Fructus Viticis Total Flavonoids (FVTF) is a novel candidate preparation that possesses anticancer activity. However, the role and mechanism of FVTF-inhibiting human hepatocellular carcinoma (HCC) cell stem properties is unclear.
Liquid chromatography (LC) in conjugation with mass spectrometer (MS) was used to identify the compounds of FVTF. Tumorsphere and soft agar colony formation ability, cancer stem marker expression levels, CD133+ cell percentage, and a xenograft model were utilized to investigate the impact of FVTF on HCC cells stemness. PCR array and qRT-PCR were conducted to identify differentially expressed cancer stem-related genes and miRNAs between FVTF-treated and untreated HCC cells, respectively. Pyrosequencing was conducted to assess the DNA methylation level of the miR-34a-5p promoter. A luciferase reporter assay was performed to verify whether FoxM1 serves as a direct target of miR-34a-5p. Additionally, immunohistochemistry of an HCC tissue microarray was carried out to assess the expression levels of DNMT1, FoxM1, and miR-34a-5p.
A total of 26 compounds, including 10 flavones, in FVTF were identified. FVTF significantly reduced the ability of tumorsphere and soft agar colony formation, the levels of CD44 protein and BMI1, OCT4 and SOX2 mRNAs in HCC cells, and in vivo tumor initiation ability of HCC cells. Mechanistically, FVTF inhibited HCC cell stem properties via targeting DNMT1/miR-34a-5p/FoxM1 axis. Clinically, DNMT1 expression was inversely correlated with miR-34a-5p expression, whereas a positive correlation was noted between DNMT1 and FoxM1 expression levels, and high DNMT1 levels, low miR-34a-5p levels, and high FoxM1 levels were associated with cancer recurrence. Furthermore, a combination of DNMT1, miR-34a-5p and FoxM1 served as an independent prognostic indicator influencing both DFS and OS in patients with HCC.
FVTF inhibits HCC cell stem properties by targeting DNMT1/miR-34a-5p/FoxM1 axis, which is associated with HCC recurrence and prognosis, and FVTF is a prospective treatment drug for human HCC.
© 2025. The Author(s).

Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcμR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition.
The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture.
The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.
Copyright © 2024 Prišlin, Butorac, Bertoša, Kunić, Ljolje, Kostešić, Vlahović, Naletilić, Turk and Brnić.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.
© 2024 Wiley Periodicals LLC.

Mesenchymal stem cells (MSCs), also referred to as "medicinal signaling cells," have gained prominence as candidates for cell-based therapy and in clinical trials owing to their regenerative and therapeutic properties. Here, we present a protocol for isolating MSCs from the decidua basalis layer of human placenta using an explant culture approach. We describe steps for collecting, disinfecting, and plating placental tissue. We then detail procedures for characterizing the isolated MSCs through flow cytometry and in vitro differentiation.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.