Stem Cells International最新文献

Background: Adipose-derived stem cells (ADSCs) have important application prospects in the field of regenerative medicine, such as adjuvant autologous fat transplantation (AFT), due to their multidirectional differentiation and immunomodulatory functions. However, functional limitation of ADSCs in hypoxic environments may affect their effectiveness in clinical applications. Hypoxic preconditioning is a potential strategy to improve the function of ADSCs by enhancing the antioxidant capacity and metabolic adaptations of the cells, but the optimal hypoxic conditions and the mechanism of action have not yet been clarified.

Methods: ADSCs were extracted and pretreated with hypoxia in order to explore its effect on the function of ADSCs. The activity, apoptosis level, proliferation ability, and antioxidant capacity of ADSCs under normoxic and hypoxic conditions were evaluated using flow cytometry (FCM), live-dead cell fluorescence assay, and apoptosis flow assay. Further, the role of METTL3-mediated m6A modification in hypoxic preconditioning was explored by real-time fluorescence quantitative PCR, protein immunoblotting assay, and m6A modification level detection. Finally, the effect of hypoxic preconditioning of ADSCs on fat graft retention was verified by the mouse AFT model.

Results: We found that the survival of ADSCs was not affected by 5% O₂ pretreatment. Moreover, the cell viability and proliferation of ADSCs were enhanced after 24 h of anoxic preconditioning. Therefore, we determined that 5% O₂ treatment for 24 h was the best hypoxic pretreatment condition for ADSCs, which enhanced the antioxidant capacity of ADSCs, reduced apoptosis. METTL3-mediated m6A modification played a critical role in hypoxic preconditioning to reduce apoptosis in ADSCs. It was verified in a mouse model that hypoxia preconditioning of ADSCs significantly improved adipose graft retention and promoted neovascularization.

Conclusions: METTL3-mediated modification of m6A enhances the function of ADSCs under hypoxic conditions to improve adipose graft retention. These findings provide a new strategy and theoretical basis for improving the clinical outcome of fat grafting, as well as new molecular targets for future research.

Background: The therapeutic potential of human umbilical cord mesenchymal stem cells (HUCMSCs) for diabetic peripheral neuropathy (DPN) and the underlying mechanisms involving transient receptor potential vanilloid 1 (TRPV1) signaling remain incompletely defined.

Objective: This study aimed to elucidate the role of the TRPV1-[Ca²⁺]i-AMPK signaling axis in mediating the beneficial effects of HUCMSCs on neuropathic pain and Schwann cell (SC) dysfunction in DPN.

Methods: A murine model of DPN was established. Mechanical allodynia and thermal hyperalgesia were assessed using Von Frey filaments and the KW-LB hot plate test, respectively. Primary mouse SCs were isolated and cultured under high glucose (HG) conditions. Intracellular calcium ([Ca²⁺]i) levels were quantified by flow cytometry. Protein expression (TRPV1, p-TRPV1, AMPK, p-AMPK, cleaved-caspase-3, Bax, Bcl-2, Drp1, PGC-1α, TFAM, Mfn2) was analyzed via Western blotting. Apoptosis and cell proliferation were evaluated using TUNEL staining and the CCK-8 assay, respectively. Specific inhibitors (AMG9810 for TRPV1 and compound C for AMPK) were employed to probe pathway involvement.

Results: HUCMSC administration significantly alleviated mechanical allodynia and thermal hyperalgesia in diabetic mice. In vitro, HUCMSC coculture counteracted HG-induced effects in SCs by: (1) increasing the p-TRPV1/TRPV1 ratio and [Ca²⁺]i influx (effects blocked by AMG9810); (2) reducing apoptosis (decreased cleaved-caspase-3/Bax, increased Bcl-2); (3) enhancing the p-AMPK/AMPK ratio (attenuated by both AMG9810 and compound C); and (4) promoting mitochondrial homeostasis, increasing PGC-1α, TFAM, and Mfn2 expression, mitochondrial membrane potential and ATP levels, and decreasing Drp1 expression. These mitochondrial improvements were reversed by compound C.

Conclusion: HUCMSCs ameliorate diabetic neuropathic pain primarily through activation of the TRPV1-[Ca²⁺]i-AMPK signaling pathway in SCs, which may provide a new molecular target for enhancing the clinical therapeutic effect of HUCMSCs on DPN.

Periodontitis is a persistent inflammatory ailment that impacts periodontal tissues. Periodontal ligament stem cells (PDLSCs), also referred to as stem cells, possess advantageous attributes for tissue engineering and regenerative medicine due to their ability to self-renew with multi-directional differentiation potential. Nevertheless, the process of cellular senescence can compromise the restoration and regeneration of tissues, thereby impairing the normal regenerative and reparative functions of the periodontium. Exendin-4 (Ex-4) has protective effects against cellular senescence and apoptosis, but the impact of Ex-4 on inflammation-induced senescence of PDLSCs is unknown. This study used lipopolysaccharide (LPS) to simulate an inflammatory microenvironment, and then assessed the effect of Ex-4 on PDLSC senescence within that environment. Initially, PDLSCs were isolated and characterized and then cultured with LPS or LPS and Ex-4. Results demonstrated that the LPS-induced inflammatory microenvironment produced premature senescence of PDLSCs, which was reversible by treatment with Ex-4. Potential mechanisms underlying the effect were evaluated with regard to senescence-associated molecular pathways, and results demonstrated senescence of PDLSCs to be associated with Sirtuin 1 down-regulation and Notch1 upregulation. Our findings suggest that Ex-4 may mitigate the inflammation-induced senescence of PDLSCs through the SIRT1/Notch1 signaling pathway.

Epithelial cells are integral to tumor composition and engage with various immune cell types within the tumor microenvironment, influencing tumor progression and metastasis. A thorough exploration of the roles and mechanisms of these epithelial cells could enhance early detection strategies and treatment modalities for lung adenocarcinoma (LUAD). This research employed single-cell analysis techniques, complemented by machine learning algorithms, to identify genes associated with epithelial cells and evaluate their prognostic significance and implications for immunotherapy in LUAD patients. By leveraging multiple datasets and applying diverse clustering methods within machine learning, we successfully crafted and validated a diagnostic model specifically for LUAD. Among the genes linked to epithelial cells, the XGBoost and random forest techniques identified PMAIP1 as the most crucial gene in terms of prognosis. Additionally, this study investigated the relationship between PMAIP1 and the infiltration of immune cells. The expression levels of PMAIP1 and its relevance in LUAD were subsequently confirmed through immunohistochemical staining and in vitro cell experiments. This analysis revealed 17 key genes associated with epithelial cells by integrating single-cell analysis with clinical data from the TCGA-LUAD dataset, underscoring their significance in diagnosis, prognostic assessment, and possible treatment avenues for LUAD patients. Importantly, PMAIP1 is strongly linked to prognosis and responses to immunotherapy in LUAD, with experimental findings indicating its heightened expression in PRAD and its connection to adverse outcomes. Furthermore, reducing PMAIP1 expression has been shown to hinder the proliferation, metastasis, and stemness of LUAD cells. In summary, our findings indicate that PMAIP1 has potential as a prognostic biomarker and a target for immunotherapy in patients with LUAD.

Introduction: Umbilical cord-derived mesenchymal stem cells (UCMSCs) are promising candidates for the treatment of myocardial infarction (MI). However, their low mobility and survival limit their clinical applicability. This study aimed to enhance the therapeutic potential of UCMSCs by preincubating them with escin, a natural medicine derived from the dried mature seeds of Aesculus wilsonii.

Methods: We characterized the functional properties of UCMSCs before and after escin preconditioning in vitro. Additionally, we performed RNA sequencing (RNA-seq) to analyze the transcriptomic differences between untreated and escin-pretreated UCMSCs (E-UCMSCs), followed by Western blot (WB) validation of the differentially expressed genes. In vivo, an MI model was established in rats, which involved permanent ligation of the left anterior descending coronary artery, followed by intravenous administration of UCMSCs and E-UCMSCs through the tail vein. The therapeutic efficacy of UCMSCs and E-UCMSCs was assessed by cardiac function measurements and Masson's trichrome staining to quantify fibrosis.

Results: No significant differences were observed in the basic characteristics of the UCMSCs before and after escin pretreatment. RNA-seq results demonstrated higher expression of intercellular adhesion molecule 1 (ICAM1) and GATA-binding protein 4 (GATA4) in E-UCMSCs than in UCMSCs. Furthermore, WB results confirmed this phenomenon. Most importantly, E-UCMSCs significantly restored myocardial contractile function and reduced infarct size in MI rats.

Conclusions: The current study demonstrates that escin upregulated ICAM1 and GATA4 gene expression in UCMSCs, thereby enhancing the therapeutic efficacy of UCMSCs in rats with MI. Therefore, pretreatment of UCMSCs with escin is a promising approach for the treatment of MI.

[This retracts the article DOI: 10.1155/2018/3272098.].

Head and neck squamous cell carcinoma (HNSC) is an aggressive malignancy whose progression is closely associated with dysregulation of programed cell death (PCD) pathways and cancer stem cell (CSC) characteristics. To systematically screen for key pathogenic genes, this study performed single-cell analysis on the GSE150321 dataset. The identified cell-specific genes were intersected with PCD- and CSC-related genes, yielding 24 candidate genes for preliminary screening. Further refinement using multiple machine learning (ML) algorithms identified PAK2 as the most central gene among these candidates. Analysis of TCGA and external datasets confirmed that PAK2 is significantly overexpressed in HNSC tissues, demonstrating good diagnostic value and strong association with poor patient prognosis. Functional studies revealed that PAK2 overexpression positively correlates with malignant phenotypes such as metabolic reprograming and tumor metastasis. Notably, PAK2 expression showed a significant negative correlation with antitumor immune status and negatively regulated the infiltration of multiple immune cell types. Spatial transcriptomics and single-cell sequencing analyses revealed PAK2's specific expression patterns within the tumor microenvironment, confirming its influence on the activity of immune-related molecules and immunomodulators. Finally, through Connectivity Map (cMAP) screening and molecular docking, we identified the small molecule compound butein as an effective agent capable of reversing PAK2-mediated procancer molecular features. Butein exhibits stable binding to the PAK2 protein, suggesting its potential as a targeted therapeutic agent. In summary, through multi-omics integration analysis, this study first reveals that PAK2 plays a central role in the pathogenesis of HNSC by regulating PCD, tumor stem cell properties, and the immune microenvironment, and provides a candidate drug for its targeted therapy.

Background: At present, healthcare facilities often face blood shortages because of the low supply of donated blood relative to the high demand. Therefore, efforts to develop red blood cell (RBC) production methods have gained traction. In this work, Lin^-CD45^-CD133⁺ cells were isolated from human umbilical cord blood (UCB) and subsequently differentiated into erythrocytes in vitro in serum-free culture medium.

Methods: Lin^-CD45^-CD133⁺ cells were prepared from mononuclear cells (MNCs) using magnetic-activated cell sorting (MACS). The characteristics of Lin^-CD45^-CD133⁺ cells were confirmed using flow cytometry analysis, colony-forming unit (CFU) assays, morphological analysis, immunocytochemistry (ICC) analysis, and real-time fluorescent quantitative polymerase chain reaction (RT-PCR). Erythrocytes were differentiated in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and FK506 for 13 days, after which autoplasma derived from UCB was added at a concentration of 5% beginning on day 14. Erythroid differentiation and maturation were examined using electron microscopy and flow cytometric analysis.

Results: Lin^-CD45^-CD133⁺ cells were successfully obtained from UCB. These cells were slightly smaller than normal RBCs and had a high nucleus-to-cytoplasm ratio. Oct-4 and Nanog were expressed at both the mRNA and protein levels in Lin^-CD45^-CD133⁺ cells. Most of the colonies were burst-forming unit-erythroid (BFU-E). After 7 days of in vitro culture, the Lin^-CD45^-CD133⁺ cells were negative for CD133 expression and positive for CD45 expression. The percentage of CD71⁺ cells gradually increased, peaked on day 10, and then started decreasing on day 13. The percentage of CD235a⁺ cells increased gradually after day 7 and peaked on day 13. CD240 expression was detected on day 18, with the highest level detected on day 20. The number of erythroid cells increased persistently during differentiation, and their morphology was consistent with that of normal erythrocytes.

Conclusion: An ex vivo culture system was developed that can generate human erythrocytes from Lin^-CD45^-CD133⁺ cells isolated from human UCB.

The development of robust and scalable culture systems is essential for the clinical-scale production of human umbilical cord (UC)-derived mesenchymal stem/stromal cells (MSCs) (UC-MSCs). While various basal and serum-free media are commercially available, systematic comparisons of their efficacy in supporting the expansion and functional properties of UC-MSCs remain limited. In this study, we conducted a comprehensive evaluation of multiple culture systems, including basal media (α-MEM, DMEM, and DMEM/F12) supplemented with human platelet lysate (HPL), and commercial serum-free media (Corning MSC Xeno-Free SFM, NutriStem XF Medium, Prime-XV MSC Expansion XSFM), for their ability to sustain UC-MSCs proliferation, maintain phenotypic properties, and support functional potency. The results demonstrated that all basal media supported cell growth, with α-MEM (Gibco) and DMEM/F12 showing superior performance over DMEM. Among serum-free formulations, Prime-XV with 2% HPL yielded the highest primary culture output and the shortest population doubling (PD) time (PDT) during passaging. Notably, cells expanded in commercial serum-free media exhibited reduced diameter and higher uniformity. Functional analyses revealed that NutriStem XF Medium supplemented with 2% HPL elicited the strongest immunomodulatory effects in mixed lymphocyte reactions (MLRs). Furthermore, all media maintained trilineage differentiation capacity and satisfied International Society for Cellular Therapy (ISCT) phenotypic criteria. Critically, no tumorigenic potential was detected in vitro or in vivo. Large-scale manufacturing using the selected medium (NutriStem XF + 2% HPL) confirmed consistent expansion kinetics, high viability, stable marker expression, and functional potency across seven production batches. This study provides a rigorous and clinically relevant framework for selecting culture media that ensure both scalability and functional integrity of UC-MSCs, highlighting the promise of serum-free systems for therapeutic manufacturing.