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Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP (StemPro, StemMacs) culture media to maintain “stemness” of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers (CD146, STRO-1, SSEA-4, CD105, CD106, CD34, K 7/8, K14, K18) variably decreased with prolonged passaging (all three media). The percentage of SA-β-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL (all three media) and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers (Nanog, SOX-2, PDX-1, OTX2, GSC, HCG) decreased with prolonged culture, indicating successive loss of “stemness”. Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions.

Although miR‐148a‐3p has been reported to function as a tumour suppressor in various cancers, the molecular mechanism of miR‐148a‐3p in regulating epithelial‐to‐mesenchymal transition (EMT) and stemness properties of pancreatic cancer (PC) cells remains to be elucidated. In the present study, we demonstrated that miR‐148a‐3p expression was remarkably down‐regulated in PC tissues and cell lines. Moreover, low expression of miR‐148a‐3p was associated with poorer overall survival (OS) in patients with PC. In vitro, gain‐of‐function and loss‐of‐function experiments showed that miR‐148a‐3p suppressed EMT and stemness properties as well as the proliferation, migration and invasion of PC cells. A dual‐luciferase reporter assay demonstrated that Wnt1 was a direct target of miR‐148a‐3p, and its expression was inversely associated with miR‐148a‐3p in PC tissues. Furthermore, miR‐148a‐3p suppressed the Wnt/β‐catenin pathway via down‐regulation of Wnt1. The effects of ectopic miR‐148a‐3p were rescued by Wnt1 overexpression. These biological functions of miR‐148a‐3p in PC were also confirmed in a nude mouse xenograft model. Taken together, these findings suggest that miR‐148a‐3p suppresses PC cell proliferation, invasion, EMT and stemness properties via inhibiting Wnt1‐mediated Wnt/β‐catenin pathway and could be a potential prognostic biomarker as well as a therapeutic target in PC.

Background Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. Methods DPSC and aBMMSC cultures ( n  = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase ( SA-β-gal ) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. Results Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions. Conclusions These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Objective: The functional role and mechanism of the long noncoding RNA (lncRNA) H19 in regulating human pancreatic cancer (PC) cell stemness and invasion have not been completely elucidated. This study aimed to evaluate the role of H19 in regulating the stemness, epithelial-mesenchymal transition (EMT), invasion and chemosensitivity of PC cells. Methods: The sphere-forming ability was assessed using serum-free floating-culture systems. Chemosensitivity was evaluated via CCK-8 and flow cytometry assays in vitro. Migration and invasion were evaluated by transwell assays. The expression of stemness and EMT markers was detected by flow cytometry, qRT-PCR and western blot analyses. Xenograft initiation, growth and sensitivity were examined; Ki-67 nuclear staining intensity was evaluated by immunohistochemistry; and in situ apoptosis was evaluated by a TUNEL assay. Results: H19 played an important role in maintaining PC cell stemness. Upregulated H19 expression in CAPAN-1 cells promoted tumor cell migration, invasion, EMT and chemoresistance. In contrast, downregulated H19 expression in PANC-1 cells yielded the opposite results. These effects were mediated by positively modulating the STAT3 pathway. Furthermore, SOCS5, an endogenous inhibitor of the STAT3 pathway, was a direct target of miR-675-3p, which was positively regulated by H19 in PC cells. Conclusions: The H19/miR-675-3p signaling axis plays a critical role in maintaining the EMT process and stemness of PC cells by directly targeting SOCS5 to activate the STAT3 pathway. These data provide new insights into the oncogenic function of H19 in human PC and reveal potential targets for the development of optimal treatment approaches for this disease.

Follicular helper T (T ) cells are essential for germinal center reactions and the maintenance of long-lived humoral immunity. Transforming growth factor-β (TGF-β) is a multifunctional cytokine implicated in immune regulation, T-cell differentiation, and the maintenance of cellular stemness. Prior studies have shown that TGF-β promotes stemness across a wide range of cell types and facilitates the differentiation of naïve CD4⁺ T cells into various T helper cell subsets. However, its precise effects on T effector function and stem-like properties remain poorly understood. The dual regulatory roles of TGF-β1 in modulating T effector functions and stem-like properties were investigated using flow cytometry-based phenotyping, co-culture assays with memory B cells, proliferation and apoptosis assays, ELISA for antibody production, and bulk RNA sequencing of naïve-derived and blood-derived T cells. We found that TGF-β1 treatment in vitro promoted human naïve CD4 T cells differentiation into CXCR3 TFH, but significantly attenuated their effector molecule expression and T -mediated memory B-cell differentiation and antibody production, whereas it enhanced the expression of stemness-associated molecules in T cells both differentiated in vitro from naïve CD4 T cells and isolated from blood. Notably, TGF-β1 promoted proliferation and reduced apoptosis of naïve-derived T cells in vitro, but suppressed proliferation and increased early apoptosis in blood-derived mature T cells. Our findings indicate that TGF-β1 tunes the balance between T effector function and stem-like properties, and show differential regulations of the early phase of T differentiation and mature T cells, which may have implications for T -driven immune pathology and disease.

Peri-implantitis—an infection caused by bacterial deposition of biofilm—is a common complication in dentistry which may lead to implant loss. Several decontamination procedures have been investigated to identify the optimal approach being capable to remove the bacterial biofilm without modifying the implant surface properties. Our study evaluated whether two different systems—Ni-Ti Brushes (Brush) and Air-Polishing with 40 µm bicarbonate powder (Bic40)—might alter the physical/chemical features of two different titanium surfaces—machined (MCH) and Ca++ nanostructured (NCA)—and whether these decontamination systems may affect the biological properties of human STRO-1+/c-Kit+ dental pulp stem cells (hDPSCs) as well as the bacterial ability to produce biofilm. Cell morphology, proliferation and stemness markers were analysed in hDPSCs grown on both surfaces, before and after the decontamination treatments. Our findings highlighted that Bic40 treatment either maintained the surface characteristics of both implants and allowed hDPSCs to proliferate and preserve their stemness properties. Moreover, Bic40 treatment proved effective in removing bacterial biofilm from both titanium surfaces and consistently limited the biofilm re-growth. In conclusion, our data suggest that Bic40 treatment may operatively clean smooth and rough surfaces without altering their properties and, consequently, offer favourable conditions for reparative cells to hold their biological properties.

TAB182 participates in DNA damage repair and radio-/chemosensitivity regulation in various tumors, but its role in tumorigenesis and therapeutic resistance in breast cancer remains unclear. In the current paper, we observed that triple-negative Breast Cancer (TNBC), a highly aggressive type of breast cancer, exhibits a lower expression of TAB182. TAB182 knockdown stimulates the proliferation, migration, and invasion of TNBC cells. Our study first obtained RNA-seq data to explore the cellular functions mediated by TAB182 at the genome level in TNBC cells. A transcriptome analysis and in vitro experiments enabled us to identify that TAB182 downregulation drives the enhanced properties of cancer stem-like cells (CSCs) in TNBC cells. Furthermore, TAB182 deletion contributes to the resistance of cells to olaparib or cisplatin, which can be rescued by silencing GLI2, a gene downstream of cancer stemness-related signaling pathways. Our results reveal a novel function of TAB182 as a potential negative regulator of cancer stem-like properties and drug sensitivity in TNBC cells, suggesting that TAB182 may be a tumor suppressor gene and is associated with increased therapeutic benefits for TNBC patients.

Mesenchymal stem cells (MSCs), derived from various tissues, are served as a promising source of cells in clinic and regenerative medicine. Umbilical cord-Wharton’s jelly (WJ-MSCs)-derived MSCs exhibit advantages over those from adult tissues, such as no ethical concerns, shorter population doubling time, broad differentiation potential, readily available non-invasive source, prolonged maintenance of stemness properties. The aim of this study was to evaluate the effect of MRI (1.5 T, 10 min) on stemness gene expression patterns (OCT-4, SOX-2, NANOG) of WJ-MSCs. Additionally, we assessed cell viability, growth kinetics and apoptosis of WJ-MSCs after MRI treatment. The quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) data showed that transcript levels of SOX-2, NANOG in MRI-treated WJ-MSCs were increased 32- and 213-fold, respectively. MTT assay was performed at 24, 48, and 72 h post-treatment and the viability was not significantly different between the two groups. The doubling time of the MRI group was markedly higher than the control group. In addition, the colony formation ability of WJ-MSCs after MRI treatment significantly increased. Furthermore, no change in apoptosis was seen before or after MRI treatment. Our results suggest that the use of MRI can improve the quality of MSCs and enhance the efficacy of mesenchymal stem cell-based therapies.

We aimed to study the regulatory roles and mechanism of circular nuclear factor IX (circNFIX) in cancer growth and stemness properties of ovarian cancer (OC). CircNFIX and SH3RF3 levels in OC tissues and cells were tested by quantitative real‐time PCR. RNase R treatment quantified circNFIX RNA stability. Molecular interaction among circNFIX, LIN28B, and SH3RF3 was predicted by bioinformatics software and validated through RNA immunoprecipitation (RIP) assay. The gain‐ or loss‐experiments of circNFIX on capabilities of metastasis and stemness in vitro were assessed using Cell Counting Kit‐8, Transwell, western blot, and sphere‐formation assays. CircNFIX and SH3RF3 were markedly elevated in OC tissues and OC cells. Knocking down circNFIX repressed the proliferation, migration, invasion, and stemness properties of A2780 and SKOV3 cells. The RIP assay verified the direct binding relationship between LIN28B, circNFIX, and SH3RF3. Additionally, overexpression of circNFIX elevated the SH3RF3 expression, while this effect was reversed by LIN28B silence. Rescue experiments demonstrated that the overexpression of SH3RF3 reversed the knockdown of circNFIX on OC cells' proliferation, metastasis, and stemness properties. CircNFIX improved the mRNA stability and translation of SH3RF3 via recruiting LIN28B, thus promoting the proliferation, invasion, and stemness properties of OC cells in vitro.

Hepatocellular carcinoma (HCC) is a major health concern worldwide. A better understanding of the mechanisms underlying the malignant phenotype is necessary for developing novel therapeutic strategies for HCC. Signaling pathways initiated by neurotransmitter receptors, such as α5-nicotinic acetylcholine receptor (CHRNA5), have been reported to be implicated in tumor progression. However, the functional mechanism of CHRNA5 in HCC remains unclear. In this study, we explored the role of CHRNA5 in HCC and found that CHRNA5 expression was increased in human HCC tissues and positively correlated with the T stage (p < 0.05) and AJCC phase (p < 0.05). The KM plotter database showed that the high expression level of CHRNA5 was strongly associated with worse survival in HCC patients. Both in vitro and in vivo assays showed that CHRNA5 regulates the proliferation ability of HCC by regulating YAP activity. In addition, CHRNA5 promotes the stemness of HCC by regulating stemness-associated genes, such as Nanog, Sox2 and OCT4. Cell migration and invasion assays demonstrated that CHRNA5 significantly enhanced the metastasis of HCC by regulating epithelial–mesenchymal transition (EMT)-associated genes. Furthermore, we found that CHRNA5 regulates the sensitivity of sorafenib in HCC. Our findings suggest that CHRNA5 plays a key role in the progression and drug resistance of HCC, and targeting CHRNA5 may be a strategy for the treatment of HCC.