Soluble Factors Research Articles

Future Perspectives of Wharton's Jelly Mesenchymal Stem Cells and their Soluble Factors in Radioprotection.

Acute radiation syndrome (ARS) is also known as triple syndrome, which develops after whole-body radiation exposure. During unforeseen exposures, these syndromes are set in depending on the dose of radiation. Cell-based therapy, especially using stem cells and their soluble factors, is gaining wide attention in the field of regenerative medicine to treat various diseases, including degenerative diseases. Stem cells attract prime attention because of their profound inherent tissue repair capability and regeneration potential. Further, stem cell therapy can be one of the promising strategies for the amelioration of ARS because of its ability to lodge in damaged tissue and release regenerative cytokines by sensing the local injury. In this regard, human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) have gained substantial attention for their applications in the treatment of various human diseases due to several advantages offered by them. This article is intended to provide future perspective on the use of WJ-MSCs for the management of accidental radiation injury in pre-clinical models, and finally, their utility in regeneration of damaged tissues and organs.

An Overview of Optic Pathway Glioma With Neurofibromatosis Type 1: Pathogenesis, Risk Factors, and Therapeutic Strategies.

Optic pathway gliomas (OPGs) are most predominant pilocytic astrocytomas, which are typically diagnosed within the first decade of life. The majority of affected children with OPGs also present with neurofibromatosis type 1 (NF1), the most common tumor predisposition syndrome. OPGs in individuals with NF1 primarily affect the optic pathway and lead to visual disturbance. However, it is challenging to assess risk in asymptomatic patients without valid biomarkers. On the other hand, for symptomatic patients, there is still no effective treatment to prevent or recover vision loss. Therefore, this review summarizes current knowledge regarding the pathogenesis of NF1-associated OPGs (NF1-OPGs) from preclinical studies to seek potential prognostic markers and therapeutic targets. First, the loss of the NF1 gene activates 3 distinct Ras effector pathways, including the PI3K/AKT/mTOR pathway, the MEK/ERK pathway, and the cAMP pathway, which mediate glioma tumorigenesis. Meanwhile, non-neoplastic cells from the tumor microenvironment (microglia, T cells, neurons, etc.) also contribute to gliomagenesis via various soluble factors. Subsequently, we investigated potential genetic risk factors, molecularly targeted therapies, and neuroprotective strategies for tumor prevention and vision recovery. Last, potential directions and promising preclinical models of NF1-OPGs are presented for further research. On the whole, NF1-OPGs develop as a result of the interaction between glioma cells and the tumor microenvironment. Developing effective treatments require a better understanding of tumor molecular characteristics, as well as multistage interventions targeting both neoplastic cells and non-neoplastic cells.

Exercise: Just What the Doctor Ordered, But Why? Elucidating Mechanisms for Women's Increased High-Density Lipoprotein Benefit From Exercise and for the Health ABC Study.

High-density lipoprotein (HDL) is protective against cardiovascular disease. Exercise can increase HDL concentration, and some evidence suggests that this effect occurs more strongly in women than in men. Both HDL and exercise are associated with inflammation. We hypothesized a sex-by-exercise interaction on HDL level, whereby women would benefit from exercise more strongly than men, and tumor necrosis factor alpha and serum soluble tumor necrosis factor receptor-2 would mediate this relationship. This study included 2,957 older adult participants (1,520 women; 41% Black, 59% White; 73.6-years-old) from the Health, Aging, and Body Composition study. Regression models revealed a positive exercise-HDL relationship in women only (sex-by-exercise interaction: β = 0.09, p = .013; exercise on HDL in women: β = 0.07, p = .015), mediated by TNFα (axb = 0.15; CI: 0.01, 0.30), suggesting that exercise may increase HDL levels in women through reduced inflammation. Given that vascular risk contributes to Alzheimer's disease risk, findings have implications for sex differences in AD risk factors.

The roles of cancer stem cell-derived secretory factors in shaping the immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide and has a poor prognosis. Although immune checkpoint inhibitors have entered a new era of HCC treatment, their response rates are modest, which can be attributed to the immunosuppressive tumor microenvironment within HCC tumors. Accumulating evidence has shown that tumor growth is fueled by cancer stem cells (CSCs), which contribute to therapeutic resistance to the above treatments. Given that CSCs can regulate cellular and physical factors within the tumor niche by secreting various soluble factors in a paracrine manner, there have been increasing efforts toward understanding the roles of CSC-derived secretory factors in creating an immunosuppressive tumor microenvironment. In this review, we provide an update on how these secretory factors, including growth factors, cytokines, chemokines, and exosomes, contribute to the immunosuppressive TME, which leads to immune resistance. In addition, we present current therapeutic strategies targeting CSC-derived secretory factors and describe future perspectives. In summary, a better understanding of CSC biology in the TME provides a rational therapeutic basis for combination therapy with ICIs for effective HCC treatment.

Mesenchymal Stem Cells from Mouse Hair Follicles Inhibit the Development of Type 1 Diabetes.

Mesenchymal stem cells (MSCs) are known for their immunosuppressive properties. Based on the demonstrated anti-inflammatory effect of mouse MSCs from hair follicles (moMSCORS) in a murine wound closure model, this study evaluates their potential for preventing type 1 diabetes (T1D) in C57BL/6 mice. T1D was induced in C57BL/6 mice by repeated low doses of streptozotocin. moMSCORS were injected intravenously on weekly basis. moMSCORS reduced T1D incidence, the insulitis stage, and preserved insulin production in treated animals. moMSCORS primarily exerted immunomodulatory effects by inhibiting CD4+ T cell proliferation and activation. Ex vivo analysis indicated that moMSCORS modified the cellular immune profile within pancreatic lymph nodes and pancreatic infiltrates by reducing the numbers of M1 pro-inflammatory macrophages and T helper 17 cells and upscaling the immunosuppressive T regulatory cells. The proportion of pathogenic insulin-specific CD4+ T cells was down-scaled in the lymph nodes, likely via soluble factors. The moMSCORS detected in the pancreatic infiltrates of treated mice presumably exerted the observed suppressive effect on CD4+ through direct contact. moMSCORS alleviated T1D symptoms in the mouse, qualifying as a candidate for therapeutic products by multiple advantages: non-invasive sampling by epilation, easy access, permanent availability, scalability, and benefits of auto-transplantation.

CAR-T Cells in Acute Myeloid Leukemia: Where Do We Stand?

Despite recent advances, the prognosis of acute myeloid leukemia (AML) remains unsatisfactory due to disease recurrence and the development of resistance to both conventional and novel therapies. Engineered T cells expressing chimeric antigen receptors (CARs) on their cellular surface represent one of the most promising anticancer agents. CAR-T cells are increasingly used in patients with B cell malignancies, with remarkable clinical results despite some immune-related toxicities. However, at present, the role of CAR-T cells in myeloid neoplasms, including AML, is extremely limited, as specific molecular targets for immune cells are generally lacking on AML blasts. Besides the paucity of dispensable targets, as myeloid antigens are often co-expressed on normal hematopoietic stem and progenitor cells with potentially intolerable myeloablation, the AML microenvironment is hostile to T cell proliferation due to inhibitory soluble factors. In addition, the rapidly progressive nature of the disease further complicates the use of CAR-T in AML. This review discusses the current state of CAR-T cell therapy in AML, including the still scanty clinical evidence and the potential approaches to overcome its limitations, including genetic modifications and combinatorial strategies, to make CAR-T cell therapy an effective option for AML patients.

Extracellular vesicles in cancer: Golden goose or trojan horse.

Intercellular communication can be mediated by direct cell-to-cell contact and indirect interactions through secretion of soluble chemokines, cytokines, and growth factors. Extracellular vesicles (EVs) have emerged as important mediators of cell-to-cell and cell-to-environment communications. EVs from tumor cells, immune cells, and stromal cells can remodel the tumor microenvironment and promote cancer cell survival, proliferation, metastasis, immune evasion, and therapeutic resistance. Most importantly, EVs as natural nanoparticles can be manipulated to serve as a potent delivery system for targeted cancer therapy. EVs can be engineered or modified to improve their ability to target tumors and deliver therapeutic substances, such as chemotherapeutic drugs, nucleic acids, and proteins, for the treatment of cancer. This review provides an overview of the biogenesis and recycling of EVs, discusses their roles in cancer development, and highlights their potential as a delivery system for targeted cancer therapy.

Hygrothermal performance of micro inhomogeneous insulation materials - EPS-based wall panel

Presence of water in liquid or vapor form leads to mould growth in building materials, weakening the building's structural integrity. Excess moisture in non-breathable materials also impacts thermal properties and performance over time. Hygrothermal analysis and understanding moisture movement within walls are crucial for comprehending wall assemblies in the enclosed spaces of buildings. Numerous commercial software packages are available for estimating moisture risk in wall assemblies. However, it's essential to recognize that these software tools are primarily designed for conventional insulation walls and may not be suitable for materials like EPS-based concrete, which incorporate expanded polystyrene particles. Since micro-level analysis of moisture flow is largely unexplored, this study used mass diffusion theory with ABAQUS-CAE. The study found that EPS beads increased moisture levels in walls by 2–6%. Additionally, due to the porous nature of concrete, moisture penetration and fluctuations were lower compared to other materials. Accumulated moisture made the wall impermeable and weakened the load-bearing material over time. To mitigate moisture effects of 20–28 %, it's recommended to use cement-fiberboard (CFB), which can be replaced if damaged without affecting structural integrity. SEM and EDX/EDAX analyses were used to show moisture accumulation at the micro level. It was observed that more oxygen elements were present at the border of the EPS-bead-mortar mix. The fuzzy-based MCDM method helped mitigate uncertainty in parameter selection, while ABAQUS model analysis considered different scenarios with variations in parameters. This research helps developers and practitioners choose materials with low concrete solubility and high concrete vapor diffusion factors for EPS-based panels, reducing moisture damage.

#1358 The association between TNFRSF1B rs1061624 polymorphism and hypertension in end-stage kidney disease patients on dialysis

Abstract Background and Aims In chronic kidney disease (CKD), hypertension (HT) is commonly found during its development and is a leading cause for its progression. HT, dyslipidemia and visceral obesity are among the components of metabolic syndrome (MetS) and seem to associate with progression of CKD to end-stage kidney disease (ESKD). Enhanced levels of soluble tumor necrosis factor receptor 2 (sTNFR2) are known to associate with progressive CKD; and this biomarker was reported as an independent predictor of all-cause mortality in ESKD patients under dialysis. Concerning the TNFRSF1B gene, the polymorphism rs1061624 (A > G), located in the 3′-untranslated region (3′-UTR), was associated with the development of HT in men and with increased risk of MetS. The aim of this study was to determine the genotype frequencies of TNFRSF1B rs1061624 in ESKD patients with and without HT, and to evaluate its relationship with the circulating levels of some MetS biomarkers. Method We studied 277 ESKD patients on dialysis (recruited between 2016-2019), 170 diagnosed with HT and 107 without HT (no-HT group); age, sex, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP) and statin therapy were recorded. Real time PCR TaqMan SNP genotyping assay was used to assess genotype frequencies of TNFRSF1B rs1061624. We also evaluated the conventional lipid profile and the levels of oxidized low-density lipoprotein, adiponectin and leptin. Results The genotype frequencies in patients with and without HT were different (P = 0.002); the AA genotype was less frequent in HT than in no-HT group, while GG and AG genotypes were more frequent. Considering the AA genotype carriers, HT ESKD patients (n = 23), compared to the no-HT group (n = 33), presented lower adiponectin levels (P = 0.025); for the AG genotype carriers, HT group (n = 89) presented a trend towards lower adiponectin concentrations (P = 0.055), and were younger than no-HT patients (n = 48; P<0.001); in both comparisons, groups were matched for sex, BMI and number of patients under statins treatment, and in the first case also for age. Considering GG genotype, HT patients (n = 58) only differed from no-HT subjects (n = 26) in SBP, that was higher (P<0.001). Among HT patients, AG genotype carriers presented higher DBP (P = 0.004), lower values of leptin (P = 0.012) and leptin/adiponectin ratio (P = 0.022) and were younger (P<0.001) than GG genotype patients; compared to AA carriers, cholesterol levels were lower (P = 0.012). For the no-HT group, AG genotype patients presented lower DBP (P = 0.008) than AA genotype carriers. Conclusion The genotype frequencies were different in ESKD patients with and without HT, being G allele more frequent in HT. The HT ESKD patients with the AA or the AG genotypes seem to present lower adiponectin levels than the ESKD no-HT patients with the same genotypes. Acknowledgements This work was financed by CESPU, through the project SNPsCKD-GI2-CESPU-2022; FCT, in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB.

#2396 The potential of human-derived macrophages to study autosomal dominant polycystic kidney disease (ADPKD)

Abstract Background and Aims Macrophages are the most prevalent immune cell type in the renal parenchyma in ADPKD. Depletion of macrophages in animal models has shown a dramatic attenuation in cystic burden, suggesting a disease promoting effect of this cell type. So far, studies investigating interaction between epithelial cells and immune cells in the field of ADPKD were performed with mice-derived myeloid cells. We present a new model using human-derived immune cells and tubular epithelial cells for the study of ADPKD. Method We established a technique to isolate CD14+ monocytes from the peripheral blood of young ADPKD patients and healthy controls. Methodology was optimized to create monocyte-derived macrophagesin vitro. Mature macrophages were checked for surface marker expression (CD14, CD11b, CD206, CD163, HLA-DR, D80, and CD86) & cytokine-release after stimulation with LPS, IL-4 and LPS + Nigericin. Co-cultures were performed using human-derived monocytes in combination with double immortalized cystic cells (ciCC) or healthy-kidney derived cell lines (HKT). Differentiation and polarization of macrophages was checked using flowcytometry. Results We observed low expression of PKD1 and PKD2 transcripts in primary, human-derived monocytes of ADPKD patients and controls. The expression of PKD1 and PKD2 increased after differentiation into macrophages. There were no clear differences between ADPKD and control-derived macrophages in the differentiation capacity, cell survival nor surface marker expression before and after differentiation. After stimulation with LPS & LPS + Nigericin ADPKD-derived macrophages showed significantly lower expression of pro-inflammatory cytokines (MIP-1α, IP-10, IL-23 and TNFα) as compared to controls. There was no difference in anti-inflammatory cytokine release. Healthy primary monocytes cultured in direct contact with conditionally immortalized cystic cells (ciCC) had a very good survival and showed differentiation into macrophages with high expression of M2-markers (CD163 and CD206) and the activation marker (HLA-DR). A similar differentiation pattern was seen when monocytes were cultured in the (cell-free) conditioned medium of the ciCC harvested after 72 h, pointing to a soluble factor produced by the cystic cells that induces monocyte differentiation and macrophage polarization. In accordance, unbiased multiplex cytokine analysis (Target 48-panel) of supernatants of ciCC, showed higher levels of macrophage colony-stimulating factor (M-CSF/CSF-1), TNF-α, hepatocyte growth factor (HGF) and MMP-1 as compared to those of HKT. Primary monocytes cultured with the conditioned medium harvested after 72 h from ciCCs (n=4) showed better overall survival than those cultured with medium from and HKT (n=4). In addition, a tendency towards higher expression of the classical M2-markers (CD163, CD206) and the activation marker (HLA-DR) in ciCCs versus HKT was seen. Conclusion There is wealth of data supporting the indispensable role of myeloid cells in disease progression of ADPKD. We established a unique in vitro fully human co-culture model for ADPKD. Cystic cells produce pro-proliferative and pro-angiogenic factors, thus actively shaping their microenvironment and creating the ideal circumstances for infiltrating monocytes/macrophages to proliferate and to destruct the kidney. This mechanism is very similar to what is seen in the cancer microenvironment. Further experiments will help to elucidate the complex interplay between different cell-types in the microenvironment in ADPKD kidneys.

Polymer Micropatches as B-Cell Engagers.

B cells, despite their several unique functionalities, remain largely untapped for use as an adoptive cell therapy and are limited to in vitro use for antibody production. B cells can be easily sourced, they possess excellent lymphoid-homing capabilities, and they can act as antigen-presenting cells (APCs), offering an alternative to dendritic cells (DCs), which have shown limited efficacy in the clinical setting. Soluble factors such as IL-4 and anti-CD40 antibody can enhance the activation, survival, and antigen-presenting capabilities of B cells; however, it is difficult to attain sufficiently high concentrations of these biologics to stimulate B cells in vivo. Micropatches as Cell Engagers (MACE) are polymeric microparticles, surface functionalized with anti-CD40 and anti-IgM, which can attach to B cells and simultaneously engage multiple B-cell receptors (BCR) and CD40 receptors. Stimulation of these receptors through MACE, unlike free antibodies, enhanced the display of costimulatory molecules on the B-cell surface, increased B-cell viability, and improved antigen presentation by B cells to T cells in vitro. B-cell activation by MACE further synergized with soluble IL-4 and anti-CD40. MACE also elicited T-cell chemokine secretion by B cells. Upon intravenous adoptive transfer, MACE-bound B cells homed to the spleen and lymph nodes, key sites for antigen presentation to T cells. Adoptive transfer of MACE-B cells pulsed with the CD4+ and CD8+ epitopes of ovalbumin significantly delayed tumor progression in a murine subcutaneous EG7-OVA tumor model, demonstrating the functional benefit conferred to B cells by MACE.

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also showthat brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-β and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta (PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. The functional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-β and suppressing Wnt/β-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

The extended transmission of cold atmospheric plasma-induced anticancer effects between cells

The application of cold atmospheric plasma (CAP) in cancer therapy has recently received extensive attention. However, researchers have mainly studied the effects of CAP on plasma-irradiated cancer cells and have not determined whether CAP affects surrounding or distant non-irradiated cancer cells. Here, the viability and intracellular reactive oxygen species levels of plasma-irradiated and non-irradiated cancer cells were monitored in vitro. Our results showed that CAP may induce plasma-irradiated cancer cells (A549 cells, A375 cells, J82 cells and HT29 cells) to secrete soluble factors into the cell culture medium, which can transmit plasma-induced anticancer effects from plasma-irradiated cancer cells to non-irradiated cancer cells. Our research confirmed that plasma-induced anticancer effects can be transmitted between cancer cells.

Ex vivo expansion in a clinical grade medium, containing growth factors from human platelets, enhances migration capacity of adipose stem cells.

Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated Supernatant Rich in Growth Factors (SRGF) can replace fetal bovine serum (FBS) to expand ASC by a clinical grade compliant protocol. The therapeutic potential of ASC is based also on their homing capacity toward inflammatory/cancer sites: oriented cell migration is a fundamental process in this scenario. We investigated the impact of SRGF on ASC migration properties. The motility/migration potential of ASC expanded in 5% SRGF was analyzed, in comparison to 10% FBS, by standard wound healing, bidimensional chemotaxis and transwell assays, and by millifluidic transwell tests. Mechanisms involved in the migration process were investigated by transient protein overexpression. In comparison to standard 10% FBS, supplementation of the cell culture medium with 5% SRGF, strongly increased migration properties of ASC along the chemotactic gradient and toward cancer cell derived soluble factors, both in static and millifluidic conditions. We showed that, independently from applied migratory stimulus, SRGF expanded ASC were characterized by far lower expression of α-smooth muscle actin (αSMA), a protein involved in the cell migration machinery. Overexpression of αSMA induced a significant and marked decrease in migration capacity of SRGF expanded ASC. In conclusion, 5% SRGF addition in the cell culture medium increases the migration potential of ASC, reasonably through appropriate downregulation of αSMA. Thus, SRGF could potentially improve the therapeutic impact of ASC, both as modulators of the immune microenviroment or as targeted drug delivery vehicles in oncology.

Acute Kidney Injury, Systemic Inflammation and Long-term Cognitive Function: ASSESS-AKI.

Cognitive dysfunction is a well-known complication of chronic kidney disease, but it is less known whether cognitive decline occurs in survivors after acute kidney injury (AKI). We hypothesized that an episode of AKI is associated with poorer cognitive function, mediated, at least in part, by persistent systemic inflammation. ASSESS-AKI enrolled patients surviving three months after hospitalization with and without AKI matched based on demographics, comorbidities, and baseline kidney function. A subset underwent cognitive testing using the modified mini-mental status examination (3MS) at 3, 12, and 36 months. We examined the association of AKI with 3MS scores using mixed linear models and assessed the proportion of risk mediated by systemic inflammatory biomarkers. Among 1538 participants in ASSESS-AKI, 1420 (92%) completed the 3MS assessment at 3 months and had a corresponding matched participant. Participants with AKI had lower 3MS scores at three years (difference -1.1 (95% CI: -2.0, -0.3) P=0.009) compared to participants without AKI. A higher proportion of AKI participants had a clinically meaningful (≥ 5 point) reduction in 3MS scores at three years compared to participants without AKI (14% vs. 10%, P=0.04). In mediation analyses, plasma soluble tumor necrosis factor receptor-1 (sTNFR-1) at three months after AKI mediated 35% (P=0.02) of the AKI related risk for 3MS scores at three years. AKI was associated with lower 3MS scores and sTNFR-1 concentrations appeared to mediate a significant proportion of the risk of long-term cognitive impairment. Further work is needed to determine if AKI is causal or a marker for cognitive impairment.

Native characterization and QC profiling of human amniotic mesenchymal stromal cell vesicular fractions for secretome-based therapy

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension.This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities.This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation.

Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation.

Role of Hypoxia in Mesenchymal Stem Cells from Dental Pulp: Influence, Mechanism and Application.

Mesenchymal stem cells (MSCs) from dental pulp (DP-MSCs), which include dental pulp stem cells (DPSCs) isolated from permanent teeth and stem cells from human exfoliated deciduous teeth (SHED), have emerged as highly promising cell sources for tissue regeneration, due to their high proliferative rate, multi-lineage differentiation capability and non-invasive accessibility. DP-MSCs also exert extensive paracrine effects through the release of extracellular vesicles (EVs) and multiple trophic factors. To be noted, the microenvironment, commonly referred to as the stem cell niche, plays a crucial role in shaping the functionality and therapeutic effects of DP-MSCs, within which hypoxia has garnered considerable attention. Extensive research has demonstrated that hypoxic conditions profoundly impact DP-MSCs. Specifically, hypoxia promotes DP-MSC proliferation, survival, stemness, migration, and pro-angiogenic potential while modulating their multi-lineage differentiation capacity. Furthermore, hypoxia stimulates the paracrine activities of DP-MSCs, leading to an increased production of EVs and soluble factors. Considering these findings, hypoxia preconditioning has emerged as a promising approach to enhance the therapeutic potential of DP-MSCs. In this comprehensive review, we provide a systematic overview of the influence of hypoxia on DP-MSCs, shedding light on the underlying mechanisms involved. Moreover, we also discuss the potential applications of hypoxia-preconditioned DP-MSCs or their secretome in tissue regeneration. Additionally, we delve into the methodologies employed to simulate hypoxic environments. This review aims to promote a comprehensive and systematic understanding of the hypoxia-induced effects on DP-MSCs and facilitate the refinement of regenerative therapeutic strategies based on DP-MSCs.

Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis

High-resolution respirometry (HRR) can assess peripheral blood mononuclear cell (PBMC) bioenergetics, but no standardized medium for PBMC preparation and HRR analysis exist. Here, we study the effect of four different media (MiR05, PBS, RPMI, Plasmax) on the count, size, and HRR (Oxygraph-O2k) of intact PBMCs. Remarkably, the cell count was 21 % higher when PBMCs were resuspended in MiR05 than in PBS or Plasmax, causing O2 flux underestimation during HRR due to inherent adjustments. Moreover, smaller cell size and cell aggregation was observed in MiR05. Based on our findings, we propose that Plasmax, PBS or RPMI is more suitable than MiR05 for HRR of intact PBMCs. We provide oxygen solubility factors for Plasmax and PBS and encourage further optimization of a standardized HRR protocol for intact PBMCs.