition; red depicts values above IC50, white corresponds to IC50, and blue illustrates the lowest viability values. Full article ">Figure 5
Bystander effect induced by MIRV into GCT cells. (A) Green fluorescent NLG-2102Ep_R_NL cells were cultured, whether alone or in coculture, with red fluorescent NLR-NOY1_R_SK cells with or without 12.5 nM MIRV. The cell number of the green 2102Ep_R_NL cells was evaluated using live-cell imaging in a kinetic proliferation assay. There was no difference between the proliferation of the FOLR1-refractory 2102Ep_R_NL cells in the coculture and the more sensitive NOY1_R_SK cells. MIRV induced only the limited inhibition of proliferation in 2102Ep_R_NL cells treated alone; however, in the coculture with MIRV-responsive NOY1_R_SK cells, there was substantial cell proliferation inhibition. (B) The endpoint evaluation of the relative fluorescence demonstrated a potent bystander cytotoxicity effect induced by MIRV in the presence of more sensitive NOY1_R_SK cells in the 2102Ep_R_NL refractory target. As an indication of viability, relative fluorescence is significantly lower in the donor cells (left panel) and significantly higher in the target cells with low FOLR1 gene expression and low sensitivity to MIRV (right panel) when cocultured together in the presence of MIRV. * p-value ≤ 0.05. Full article ">Figure 6
Immunohistochemical staining of FOLR1 protein in cisplatin-resistant GCT cell line xenografts TCam2_R_SK (A), NOY1_R-SK (B), JAR_R_SK (C), JEG3_R_SK (D), NT2_R_SK (E), and NCCIT_R_SK (F). Original magnification: ×200; scale bar: 100 μm. Visualization with 3,3′-diaminobenzidine. Full article ">Figure 7
Immunohistochemical analysis of FOLR1 in TGCT patient samples. The FOLR1 level in postchemotherapy-viable tumors was significantly higher than in chemotherapy-naïve TGCTs. There was a significant association between FOLR1 levels in tumor tissues and unfavorable treatment responses when stages IA and IB were excluded or in patients with intermediate/high risk according to their IGGCCG scores. Full article ">

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Cells, Volume 14, Issue 4 (February-2 2025) – 80 articles

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