Magnetic-activated Cell Sorting Research Articles

ApTOLL: A new therapeutic aptamer for cytoprotection and (re)myelination after multiple sclerosis.

ApTOLL is an aptamer selected to antagonize toll-like receptor 4 (TLR4), a relevant actor for innate immunity involved in inflammatory responses in multiple sclerosis (MS) and other diseases. The currently available therapeutic arsenal to treat MS is composed of immunomodulators but, to date, there are no (re)myelinating drugs available in clinics. In our present study, we studied the effect of ApTOLL on different animal models of MS. The experimental autoimmune encephalomyelitis (EAE) model was used to evaluate the effect of ApTOLL on reducing the inflammatory component. A more direct effect on oligodendroglia was studied with the cuprizone model and purified primary cultures of murine and human oligodendrocyte precursor cells (OPCs) isolated through magnetic-activated cell sorting (MACS) from samples of brain cortex. Also, we tested these effects in an ex vivo model of organotypic cultures demyelinated with lysolecithin (LPC). ApTOLL treatment positively impacted the clinical symptomatology of mice in the EAE and cuprizone models, which was associated with better preservation plus restoration of myelin and oligodendrocytes in the demyelinated lesions of animals. Restoration was corroborated on purified cultures of rodent and human OPCs. Our findings reveal a new therapeutic approach for the treatment of inflammatory and demyelinating diseases such as MS. The molecular nature of the aptamer exerts not only an anti-inflammatory effect but also neuroprotective and remyelinating effects. The excellent safety profile demonstrated by ApTOLL in animals and humans opens the door to future clinical trials in MS patients.

Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation.

Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

In cases with raised sperm DNA fragmentation, can sperm selection by magnetic-activated cell sorting or testicular sperm aspiration help improve reproductive outcomes?

To evaluate the efficacy of magnetic-activated cell sorting (MACS) or testicular sperm aspiration (TESA) to improve reproductive outcomes in cases with elevated sperm DNA fragmentation undergoing assisted reproduction. This randomized controlled trial included couples with failed IVF cycles and sperm DNA fragmentation > 30%. Sperm DNA fragmentation was assessed using the sperm chromatin structure assay (SCSA) method. Participants were randomly assigned to either the MACS or TESA group. Testicular sperm retrieval was performed for the TESA group, while MACS involved sperm selection using magnetic beads. Extended blastocyst culture, freeze all policy of blastocysts by vitrification, and frozen embryo transfer were undertaken as per clinic's standard operating protocols. Blastocyst formation rate, implantation rate, miscarriage rate, multiple pregnancy rate, and live birth rate were analyzed and compared between MACS and TESA groups. There were no significant differences in female age, male age, or sperm DNA fragmentation index (DFI) between the MACS and TESA groups. The blastocyst conversion rate was slightly higher in the TESA group (39%) compared to the MACS group (32%). However, the MACS group had a higher implantation rate (50%) than the TESA group (35%). Miscarriage rates, multiple pregnancy rates, and live birth rates did not show statistically significant differences between the groups. A chi-squared test was conducted to compare categorical variables, and t-tests were done to compare continuous variables. In cases with raised sperm DNA fragmentation, sperm selection by MACS or TESA seems to offer comparable reproductive outcomes. There seems no superiority of one intervention over the other in cases with raised sperm DNA fragmentation undergoing assisted reproduction. Both interventions seem to be beneficial for couples seeking assisted reproduction with raised sperm DNA fragmentation.

Characterization of Ovarian Progenitor Cells for Their Potentials to Generate Steroidogenic Theca Cells in Vitro.

Adult mammalian ovaries contain stem/progenitor cells necessary for folliculogenesis and ovulation-related tissue rupture repair. Theca cells are recruited and developed from progenitors during the folliculogenesis. Theca cell progenitors were not well-defined. The aim of current study is to compare the potential of four ovarian progenitors with defined markers (LY6A, EPCR, LGR5and PDGFRA) to form steroidogenic theca cells in vitro. Ovarian progenitors were identified by the above four makers reported previously. The location of the cells was determined by immunohistochemistry and immunofluorescence staining of ovarian sections of adult mice. Different progenitor populations were purified by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) techniques from ovarian cell preparation and were tested for their abilities to generate steroidogenic theca cells in vitro. The cells were differentiated with a medium containing LH, ITS and DHH agonist for 12 days. EPCR+ and LGR5+ cells primarily distributed along ovarian surface epitheliums (OSE), while LY6A+ cells distributed in both OSE and parenchyma. However, PDGFRA+ cells were exclusively located in interstitial compartment. When the progenitors were purified by these markers and differentiated in vitro, LY6A+ and PDGFRA+ cells formed steroidogenic cells expressing both CYP11A1 and CYP17A1 and primarily producing androgens, showing characteristics of theca-like cells, while LGR5+ cells generated steroidogenic cells devoid of CYP17A1 expression and androgen production, showing a characteristic of progesterone-producing cells (granulosa- or lutea-like cells). Progenitors from both OSE and parenchyma of adult mice are capable of generating steroidogenic cells with different steroidogenic capacities, showing a possible lineage preference.

Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 genein induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations onplasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.

Magnetic CAR T cell purification using an anti-G4S linker antibody

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Abstract 4264: The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment

Abstract High grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy but a majority relapse with chemoresistant disease. Research supports the notion that tumor-initiating cells (TICs), a subpopulation of drug resistant stem-like tumor cells, are responsible for facilitating relapse and therapies targeting these fecund cells may prolong remission. The tumor microenvironment (TME) plays a crucial role in therapy resistance, and tumor relapse. Cytotoxic chemotherapies have been shown to modify the stroma of the TME which may favor the survival of TICs displaying specific adhesion proteins. We and others have shown that NF-kB transcription factors RelA and RelB respond to signals from the ovarian TME to promote proliferation, chemoresistance, and survival. Our results further show that RelA and RelB regulate expression of specific integrins. Recently we found that TNF-like inducer of apoptosis (TWEAK), a cytokine involved in tissue repair, and its only known receptor Fn14, are enriched following chemotherapy, leading to enhanced chemoresistance and survival of TICs. TWEAK also induces expression of specific integrins in ovarian TICs. Therefore, we hypothesize that NF-kB mediated integrin expression enables adhesion and survival of TICs to a chemotherapy modified extracellular matrix (ECM) to facilitate tumor repopulation and relapse. Using magnetic activated cell sorting (MACS) for CD117+ TICs, we found that a significant majority are enriched for integrin AVB3. Supporting a role for TWEAK-Fn14-NF-kB in mediating integrin expression we further show that integrin AVB3, but not AVB5 or A5B1, was significantly increased in CD117+ TICs in the presence of TWEAK and was dependent on Fn14 as demonstrated through knockdown experiments. RNA-sequencing analysis of tumor cells grown in spheroid conditions with RelA or RelB shRNA knockdown relative to control, show ITGAV is dependent on RelA while ITGB3 expression is dependent on RelB. Taken together, our findings suggest CD117+ TICs have enhanced expression of AVB3 and is further induced by TWEAK mediated NF-kB activity for adaptation to a stressful post-chemotherapy environment. Ongoing in vitro and in vivo studies are investigating the therapeutic benefit of targeting AVB3 or TWEAK in CD117+ TICs for prevention of adhesion and tumor repopulation following chemotherapy. These studies will uncover interactions between TICs and ECM through their unique expression of integrins and clarifies the role of NF-kB transcription factors RelA and RelB in regulating TIC survival in the post-chemotherapy TME. Citation Format: Omar Lujano Olazaba, Mikella Robinson, Cecilia Gallo, Mena Shammas, Katelyn Shelby, Luisjesus Cruz, Samuel F. Gilbert, Carrie D. House. The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4264.

Methods to Isolate Muscle Stem Cells for Cell-Based Cultured Meat Production: A Review.

Cultured meat production relies on various cell types, including muscle stem cells (MuSCs), embryonic stem cell lines, induced pluripotent cell lines, and naturally immortalized cell lines. MuSCs possess superior muscle differentiation capabilities compared to the other three cell lines, making them key for cultured meat development. Therefore, to produce cultured meat using MuSCs, they must first be effectively separated from muscles. At present, the methods used to isolate MuSCs from muscles include (1) the pre-plating method, using the ability of cells to adhere differently, which is a biological characteristic of MuSCs; (2) the density gradient centrifugation method, using the intrinsic density difference of cells, which is a physical characteristic of MuSCs; and (3) fluorescence- and magnetic-activated cell sorting methods, using the surface marker protein on the cell surface of MuSCs, which is a molecular characteristic of MuSCs. Further efficient and valuable methods for separating MuSCs are expected to be required as the cell-based cultured meat industry develops. Thus, we take a closer look at the four methods currently in use and discuss future development directions in this review.

Mechanical overload-induced release of extracellular mitochondrial particles from tendon cells leads to inflammation in tendinopathy

Tendinopathy is one of the most common musculoskeletal diseases, and mechanical overload is considered its primary cause. However, the underlying mechanism through which mechanical overload induces tendinopathy has not been determined. In this study, we identified for the first time that tendon cells can release extracellular mitochondria (ExtraMito) particles, a subtype of medium extracellular particles (mEPs), into the environment through a process regulated by mechanical loading. RNA sequencing systematically revealed that oxygen-related reactions, extracellular particles, and inflammation were present in diseased human tendons, suggesting that these factors play a role in the pathogenesis of tendinopathy. We simulated the disease condition by imposing a 9% strain overload on three-dimensional mouse tendon constructs in our cyclic uniaxial stretching bioreactor. The three-dimensional mouse tendon constructs under normal loading with 6% strain exhibited an extended mitochondrial network, as observed through live-cell confocal laser scanning microscopy. In contrast, mechanical overload led to a fragmented mitochondrial network. Our microscopic and immunoblot results demonstrated that mechanical loading induced tendon cells to release ExtraMito particles. Furthermore, we showed that mEPs released from tendon cells overloaded with a 9% strain (mEP9%) induced macrophage chemotaxis and increased the production of proinflammatory cytokines, including IL-6, CXCL1, and IL-18, from macrophages compared to mEP0%, mEP3%, and mEP6%. Partial depletion of the ExtraMito particles from mEP9% by magnetic-activated cell sorting significantly reduced macrophage chemotaxis. N-acetyl-L-cysteine treatment preserved the mitochondrial network in overloaded tendon cells, diminishing overload-induced macrophage chemotaxis toward mEP9%. These findings revealed a novel mechanism of tendinopathy; in an overloaded environment, ExtraMito particles convey mechanical response signals from tendon cells to the immune microenvironment, culminating in tendinopathy.

Single Cell Microgels for High‐Throughput Magnetic Sorting and Sequencing of Antigen‐Specific Antibodies

AbstractProgress in the discovery of potent antibodies for therapeutic and research purposes has been accelerated by mining the antibody repertoire of plasma cells and plasmablasts. Magnetic activated cell sorting (MACS) is a well recognized method for cell sorting, however, secreted IgGs rapidly diffuse away from cells, therefore requiring specialized technique to compartmentalize individual cells and capture their secretions for MACS isolation. To this end, a scalable method to prepare hydrogel‐based microcontainers for single cell culture and subsequent sorting based on their secreted IgG products at high‐throughput level are described. In situ crosslinkable microgels featuring tunable viscosity are fabricated to act as soft compartments that accommodate delicate primary cells and capture secreted proteins, while providing solid supports after solgel transition to enable immune recognition with chemically functionalized magnetic nanorods in aqueous medium for MACS separation. Splenocytes are sorted based on antigen‐specific IgG production from mice immunized with an antibiotic target ceftriaxone sodium, enriching a population of 220 000 000 to 800 000 prevalence following sorting. This enrichment ratio is substantially higher than conventional fluorescence activated cell sorting (FACS) methods, likely due to the multivalency of the antigen‐conjugated magnetic nanorods.

Stemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton's jelly mesenchymal stem/stromal cells.

Background: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due to different degrees of differentiation of cell subpopulations poses a considerable challenge in preclinical studies. The cells at a pluripotent-like stage represent a stem cell population of interest for many researchers worldwide, which is worthy of identification, isolation, and functional characterization. In the current study, we asked whether Wharton's jelly-derived MSCs (WJ-MSCs) which express stage-specific embryonic antigen-4 (SSEA-4) can be considered as a pluripotent-like stem cell population. Methods: SSEA-4 expression in different culture conditions was compared and the efficiency of two cell separation methods were assessed: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After isolation, SSEA-4+ cells were analyzed for the following parameters: the maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-related gene expression, proliferation potential, clonogenicity, secretome profiling, and the ability to form spheres under 3D culture conditions. Results: FACS allowed for the enrichment of SSEA-4+ cell content in the population that lasted for six passages after sorting. Despite the elevated expression of stemness-related genes, SSEA-4+ cells neither differed in their proliferation and clonogenicity potential from initial and negative populations nor exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to form smaller spheroids and exhibited increased survival under 3D conditions. Conclusion: Despite the transient expression of stemness-related genes, our findings could not fully confirm the undifferentiated pluripotent-like nature of the SSEA-4+ WJ-MSC population cultured in vitro.

Dimethyl fumarate improves cognitive impairment and neuroinflammation in mice with Alzheimer's disease.

Neuroinflammation substantially contributes to the pathology of Alzheimer's disease (AD), the most common form of dementia. Studies have reported that nuclear factor erythroid 2-related factor 2 (Nrf2) attenuates neuroinflammation in the mouse models of neurodegenerative diseases, however, the detailed mechanism remains unclear. The effects of dimethyl fumarate (DMF), a clinically used drug to activate the Nrf2 pathway, on neuroinflammation were analyzed in primary astrocytes and AppNL-G-F (App-KI) mice. The cognitive function and behavior of DMF-administrated App-KI mice were evaluated. For the gene expression analysis, microglia and astrocytes were directly isolated from the mouse cerebral cortex by magnetic-activated cell sorting, followed by quantitative PCR. DMF treatment activated some Nrf2 target genes and inhibited the expression of proinflammatory markers in primary astrocytes. Moreover, chronic oral administration of DMF attenuated neuroinflammation, particularly in astrocytes, and reversed cognitive dysfunction presumably by activating the Nrf2-dependent pathway in App-KI mice. Furthermore, DMF administration inhibited the expression of STAT3/C3 and C3 receptor in astrocytes and microglia isolated from App-KI mice, respectively, suggesting that the astrocyte-microglia crosstalk is involved in neuroinflammation in mice with AD. The activation of astrocytic Nrf2 signaling confers neuroprotection in mice with AD by controlling neuroinflammation, particularly by regulating astrocytic C3-STAT3 signaling. Furthermore, our study has implications for the repositioning of DMF as a drug for AD treatment.

Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment.

Obtaining an enriched and phenotypically pure cell population from heterogeneous cell mixtures is important for diagnostics and biosensing. Existing techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require preincubation with antibodies (Ab) and specialized equipment. Cell immunopanning removes the need for preincubation and can be done with no specialized equipment. The majority of the available antibody-mediated analyte capture techniques require a modification to the Abs for binding. In this work, no antibody modification is used because we take advantage of the carbohydrate chain in the Fc region of Ab. We use boronic acid as a cross-linker to bind the Ab to a modified surface. The process allows for functional orientation and cleavable binding of the Ab. In this study, we created an immunoaffinity matrix on polystyrene (PS), an inexpensive and ubiquitous plastic. We observed a 37% increase in Ab binding compared with that of a passive adsorption approach. The method also displayed a more consistent antibody binding with 17 times less variation in Ab loading among replicates than did the passive adsorption approach. Surface topography analysis revealed that a dextran coating reduced nonspecific antibody binding. Elemental analysis (XPS) was used to characterize the surface at different stages and showed that APBA molecules can bind upside-down on the surface. While upside-down antibodies likely remain functional, their elution behavior might differ from those bound in the desired way. Cell capture experiments show that the new surface has 43% better selectivity and 2.4-fold higher capture efficiency compared to a control surface of passively adsorbed Abs. This specific surface chemistry modification will allow the targeted capture of cells or analytes with the option of chemical detachment for further research and characterization.

Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galβ1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Blockade of V-domain immunoglobulin suppressor of T-cell activation reprograms tumour-associated macrophages and improves efficacy of PD-1 inhibitor in gastric cancer.

In gastric cancer, the response rate of programmed cell death protein-1 (PD-1) inhibitor is far from satisfactory, indicating additional nonredundant pathways might hamper antitumour immunity. V-domain immunoglobulin suppressor of T-cell activation (VISTA) has been reported in several malignancies as a novel immune-checkpoint. Nevertheless, the role of VISTA in gastric cancer still remains obscure. Our purpose is to explore the clinical significance and potential mechanism of VISTA in affecting gastric cancer patients' survival and immunotherapeutic responsiveness. Our study recruited eight independent cohorts with a total of 1403 gastric cancer patients. Immunohistochemistry, multiplex immunofluorescence, flow cytometry or intracellular flow cytometry, quantitative polymerase chain reaction, western blotting, fluorescence-activated cell sorting, magnetic-activated cell sorting, smart-seq2, in vitro cell co-culture and ex vivo tumour inhibition assays were applied to investigate the clinical significance and potential mechanism of VISTA in gastric cancer. VISTA was predominantly expressed on tumour-associated macrophages (TAMs), and indicated poor clinical outcomes and inferior immunotherapeutic responsiveness. VISTA+ TAMs showed a mixed phenotype. Co-culture of TAMs and CD8+ T cells indicated that VISTA+ TAMs attenuated effective function of CD8+ T cells. Blockade of VISTA reprogrammed TAMs to a proinflammatory phenotype, reactivated CD8+ T cells and promoted apoptosis of tumour cells. Moreover, blockade of VISTA could also enhance the efficacy of PD-1 inhibitor, suggesting that blockade of VISTA might synergise with PD-1 inhibitor in gastric cancer. Our data revealed that VISTA was an immune-checkpoint associated with immunotherapeutic resistance. Blockade of VISTA reprogrammed TAMs, promoted T-cell-mediated antitumour immunity, and enhanced efficacy of PD-1 inhibitor, which might have implications in the treatment of gastric cancer.

Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells.

Airway epithelial cells (AECs) play a key role in maintaining lung homeostasis, epithelium regeneration and the initiation of pulmonary immune responses. To isolate and study murine AECs investigators have classically used short and hot (1h 37°C) digestion protocols. Here, we present a workflow for efficient AECs isolation and culture, utilizing long and cold (20h 4°C) dispase II digestion of murine lungs. This protocol yields a greater number of viable AECs compared to an established 1h 37°C dispase II digestion. Using a combination of flow cytometry and immunofluorescent microscopy, we demonstrate that compared to the established method, the cold digestion allows for recovery of a 3-fold higher number of CD45-CD31-EpCAM+ cells from murine lungs. Their viability is increased compared to established protocols, they can be isolated in larger numbers by magnetic-activated cell sorting (MACS), and they result in greater numbers of distal airway stem cell (DASC) KRT5+p63+ colonies in vitro. Our findings demonstrate that temperature and duration of murine lung enzymatic digestion have a considerable impact on AEC yield, viability, and ability to form colonies in vitro. We believe this workflow will be helpful for studying lung AECs and their role in the biology of lung.

Microfluidics-assisted Tumor Cell Separation Approaches for Clinical Applications: An Overview on Emerging Devices.

Recent advances in science and technology have led to revolutions in many different scientific and industrial fields. The term lab on a chip, or in other word, performing a variety of complex analyzes in just a short time and in a very small space, is a term that has become very common in recent years, and what used to be a dream has now come to life in practice. In this paper, we has been tried to investigate a specific type of lab technology on a chip, which is of course one of the most common, namely the knowledge and technology of cell separation by using microfluidic technique that can be separated based on size and deformation, adhesion and electrical properties. The tissue of the human body is degraded due to injury or aging, which is often tried to treat this tissue disorder by using drugs, but they are not always enough. Stem cell-based medicine is a novel form of medicine which promises the restoration or regeneration of tissues and functioning organs. Although many models of microfluidic systems have been designed for cell separation, the choice of appropriate device to achieve a reliable result is presented as a challenge. So, in this study, Fluorescence Activated Cell Sorting (FACS), Dielectrophoresis (DEP), Magnetic Activated Cell Sorting (MACS) and Acoustic microfluidic system are four distinct categories of active microfluidic systems explored. Also, the advantages, disadvantages and the current status of the mentioned devices in these methods, are reviewed.

The Neural Progenitor Cell-Associated Transcription Factor FoxG1 Regulates Cardiac Epicardial Cell Proliferation.

The epicardium is a layer of mesothelial cells that covers the surface of the heart. During development, epicardial cells undergo epithelial-to-mesenchymal transition (EMT) to form multipotent precursors that migrate into the heart and contribute to the coronary vasculature by differentiating into adventitial fibroblasts, smooth muscle cells, and endothelial cells. Epicardial cells also provide paracrine signals to cardiac myocytes that are required for appropriate heart growth. In adult hearts, a similar process of epicardial cell EMT, migration, and differentiation occurs after myocardial infarction (MI, heart attack). Pathological cardiac hypertrophy is associated with fibrosis, negative remodeling, and reduced cardiac function. In contrast, aerobic exercises such as swimming and running promote physiological (i.e., beneficial) hypertrophy, which is associated with angiogenesis and improved cardiac function. As epicardial cell function(s) during physiological hypertrophy are poorly understood, we analyzed and compared the native epicardial cells isolated directly from the hearts of running-exercised mice and age-matched, nonrunning littermates. To obtain epicardial cells, we enzymatically digested the surfaces of whole hearts and performed magnetic-activated cell sorting (MACS) with antibodies against CD104 (integrin β4). By cDNA microarray assays, we identified genes with increased transcription in epicardial cells after running exercise; these included FoxG1, a transcription factor that controls neural progenitor cell proliferation during brain development and Snord116, a small noncoding RNA that coordinates expression of genes with epigenetic, circadian, and metabolic functions. In cultured epicardial cells, shRNA-mediated FoxG1 knockdown significantly decreased cell proliferation, as well as Snord116 expression. Our results demonstrate that FoxG1 regulates epicardial proliferation, and suggest it may affect cardiac remodeling.

The Use of Non-Apoptotic Sperm Selected by Magnetic Activated Cell Sorting (MACS) to Enhance Reproductive Outcomes: What the Evidence Says.

Sperm selection of the most competent sperm is a promising way to enhance reproductive outcomes. Apoptosis is the programmed cell death process to maintain tissue homeostasis, and MACS sperm selection of non-apoptotic cells enables the removal of apoptotic sperm from an ejaculate, thus leaving the non-apoptotic available to be microinjected, but given the associated costs of adding these sperm selection steps to the routine practice, there is a need for a careful examination of the literature available to answer questions such as who can benefit from this MACS, how significant this improvement is, and how robust the evidence and data available supporting this choice are. Thus, the aim of this narrative review was to objectively evaluate the available evidence regarding the potential benefits of the use of MACS. From the literature, there are controversial results since its implementation as an in vitro fertilization add-on, and this may be explained in part by the low quality of the evidence available, wrong designs, or even inadequate statistical analyses. We concluded that the benefits of adding MACS are unclear, and further methodologically sound research on specific populations is much needed before offering it clinically.