Human Cell Cultures Research Articles

MiR‐124 affects dendritic cells function in CRSwNP by regulating exosomes secretion in nasal mucosal epithelial cells

AbstractThe intricate immunomodulatory mechanism underlying the onset and progression of chronic rhinosinusitis with nasal polyps (CRSwNP) in chronic sinusitis with nasal polyps remains poorly understood, particularly regarding the regulatory role of dendritic cells (DCs). MicroRNA has been identified as a modulator of DC function. Primary cells have a short time in vitro and their biological characteristics have not changed greatly which can better reflect the growth state of cells in the body, so as to obtain data closer to the physiological function in the body. Our research group has been engaged in the culture of human nasal epithelial progenitor cells (hNEPCs) for a long time, and in previous investigations, we observed downregulation of microRNA‐124 (miR‐124) in patients with chronic sinusitis and nasal polyps. Thus, our current study aims to elucidate the regulatory mechanism of DCs in chronic sinusitis with polyps by examining the impact of miR‐124 on DCs in this context. To accomplish this, we took the nasal mucosa of CRSwNP patients and normal nasal mucosa of non‐rhinitis patients for primary culture. We isolated exosomes from these cells for co‐culture experiments with DCs. Flow cytometry analysis was employed to assess the maturation phenotypes (MHC II, CD80 and CD86), cell cycle and apoptosis levels of DCs. Furthermore, enzyme‐linked immunosorbent assay was utilized to measure the levels of pro‐inflammatory cytokines (IL‐10, IL‐12 and IL‐23). The TLR4/NF‐κB signalling pathway in co‐cultured DCs was evaluated using the western blot assay. Through querying the Targetscan website, we predicted Rab27a as a target gene of miR‐124, which was subsequently validated through double luciferase assay, flow cytometry and other experimental approaches. Our findings demonstrate that MiR‐124 modulates the secretion of CRSwNP epithelial exosomes, thereby influencing DC function via its interaction with the target gene Rab27a. These results offer novel insights into potential therapeutic targets for the treatment of CRSwNP.

Targeting TRPV1 signaling: Galangin improves ethanol-induced gastric mucosal injury

Galangin, a bioactive compound extracted from Alpinia officinarum Hance (Zingiberaceae), a plant with significant ethnopharmacological importance, has been used for thousands of years as a spice, condiment, and medicinal agent for various conditions, including gastrointestinal disorders. Although there is evidence suggesting its potential to improve gastric ulcers, the molecular mechanisms underlying its anti-ulcer properties are not fully understood. of the Study: This study aimed to investigate the effects of galangin on ethanol-induced acute gastric mucosal injury (AGMI) in mice and elucidate its molecular mechanisms. Sixty BALB/c mice were randomly assigned into two main groups: a normal control group (n=10) and an ethanol-induced group (n=50). After establishing the AGMI model in mice using a combination of 40% ethanol and anhydrous ethanol, the ethanol-induced group was further subdivided into five subgroups (n=10): an omeprazole control group (20mg/kg), an untreated ethanol group, and three treatment groups receiving high-dose (50mg/kg) or low-dose (25mg/kg) galangin or capsazepine (CPZ, 2mg/kg). The protective effects of galangin were evaluated through mucosal injury indices, hematoxylin and eosin staining, and quantification of inflammatory markers (IL-1β, IL-6, IL-8, and TNF-α). Oxidative stress levels and matrix metalloproteinase activity were measured using specific assay kits. Molecular docking was conducted to assess the binding affinity of galangin to key proteins within the transient receptor potential vanilloid 1 (TRPV1) pathway. Real-time fluorescence quantitative PCR (qPCR) was used to determine mRNA expression levels of TRPV1, calmodulin (CaM), substance P (SP), and CGRP in gastric tissues. Protein expression levels of TRPV1, nerve growth factor (NGF), tropomyosin receptor kinase A (TRKA), transforming growth factor beta (TGF-β), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) were assessed through Western blot analysis. In cellular experiments, Culture of Human Gastric Epithelial Cells (GES-1) were treated with various concentrations of galangin after 7% ethanol induction. Cell proliferation, apoptosis, and migration were evaluated using Hoechst 33258 staining and transwell migration assays. TRPV1 protein expression was detected using immunofluorescence, and the expression levels of Bcl-2, BCL2-Associated X (BAX), and Caspase-3 were quantified by qPCR. Additionally, specific probe kits were used to measure intracellular calcium ions (Ca2+) and mitochondrial membrane potential. The findings indicate that galangin significantly improved mucosal pathology by reducing ulcer indices and inflammatory levels, while enhancing superoxide dismutase (SOD) activity and decreasing malondialdehyde (MDA) concentration. Galangin also reduced matrix metalloproteinase-2 (MMP-2), m metalloproteinase-9 (MMP-9) levels, promoting mucosal repair. At the cellular level, galangin decreased intracellular calcium ion concentration and mitigated the decline in mitochondrial membrane potential, enhance the restoration of mucosal cells, increased migration and proliferation, and reduced apoptosis. Molecularly, galangin demonstrated favorable binding to TRPV1, NGF, TRKA, TGF-β, COX-2, and NF-κB, and reversed the elevated expression of these proteins. Additionally, galangin downregulated the mRNA expression of TRPV1, CaM, SP, CGRP, BAX, and Caspase-3 in gastric tissues/cells, while upregulating Bcl-2 mRNA expression. Galangin mitigates AGMI by inhibiting the overactivation of the TRPV1 pathway, thereby blocking aberrant signal transduction. This study suggests that galangin has therapeutic potential against ethanol-induced AGMI and may be a viable alternative for the treatment of alcohol-induced gastric mucosal injuries.

Uniform Suspension of Heat-Killed Staphylococcus aureus for a Positive Control Used in the Monocyte-Activation Test.

Pyrogens, classified as bacterial endotoxins and non-endotoxin pyrogens (NEPs), induce fever or shock when released into the bloodstream or spinal fluid. Recently, a monocyte-activation test (MAT) involving human cell culture has been developed to detect pyrogens in injectable products. To evaluate the sensitivity of MAT, a reference standard endotoxin was used as a positive control; however, the reactivity differed between the endotoxins and NEPs, necessitating positive controls for NEPs. This study aimed to explore a preparation method for heat-killed Staphylococcus aureus (HKSA) as a positive control for NEPs in MAT. Because S. aureus forms grape-like clusters, nine types of glass filters with pore sizes of 0.5-2.7 µm were evaluated to obtain a uniform bacterial suspension. The suspension was then heat-treated to kill the bacteria, resulting in HKSA samples. Serial dilutions of HKSA were tested by MAT using peripheral blood mononuclear cells. The interleukin-6 concentrations in the culture supernatant were measured by enzyme-linked immuno-sorbent assay to assess pyrogenic activities of HKSA. The pore sizes of the glass filters affected the uniformity of HKSA, and GF/C filter was selected for HKSA preparation. Repeated filtration improved uniformity, and a uniform suspension of HKSA was obtained through double filtration using a GF/C filter. Despite the decrease in HKSA activity as filtration frequency increased, the detection limit remained consistently unchanged. This suggests that repeated filtration can adjust the activity of HKSA to a baseline level and that a uniform suspension of HKSA exhibiting low variation is suitable as a positive control in MAT.

Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

Synthesis, crystal structure and cytotoxic activity of 2,2′-bipyridine / 1,10-Phenanthroline based Copper(II) complexes with diphenylphosphinic acid

Mixed-ligand 2,2′-bipyridine, 1,10-phenanthroline based copper(II) complexes are often considered as potential antitumor agents. This research was aimed at synthesis and finding the cytotoxic potential of copper(II) complexes with diphenylphosphinic acid (HL) and 2,2′-bipyridine, 1,10-phenanthroline derivatives – [Cu(phen)(H2O)L2]·H2O (1), [Cu(dmphen)(H2O)L2] (2), [Cu(Cl-phen)(H2O)2L]L·EtOH (3), [Cu(bipy)(H2O)L2] (4) and Cu(dmbipy)(H2O)L2 (5) (phen – 1,10-phenanthroline, dmphen – 4,7-dimethyl-1,10-phenanthroline, Cl-phen – 5-chloro-1,10-phenanthroline, bipy – 2,2′-bipyridine, dmbipy – 4,4′-dimethyl-2,2′-bipyridine). Obtained mononuclear compounds have been characterized by EPR and IR-spectroscopy, elemental, thermogravimetric, single-crystal and powder X-ray diffraction analyses. According to single-crystal X-ray diffraction data, complexes possess distorted trigonal bipyramidal or square pyramidal geometry. 2,2′-Bipyridine and 1,10-phenanthroline derivatives have been shown to be chelating agents in these complexes, while anion of diphenylphosphinic acid acts as a monodentate ligand. Complexes 1–5 have been monitored for their solution stability using UV–vis spectroscopy. In the present study, cytotoxic activity of the complexes has been investigated on 2D human cell culture models (larynx carcinoma Hep2, hepatocellular carcinoma HepG2, breast carcinoma MCF-7 and non-tumor lung fibroblasts MRC5) and 3D HepG2 human cell model. 1,10-Phenanthroline based complexes have demonstrated the highest cytotoxicity superior to the reference drug cisplatin. The level of reactive oxygen species generation in Hep2 cells has been shown to increase after incubation with complexes.

Characterization of the TLR9-Activating Potential of LNA-Modified Antisense Oligonucleotides.

Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.

Primary human mesenchymal stem cell culture under xeno-free conditions using surface-modified cellulose nanofiber scaffolds

Stem cell culture often requires various animal-derived components such as serum and collagen. This limits its practical use. Therefore, xeno-free (xenogeneic component-free) culture systems are receiving increased attention. Herein, we propose xeno-free, plant-derived cellulose nanofibers (CNFs) with different surface chemistry: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized CNFs (TOCNFs) with carboxy groups and surface-sulfated CNFs (S-CNFs) for the proliferation of human mesenchymal stem cells (hMSCs) under various serum conditions. We cultured bone marrow-derived hMSCs on CNF scaffolds with various fiber lengths and functional group contents. Original CNFs were bioinert materials that did not contribute to cell adhesion. In contrast, the surface-modified CNFs facilitated the proliferation of immortalized hMSCs under normal and low-serum conditions. The TOCNFs (COONa: 1.47 mmol g−1; length: 0.53 μm), the S-CNFs (OSO3Na: 0.64 mmol g−1; 0.61 μm), and a combination of the two (1:1 by weight) enabled immortalized hMSCs to maintain their multipotency, even under serum-free conditions. Primary cultured hMSCs proliferated well on the TOCNF/S-CNF scaffolds in a completely serum-free medium, comparable to animal-derived type I collagen, although few hMSCs adhered to the standard polystyrene substrate. Our strategy of using surface-modified CNFs will inform the development of xeno-free culture systems to avoid the use of animal-derived materials for both cell culture media and scaffolds.

Enzymatic Acrolein Production System and Its Impact on Human Cells.

Acrolein is an environmental toxicant and is also generated by microbial metabolism in the intestinal tract. Aqueous acrolein rapidly dissipates from standard human cell culture media with nondetectable levels after 8 h, hindering cell-based studies to understand its biological impacts. Thus, we developed an extracellular acrolein biosynthesis system to continuously produce acrolein compatible with human cell culture conditions. The approach uses spermine as a precursor, amine oxidase found in fetal calf serum, and catalase to remove the hydrogen peroxide byproduct. We confirmed amine oxidase activity of calf serum using a colorimetric assay and further tested the requirement for catalase in the system to mitigate hydrogen peroxide-induced cytotoxicity. We calibrated responses of human colon cells to this enzymatic acrolein production system by comparing transcriptional responses, DNA adduct formation and cytotoxicity responses to either this system or pure acrolein exposures in a human colon cell line. Several genes related to oxidative stress including HMOX1, and the colorectal cancer-related gene SEMA4A were upregulated similarly between the enzymatic acrolein production system or pure acrolein. The acrolein-DNA adduct γ-OH-Acr-dG increased in a dose-dependent manner with spermine in the enzymatic acrolein production system, producing a maximum of 1065 adducts per 108 nucleosides when 400 μM spermine was used. This biosynthetic production method provides a relevant model for controlled acrolein exposure in cultured human cells and overcomes current limitations due to its physical properties and limited availability.

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

Stem Cell Studies of Professor Doctor Sureyya Tahsin Aygun

Stem cells are unspecialized cells in the human body. They can differentiate into any cell of the organism and have the ability to renew themselves. Thus, they contribute to the formation of all mature cells in the body and form the basis of every cell, tissue and organ. A stem cell can ensure both self-renewal and differentiation. Süreyya Tahsin Aygün received his Ph.D. from the Berlin School of Veterinary Medicine in 1926. He focused on cell culture and conducted studies on cardiac stem cells. He observed the regenerative and proliferative capacity of cardiac stem cells. Aygün concluded that young, immature, homogeneous human cells have an unexpected medical effect because animal cells are suitable for animal organism and human cells are suitable for human organism. Aygün's treatment with human cell cultures can be used for many diseases such as heart, kidney, liver and circulatory diseases, stroke, tumors, schizophrenia and multiple sclerosis. Even prolongation of physiological age and life expectancy combined with visible rejuvenation has come into the realm of possibility as pathophysiologically aging cells regress and are replaced by young, viable human cultured cells. Süreyya Tahsin Aygün is the first Turkish scientist to work on stem cells. Süreyya Tahsin Aygün's studies in Germany and Turkey demonstrate his contribution to stem cell and regenerative medicine. Looking at Aygün's other studies, one better understands the importance of joint research between veterinary medicine and medicine. Ord. prof. Aygün's work "Die Human-Zellkultur-Therapie, Neue erfolgreiche Möglichkeiten zur Therapie des Mongolismus und anderer Krankheiten" and other works should be reissued in view of their contribution to today's medical developments.

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Serum from patients with cirrhosis undergoing liver transplantation induces permeability in human pulmonary microvascular endothelial cells ex vivo.

Patients with cirrhosis undergoing liver transplantation frequently exhibit systemic inflammation, coagulation derangements, and edema, indicating endothelial dysfunction. This syndrome may worsen after ischemia-reperfusion injury of the liver graft, coincident with organ dysfunction that worsens patient outcomes. Little is known about changes in endothelial permeability during liver transplantation. We hypothesized that sera from these patients would increase permeability in cultured human endothelial cells ex vivo. Adults with cirrhosis presenting for liver transplantation provided consent for blood collection during surgery. Sera were prepared at five time points spanning the entire operation. The barrier function of human pulmonary microvascular endothelial cells in culture was assessed by transendothelial resistance measured using the ECIS ZΘ system. Confluent cells from two different endothelial cell donors were stimulated with human serum from liver transplant patients. Pooled serum from healthy men and purified inflammatory agonists served as controls. The permeability response to serum was quantified as the area under the normalized resistance curve. Responses were compared between time points and analyzed for associations with clinical characteristics of liver transplant patients and their grafts. Liver transplant sera from all time points during surgery-induced permeability in both endothelial cell lines. The magnitude of permeability change was heterogeneous between patients, and there were differences in the effects of sera on the two endothelial cell lines. In one of the cell lines, the severity of liver disease was associated with greater permeability at the start of surgery. In the same cell line, serum collected 15 min after liver reperfusion induced significantly more permeability as compared to that collected at the start of surgery. Early postreperfusion sera from patients undergoing living donor transplants induced more permeability than sera from deceased donor transplants. Sera from two exemplary cases of patients on preoperative dialysis, and one patient with an unexpectedly long warm ischemia time of the liver graft, induced exaggerated and prolonged endothelial permeability. Serum from patients with cirrhosis undergoing liver transplantation induces permeability of cultured human pulmonary microvascular endothelial cells. Increased endothelial permeability during liver transplantation may contribute to organ injury and present a target for future therapeutics.

P-800 Best matrix match: Laminin-511 improves human trophoblast stem cell identity in vitro

Abstract Study question Can alternative basement membranes support human trophoblast stem cell (hTSC) culture and recapitulate the in vivo environment more effectively than Collagen-IV (Col-IV)? Summary answer Laminin-511 (Lam-511) is the optimal alternative xeno-free matrix that enhances the stem cell state and more closely recapitulates the in vivo hTSC niche. What is known already Our knowledge on early placentation is limited due to a historical lack of robust in vitro models. Recently, hTSCs isolated from human blastocysts and first trimester placentas were successfully cultured on Col-IV, recapitulating cytotrophoblast cell self-renewal and differentiation. Even though Col-IV has been detected in the cytotrophoblast basement membrane, previously published RNA-seq analysis does not detect transcripts encoding Col-IV in the human trophectoderm. Attachment of adhesion molecules to the ECM can have an effect on cell proliferation, differentiation, polarity and gene expression. Using the correct matrix ensures a stable cell growth, as it can influence cell behaviour and identity. Study design, size, duration This was a prospective study carried out over a 6-month period. Patient-derived hTSC lines, BTS5 and CT27, obtained from RIKEN BRC Cell Bank were cultured according to the previously published protocol (1). hTSCs were maintained on culture plates coated with Col-IV, Lam-511, laminin-521 and fibronectin. Experiments were performed with biological replicates of both cell lines (n = 4), grown in every condition. Cells grown on Col-IV were used as a positive control. Participants/materials, setting, methods hTSCs were maintained on the candidate matrices for minimum of 5 passages and then were used for subsequent experiments. hTSC identity was determined by immunofluorescence analysis for known hTSC markers, GATA2 and KRT18. qRT-PCR analyses were also performed to detect additional markers EGFR, ITGA6, TEAD4, TP63, and expression from the chromosome 19 miRNA cluster (C19MC). The self-renewal capacity was assessed by cell counting and Ki67-staining. The differentiation potential was examined by performing previously published protocols. Main results and the role of chance RNA-seq analysis identified expression of ECM ligand and receptor transcripts encoding Lam-511, laminin-521 and fibronectin, which were used as candidate matrices to test. hTSC identity was maintained in all conditions as confirmed by morphological assessment, expression of known hTSC markers at the transcript and protein level, and detection of expression of C19MC miRNAs. The highest hTSC marker expression was found in cells grown on laminins. However, interestingly it was observed that only Lam-511 was capable of supporting hTSCs for more than 15 passages. Compared to Col-IV, hTSCs grown on Lam-511 appear to be more proliferative as there was a higher cell count over a 6-day period and a higher proportion of Ki-67-positive proliferative cells (p = 0.03). Additionally, preliminary data suggests that differentiation ability of hTSCs grown on Lam-511 into syncytiotrophoblasts and extravillous cytotrophoblasts is retained. Therefore, Lam-511 is an improvement to the conventional model as it enhances the stem-cell identity and closely recapitulates the in vivo niche. It can also be used as an alternative xeno-free matrix in hTSC culture in order to overcome current supply chain issues and reduce batch-to-bath variability associated with Col-IV. Limitations, reasons for caution During the 6-month period of this study, two contaminations occurred that led to slower progression of the experiments and smaller sample size. Also, there was low efficiency of the extravillous cytotrophoblast differentiation which was due to the fact that the previously published protocol performed has not yet been optimised. Wider implications of the findings Recapitulating the in vivo niche more closely will allow a deeper understanding of early placentation and help create more accurate 3D models to study the pathomechanisms of various pregnancy complications. Furthermore, utilising the xeno-free Lam-511 as an alternative matrix is a step closer to building a GMP-compliant hTSC culture. Trial registration number Not applicable

An expedited screening platform for the discovery of anti-ageing compounds in vitro and in vivo

BackgroundRestraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan.MethodsUsing Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture.ResultsWe developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo.ConclusionsOur method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

SARS-CoV-2-related peptides induce endothelial-to-mesenchymal transition in endothelial capillary cells derived from different body districts: focus on membrane (M) protein.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19, may lead to multiple organ dysfunctions and long-term complications. The induction of microvascular dysfunction is regarded as a main player in these pathological processes. To investigate the possible impact of SARS-CoV-2-induced endothelial-to-mesenchymal transition (EndMT) on fibrosis in "long-COVID" syndrome, we used primary cultures of human microvascular cells derived from the lungs, as the main infection target, compared to cells derived from different organs (dermis, heart, kidney, liver, brain) and to the HUVEC cell line. To mimic the virus action, we used mixed SARS-CoV-2 peptide fragments (PepTivator®) of spike (S), nucleocapsid (N), and membrane (M) proteins. TGFβ2 and cytokine mix (IL-1β, IL-6, TNFα) were used as positive controls. The percentage of cells positive to mesenchymal and endothelial markers was quantified by high content screening. We demonstrated that S+N+M mix induces irreversible EndMT in all analyzed endothelial cells via the TGFβ pathway, as demonstrated by ApoA1 treatment. We then tested the contribution of single peptides in lung and brain cells, demonstrating that EndMT is triggered by M peptide. This was confirmed by transfection experiment, inducing the endogenous expression of the glycoprotein M in lung-derived cells. In conclusion, we demonstrated that SARS-CoV-2 peptides induce EndMT in microvascular endothelial cells from multiple body districts. The different peptides play different roles in the induction and maintenance of the virus-mediated effects, which are organ-specific. These results corroborate the hypothesis of the SARS-CoV-2-mediated microvascular damage underlying the multiple organ dysfunctions and the long-COVID syndrome.

Synthesis of Mesylated and Tosylated α-Hydroxy-Benzylphosphonates; Their Reactivity and Cytostatic Activity.

α-Hydroxyphosphonates and their acylated and phosphorylated derivatives may be of significant biological activity including cytotoxic effects. To extend the pool of the potentially bioactive species, new methane- and arenesulfonyloxyphosphonates were synthesized by the sulfonylation of differently substituted α-hydroxy-benzylphosphonates using methanesulfonyl chloride or p-toluenesulfonyl chloride at 25 °C in the presence of triethylamine in toluene. The new sulfonyl derivatives were obtained in 54-80% yields. In the case of the 4- or 2-methoxy substituent in the aromatic ring, surprisingly the corresponding α-chlorophosphonates were the exclusive products, whose formation was explained assuming a quinoid intermediate and supported by theoretical calculations. With a 3-methoxyphenyl substituent, the expected mesylation of the hydroxy group took place. Attempted alcoholyses of the diethyl α-methanesulfonyloxy-benzylphosphonates with different substituents in the benzyl ring at ∼140 °C in the presence of triethylamine under microwave irradiation left the P-function intact under the conditions applied, instead, the mesyloxy group was substituted by an alkoxy unit in a selective new reaction. The α-alkoxy-benzylphosphonates were isolated in 60-77% yields. While α-chloro- or α-bromo-benzylphosphonates proved to be rather inefficient in the Michaelis-Arbuzov reaction with triethyl phosphite, according to a new possibility, the α-methansulfonyloxy-benzylphosphonates underwent an efficient Arbuzov fission using the phosphite in excess at 135 °C. The arylmethylenebisphosphonates were obtained in yields of 76-81%. Bioactivity studies with the members of the phosphonate library revealed pronounced in vitro cytostatic effect of the α-hydroxy- and α-mesyloxy-3,5-di-tert-butylbenzylphosphonates on human breast carcinoma cell culture with IC50 values of 16.4 and 28.0 μM, respectively. The mesyloxy species was also cytostatic on melanoma cells (IC50 = 34.9).

Fibroblast growth factor 23-mediated regulation of osteoporosis: Assessed via Mendelian randomization and invitro study.

Despite numerous investigations on the influence of fibroblast growth factor 23 (FGF23), α-Klotho and FGF receptor-1 (FGFR1) on osteoporosis (OP), there is no clear consensus. Mendelian randomization (MR) analysis was conducted on genome-wide association studies (GWASs)-based datasets to evaluate the causal relationship between FGF23, α-Klotho, FGFR1 and OP. The primary endpoint was the odds ratio (OR) of the inverse-variance weighted (IVW) approach. Furthermore, we stably transfected FGF23-mimic or siRNA-FGF23 into human bone marrow mesenchymal stem cells (hBMSCs) in culture and determined its cell proliferation and the effects on osteogenic differentiation. Using MR analysis, we demonstrated a strong correlation between serum FGF23 levels and Heel- and femoral neck-BMDs, with subsequent ORs of 0.919 (95% CI: 0.860-0.983, p = 0.014) and 0.751 (95% CI: 0.587-0.962; p = 0.023), respectively. The expression levels of FGF23 were significantly increased in femoral neck of patients with OP than in the control cohort (p < 0.0001). Based on our invitro investigation, after overexpression of FGF23, compared to the control group, the BMSC's proliferation ability decreased, the expression level of key osteogenic differentiation genes (RUNX2, OCN and OSX) significantly reduced, mineralized nodules and ALP activity significantly decreased. After silencing FGF23, it showed a completely opposite trend. Augmented FGF23 levels are causally associated with increased risk of OP. Similarly, FGF23 overexpression strongly inhibits the osteogenic differentiation of hBMSCs, thereby potentially aggravating the pathological process of OP.

Transcriptional insights from the human embryo identify laminin-511 as a suitable matrix for human trophoblast stem cell culture

The establishment of culture conditions to propagate self-renewing human trophoblast stem cells in long-term culture provides a paradigm for in vitro modelling of trophoblast. The extracellular matrix (ECM) is a critical determinant of cell identity and behaviour. Therefore, models aiming to reproduce cells in vitro should recapitulate the native cell-ECM microenvironment. Here, we mine human embryo transcriptional datasets to identify ECM components and cognate receptors expressed in the trophectoderm. Following, we identify laminin-511-E8 protein fragment as a physiologically relevant ECM capable of maintaining hTSCs in the stem cell state and retaining differentiation ability.

Biocompatibility Testing of UV-Curable Polydimethylsiloxane for Human Umbilical Vein Endothelial Cell Culture on-a-Chip.

Polydimethylsiloxane (PDMS) is extensively used to fabricate biocompatible microfluidic systems due to its favorable properties for cell culture. Recently, ultraviolet-curable PDMS (UV-PDMS) has shown potential for enhancing manufacturing processes and final optical quality while retaining the benefits of traditional thermally cured PDMS. This study investigates the biocompatibility of UV-PDMS under static and flow conditions using human umbilical vein endothelial cells (HUVECs). UV-PDMS samples were treated with oxygen plasma and boiling deionized water to assess potential improvements in cell behavior compared with untreated samples. We evaluated HUVECs adhesion, growth, morphology, and viability in static cultures and microchannels fabricated with UV-PDMS to test their resistance to flow conditions. Our results confirmed the biocompatibility of UV-PDMS for HUVECs culture. Moreover, plasma-oxygen-treated UV-PDMS substrates exhibited superior cell growth and adhesion compared to untreated UV-PDMS. This enhancement enabled HUVECs to maintain their morphology and viability under flow conditions in UV-PDMS microchannels. Additionally, UV-PDMS demonstrated improved optical quality and more efficient handling and processing, characterized by shorter curing times and simplified procedures utilizing UV light compared to traditional PDMS.

An antibody to IL-1 receptor 7 protects mice from LPS-induced tissue and systemic inflammation.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.