Isolation Of Cell Populations Research Articles

Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium.

There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.

Understanding virus-host interactions in tissues.

Although virus-host interactions are usually studied in a single cell type using in vitro assays in immortalized cell lines or isolated cell populations, it is important to remember that what is happening inside one infected cell does not translate to understanding how an infected cell behaves in a tissue, organ or whole organism. Infections occur in complex tissue environments, which contain a host of factors that can alter the course of the infection, including immune cells, non-immune cells and extracellular-matrix components. These factors affect how the host responds to the virus and form the basis of the protective response. To understand virus infection, tools are needed that can profile the tissue environment. This Review highlights methods to study virus-host interactions in the infection microenvironment.

Separation of epithelial and immune cells from biopsy samples.

Celiac disease (CD) is a chronic and autoimmune disease that develops in genetically predisposed individuals upon exposure to dietary gluten. The availability of the target tissue for research has made it possible to identify alterations in the transcriptome and methylome in the celiac gut. However, gene expression and methylation is highly variable among different cell types, and separation of cellular populations in target tissue must be considered for the understanding of the specific cellular and immune responses to gluten. In this context, a few studies have demonstrated that focusing on an isolated cell population, novel candidate genes involved in the pathogenesis of the disease can be identified. Here, we describe a method to separate epithelial and immune cells from biopsy samples for DNA and RNA isolation. With minor variations, the same technique can be applied to other tissues and cell types.

Single-cell transcriptomics reveals peripheral immune responses in non-segmental vitiligo.

Vitiligo is a common autoimmune depigmented dermatology due to destruction of melanocytes. Much evidence suggests that vitiligo is associated with systemic immune activation. Previous studies have focused on immune cell infiltration in and around lesion areas, but few studies have investigated the cell types and function of circulating immune cells in peripheral blood. Here, single cell RNA-sequencing (scRNA-seq) was used to investigate the mechanisms of peripheral immune responses in vitiligo patients. Peripheral blood was collected from five patients with progressive non-segmental vitiligo and three healthy controls. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Paque density gradient centrifugation, and scRNA-seq was performed on isolated cell populations to obtain single cell transcriptomes and characterize important genes and intracellular signaling pathways. The key findings were validated with qPCR and flow cytometry assays. We identified 10 major cell types by scRNA-seq. Among these cell types, neutrophils were specifically observed in our scRNA-seq data from PBMCs. Peripheral blood effector CD8+ T cells from vitiligo patients did not show significant differences at the transcriptome level compared with healthy controls, whereas regulatory T cells showed pro-inflammatory TH1-like properties. Innate immune cells, including natural killer cells and dendritic cells, showed increased antigen processing and presentation as well as upregulated interferon responses. B cells, monocytes, and neutrophils all showed activation. B cells, especially memory B cells, had upregulated expression of genes related to humoral immunity. Monocytes showed production of proinflammatory cytokines and chemokines. Neutrophils showed strong chemokine ligand-receptor (L-R) pair (CXCR8-CXCR2) autocrine signaling pathway. This study revealed the genetic profile and signaling pathway characteristics of peripheral blood immune cells in vitiligo patients, providing new insights into its pathogenesis, which may facilitate identification of potential therapeutic targets.

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Cellular heterogeneity and repolarisation across the atria: an in silico study

Mechanisms of atrial fibrillation and the susceptibility to reentries can be impacted by the repolarization across the atria. Studies into atrial fibrillation ignore cell-to-cell heterogeneity due to electrotonic coupling. Recent studies show that cellular variability may have a larger impact on electrophysiological behaviour than assumed. This paper aims to determine the impact of cellular heterogeneity on the repolarization phase across the AF remodelled atria. Using a population of models approach, 10 anatomically identical atrial models were created to include cellular heterogeneity. Atrial models were compared with an equivalent homogenous model. Activation, APD90, and repolarization maps were used to compare models. The impact of electrotonic coupling in the tissue was determined through a comparison of RMP, APD20, APD50, APD90, and triangulation between regional atrial tissue and the single cell populations. After calibration, cellular heterogeneity does not impact atrial depolarization. Repolarization patterns were significantly impacted by cellular heterogeneity, with the APD90 across the LA increasing due to heterogeneity and the reverse occurring in the RA. Electrotonic coupling caused a reduction in variability across all biomarkers but did not fully remove variability. Electrotonic coupling resulted in an increase in APD20 and APD50, and reduced triangulation compared to isolated cell populations. Heterogeneity also caused a reduction in triangulation compared with regionally homogeneous atria. Graphical abstract

Abstract 199: A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments

Abstract Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 199.

Abstract 5324: Isolation of small number of tumor cells for proliferation studies using DEPArray technology

Abstract Cancer is universally recognized as a heterogeneous disease. The different cell clones present in a tumor can display distinct molecular and phenotypic profiles, which may influence proliferation capabilities, metastatic potential and response to therapy. To better understand cancer progression and possibly design effective therapeutic strategies, it is fundamental to design cell-sorting technologies able to isolate rare, viable tumor cell clones from tissue or liquid biopsies. Fluorescence-activated cell sorting (FACS) is the most widely used technique to isolate cell populations from heterogeneous samples. However, FACS analysis presents technical limitations when applied to rare cell populations and/or samples with low cellularity and limitations associated with the low total number of target cells in the sample. DEParray™ PLUS (DA) is a highly automated image-based cell sorting technology capable of isolating pure, single and rare cells from heterogeneous samples. Here we evaluate the capabilities of the DA system to isolate a small number of viable tumor cells and assess the proliferation capacity of DA-sorted cells in comparison with FACS-sorted cells. Human Jurkat T lymphoblast cell line were cultured in RPMI medium and a double staining procedure was optimized to label cells with CellTraceFarRed (CTFR), division tracking dye and Live/Dead (LD) Fixable marker. CTFR/LD staining was performed for each experiment and, at days 0 and 7 of culture, CTFR and LD fluorescences were analyzed by means of BD CANTO II Flow cytometry. At day 0, FACS analysis discriminate dead from live cells, while at day 7 evaluate cell proliferation and viability after 7 days in culture. Comparison tests were carried out following two different recovery schemes: 1 recovery of 100 CTFR+/LD- cells pool and 10 recoveries of 10 CTFR+/LD- cells pool using DA Plus and BD FACSAria Fusion. For each experiment, the results were compared to cells seeded manually, which represent our unsorted control. When comparing the results obtained with a 100-cells pool, no significant difference in cell viability and proliferation were observed between DA-sorted and FACSAria-sorted cells after 7 days in culture. On the other hand, the 10 cells pool sorted with DA PLUS showed a significantly higher viability and proliferation rate compared to those sorted with FACSAria at day 7. Moreover, cells sorted by DA-technology showed less variation between experiments, highlighting a more robust performance when sorting a small number of cells. Overall, the results confirm the unique capability of the DA system to sort a small number of cells, which maintain a higher cell viability and proliferation capacity compared to those sorted with FACS. This opens up the possibility to isolate rare tumor cell clones for functional studies that can help to better understand tumor progression and response to therapy. Citation Format: Maximilian Sergio, Chiara Rossi, Valeria Stivani, Ilaria Maffei, Marco Rossi, Gianpiero Parisi, Gianni Medoro. Isolation of small number of tumor cells for proliferation studies using DEPArray technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5324.

Isolation of single cells from human uterus in the third trimester of pregnancy: myometrium, decidua, amnion and chorion.

During pregnancy, interactions between uterine immune cells and cells of the surrounding reproductive tissues are thought to be vital for regulating labour. The mechanism that specifically initiates spontaneous labour has not been determined, but distinct changes in uterine immune cell populations and their activation status have been observed during labour at term gestation. To understand the regulation of human labour by the immune system, the ability to isolate both immune cells and non-immune cells from the uterus is required. Here, we describe protocols developed in our laboratory to isolate single cells from uterine tissues, which preserve both immune and non-immune cell populations for further analysis. We provide detailed methods for isolating immune and non-immune cells from human myometrium, chorion, amnion and decidua, together with representative flow cytometry analysis of isolated cell populations present. The protocols can be completed in tandem and take approximately 4-5 h, resulting in single-cell suspensions that contain viable leucocytes, and non-immune cells in sufficient numbers for single-cell analysis approaches such as flow cytometry and single cell RNA sequencing (scRNAseq).

ScGate: marker-based purification of cell types from heterogeneous single-cell RNA-seq datasets.

SummaryA common bioinformatics task in single-cell data analysis is to purify a cell type or cell population of interest from heterogeneous datasets. Here, we present scGate, an algorithm that automatizes marker-based purification of specific cell populations, without requiring training data or reference gene expression profiles. scGate purifies a cell population of interest using a set of markers organized in a hierarchical structure, akin to gating strategies employed in flow cytometry. scGate outperforms state-of-the-art single-cell classifiers and it can be applied to multiple modalities of single-cell data (e.g. RNA-seq, ATAC-seq, CITE-seq). scGate is implemented as an R package and integrated with the Seurat framework, providing an intuitive tool to isolate cell populations of interest from heterogeneous single-cell datasets.Availability and implementationscGate is available as an R package at https://github.com/carmonalab/scGate (https://doi.org/10.5281/zenodo.6202614). Several reproducible workflows describing the main functions and usage of the package on different single-cell modalities, as well as the code to reproduce the benchmark, can be found at https://github.com/carmonalab/scGate.demo (https://doi.org/10.5281/zenodo.6202585) and https://github.com/carmonalab/scGate.benchmark. Test data are available at https://doi.org/10.6084/m9.figshare.16826071.Supplementary information Supplementary data are available at Bioinformatics online.

The pituitary tumour-transforming gene 1/delta-like homologue 1 pathway plays a key role in liver fibrogenesis.

Background and Aims PTTG1 is almost undetectable in adult livers but is highly expressed in hepatocarcinoma. While little is known about its involvement in liver fibrosis, PTTG1 expression is associated with DLK1. We assessed the role of the PTTG1/DLK1 pathway in fibrosis progression and the potential therapeutic effect of PTTG1 silencing in fibrosis.Methods Pttg1 and Dlk1 were studied in liver and isolated cell populations of control and fibrotic rats and in human liver biopsies. The fibrotic molecular signature was analysed in Pttg1 −/− and Pttg1 +/+ fibrotic mice. Finally, Pttg1 silencing was evaluated in rats as a novel antifibrotic therapy.Results Pttg1 and Dlk1 mRNA selectively increased in fibrotic rats paralleling fibrosis progression. Serum DLK1 concentrations correlated with hepatic collagen content and systemic and portal haemodynamics. Human cirrhotic livers showed greater PTTG1 and DLK1 transcript abundance than non‐cirrhotic, and reduced collagen was observed in Pttg1 Pttg1 −/− mice. The liver fibrotic molecular signature revealed lower expression of genes related to extracellular matrix remodelling including Mmp8 and 9 and Timp4 and greater eotaxin and Mmp13 than fibrotic Pttg1 +/+ mice. Finally, interfering Pttg1 resulted in reduced liver fibrotic area, lower α‐Sma and decreased portal pressure than fibrotic animals. Furthermore, Pttg1 silencing decreased the transcription of Dlk1, collagens I and III, Pdgfrβ, Tgfrβ, Timp1, Timp2 and Mmp2.Conclusions Pttg1/Dlk1 are selectively overexpressed in the cirrhotic liver and participate in ECM turnover regulation. Pttg1 disruption decreases Dlk1 transcription and attenuates collagen deposition. PTTG1/DLK1 signalling is a novel pathway for targeting the progression of liver fibrosis.

Identification and isolation of slow-cycling glioma stem cells.

Cancer stem cells are defined as low-abundance, quiescent cells and are considered a major cellular source of tumor recurrence following therapy, which identifies these cells as important therapeutic targets for difficult-to-treat cancers, including high-grade gliomas. By contrast to the highly proliferative bulk tumor cells, glioma stem cells (GSC) are slow-cycling, and therefore less sensitive to DNA damaging cytotoxic drugs. GSC are also less reliant on aerobic glycolytic metabolism, leading to inadequate clearing of GSC by chemotherapy and radiotherapy. The definition of GSC is based on the expression of specific stem cell protein markers. This method of GSC isolation is successful in isolating cell populations that can reliably recapitulate the tumor. However, cell populations that lack stem marker expression may also be capable of tumor recapitulation. Therefore, robust, reproducible methods for isolating GSC are required to identify and isolate cells with stem cell characteristics. Here, we provide a comprehensive and reproducible protocol for the isolation of slow-cycling GSC. Using this method, GSC isolated retain key characteristics of the cells in situ, including expression of genes associated with cell quiescence and invasive potential, compared to non-quiescent cell populations. Thus, isolation of GSC gated on cell proliferation offers a reliable alternative method for in vitro GSC identification, that adequately mirrors the physiological properties of GSC seen in vivo.

Isolation of tdTomato Expressing Inter-follicular Epidermal Melanocytes or Keratinocytes from Mouse Tail Skin.

The epidermis is the outermost layer of the skin. It is made up of mostly keratinocytes along with a small number of melanocytes and Langerhans cells. Melanocytes produce a pigment called melanin, which is transferred to the keratinocytes, and protects these cells from damage from UV radiation, as well as generating hair and skin colours. In this important relationship, keratinocytes exert control over melanocytes. Many questions regarding keratinocyte-melanocyte interactions have yet to be answered, and would benefit from study in model systems, to address diseases such as vitiligo and cutaneous melanoma. Most of the mouse is covered in fur and these areas lack the skin pigmenting inter-follicular epidermal (IFE) melanocytes. However, the mouse tail is pigmented analogously to human skin. Here, we present a method for isolating IFE melanocytes or keratinocytes expressing the tdTomato marker from the mouse tail, using fluorescence-activated cell sorting (FACS). The method involves firstly separating the tail skin epidermis from the dermis, and then digesting the epidermis to produce dissociated cells, which can then be sorted. These isolated cell populations can be studied using RNAseq or cultured in vitro. This protocol isolates IFE melanocytes or keratinocytes and immediately provides reasonable yields of cells, without the need to stain the cells for cell specific markers.

The Osteocyte Transcriptome: Discovering Messages Buried Within Bone.

Osteocytes are cells embedded within the bone matrix, but their function and specific patterns of gene expression remain only partially defined; this is beginning to change with recent studies using transcriptomics. This unbiased approach can generate large amounts of data and is now being used to identify novel genes and signalling pathways within osteocytes both at baseline conditions and in response to stimuli. This review outlines the methods used to isolate cell populations containing osteocytes, and key recent transcriptomic studies that used osteocyte-containing preparations from bone tissue. Three common methods are used to prepare samples to examine osteocyte gene expression: digestion followed by sorting, laser capture microscopy, and the isolation of cortical bone shafts. All these methods present challenges in interpreting the data generated. Genes previously not known to be expressed by osteocytes have been identified and variations in osteocyte gene expression have been reported with age, sex, anatomical location, mechanical loading, and defects in bone strength. A substantial proportion of newly identified transcripts in osteocytes remain functionally undefined but several have been cross-referenced with functional data. Future work and improved methods (e.g. scRNAseq) likely provide useful resources for the study of osteocytes and important new information on the identity and functions of this unique cell type within the skeleton.

RNA-Sequencing of Isolated Cell Populations Expressing Human APOL1 Risk Variants Reveals Molecular Correlates of Sickle Cell Nephropathy in Zebrafish Podocytes

Background: Kidney failure occurs in 5-13% of individuals with sickle cell disease (SCD), and is associated with early mortality. We have shown previously that genetic variation in apolipoprotein L1 (APOL1) is associated with increased risk for developing SCD nephropathy (Ashley-Koch et al, 2011). Two APOL1 alleles (G1 and G2) have been identified as risk factors for SCD nephropathy, as well as chronic kidney disease (CKD) in African Americans without SCD (Limou et al, 2014). Both risk alleles are highly prevalent in individuals of African descent. Despite the strong association of G1 and G2 with kidney dysfunction, the mechanisms by which these variants contribute to increased CKD risk remain poorly understood. However, the observation of podocyte effacement in CKD patients suggests that podocytes and/or endothelial cells are likely implicated in pathogenesis. We established zebrafish models of these human risk alleles previously for the purpose of better understanding the underlying pathology contributing to SCD nephropathy (Anderson et al, 2015). Here, we have utilized these models to explore further the cell-type specific transcriptomic perturbations contributing to nephropathy.Methods: To investigate the effects of the APOL1 G2 risk variant on cell-type specific transcriptomes, we injected human APOL1 mRNA into embryos from 3 different zebrafish lines expressing cell-type specific reporters to permit the isolation of podocytes (pod:NTR-mCherry), endothelial cells (fli1:egfp), or peripheral neurons (hb9:gfp; control). Three populations from each line were utilized: (1) uninjected controls; (2) embryos injected with canonical human APOL1 (G0); and (3) embryos injected with human APOL1 (G2) mRNA. Larvae were dissociated at 4 days post-fertilization (4 dpf), and fluorescent cell types were isolated via fluorescence activated cell-sorting (FACS). Messenger RNA was extracted from sorted cell populations (n=3 FACS sorted cell populations per condition) and subsequently amplified to obtain a sufficient amount of RNA for sequencing. The amplified RNA was prepared into cDNA libraries, and sequenced on an Illumina HiSeq 2500. We performed pairwise comparisons between uninjected and G0 injected larvae in each cell type to ascertain the effects of canonical human APOL1 on the zebrafish kidney transcriptome. Additionally, to measure the effects of APOL1 G2 expression on the transcriptome, we compared APOL1 G0 and APOL1 G2 samples. Transcripts were considered to be differentially expressed if they achieved an FDR-adjusted q-value of less than 0.05. To understand better the biological processes altered by injection condition, we conducted gene ontology enrichment analyses (GOEA) on sets of differentially expressed genes.Results: Our analysis of uninjected and APOL1 G0 libraries identified 38, 154, and 204 differentially expressed (DE) genes in endothelial cells, podocytes, and motor neurons, respectively. No significant enrichment of gene ontology terms was detected, suggesting that canonical G0 APOL1 did not have a substantial impact on the zebrafish transcriptome. In contrast, the comparison of APOL1 G0 and APOL1 G2 samples found 95, 7400 (Figure below), and 652 DE genes in endothelial cells, podocytes, and motor neurons, respectively. GOEA of the podocyte DE gene list showed enrichment for several autophagy-associated terms, including “Lysosome” and “Phagosome”. Close inspection of these autophagy-associated genes revealed that the majority are up-regulated in G2 relative to G0 (68/75 genes for “Lysosome” and 52/78 genes for “Phagosome”). Thus, the G2 allele appears to induce transcription of genes involved in autophagy in podocytes.Summary: Taken together, these data suggest that podocytes are the predominant cell type affected by the expression of APOL1 G2. The comparison of podocytes injected with APOL1 G0 or APOL1 G2 yielded an order of magnitude more DE transcripts than any other comparison. The GOEA results of the podocyte comparison are consistent with a recent study of transgenic mice expressing the G1 or G2 APOL1 high risk variants, which showed alterations in endocytic vesicle content and autophagic flux (Beckerman et al 2017). Ongoing work includes molecular analysis to characterize the effects of the APOL1 G1 allele. This work will continue to shed light on the molecular pathology of SCD nephropathy and provide insight into new therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activation of human γδ T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells.

Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-, γδ T-, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST-1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST-1. SE-induced IFN-γ expression in MAIT-, γδ T-, and NK cells was clearly reduced by neutralization of IL-12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore, co-cultures of isolated cell populations revealed that the ability of SEA to activate γδ T- and NK cells was fully dependent on the presence of both monocytes and αβ T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections.

Visualising and understanding cGMP signals in the cardiovascular system.

cGMP is an important signalling molecule in humans. Fluorescent cGMP biosensors have emerged as powerful tools for the sensitive analysis of cGMP pathways at the single-cell level. Here, we briefly outline cGMP's multifaceted role in (patho)physiology and pharmacotherapy. Then we summarise what new insights cGMP imaging has provided into endogenous cGMP signalling and drug action, with a focus on the cardiovascular system. Indeed, the use of cGMP biosensors has led to several conceptual advances, such as the discovery of local, intercellular and mechanosensitive cGMP signals. Importantly, single-cell imaging can provide valuable information about the heterogeneity of cGMP signals within and between individual cells of an isolated cell population or tissue. We also discuss current challenges and future directions of cGMP imaging, such as the direct visualisation of cGMP microdomains, simultaneous monitoring of cGMP and other signalling molecules and, ultimately, cGMP imaging in tissues and animals under close-to-native conditions. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Absence of Both Thyroid Hormone Transporters MCT8 and OATP1C1 Impairs Neural Stem Cell Fate in the Adult Mouse Subventricular Zone.

SummaryAdult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains.

Comparative study of coelomocytes from Arbacia lixula and Lythechinus variegatus: Cell characterization and in vivo evidence of the physiological function of vibratile cells

The knowledge on echinoderm coelomocytes has increased in recent years, but researchers still face a complex problem: how to obtain purified cells. Even flow cytometry being useful to address coelomocytes in suspension, the need for a method able to provide isolated cells is still noteworthy. Here, we use Imaging Flow Cytometry (IFC) to characterize the coelomocytes of two sea urchin species – Arbacia lixula and Lytechinus variegatus – and obtain gates to isolate cell populations. Then, we used these gates to study the physiological response of A. lixula coelomocytes during an induced immune challenge with Escherichia coli. An analysis of area and aspect ratio parameters of the flow cytometer allowed the identification of two main cell populations in the coelomic fluid: circular and elongated cells. A combination of this method with nucleus labeling using propidium iodide allowed the determination of gates containing isolated subpopulations of vibratile cells, red spherulocytes, and two phagocytes subpopulations in both species. We observed that during an induced bacterial immune challenge, A. lixula was able to modulate coelomocyte frequencies, increasing the phagocytes and decreasing red spherulocytes and vibratile cells. These results indicate that vibratile cells and red spherulocytes act by immobilizing and stoping bacterial growth, respectively, cooperating with phagocytes in the immune response. The use of IFC was fundamental not only to identify specific gates for the main coelomic subpopulations but also allowed the investigation on how echinoids modulate their physiological responses during immune challenges. Furthermore, we provide the first experimental evidence about the role of vibratile cells, corroborating its involvement with the immune system.

Long-term in vitro persistence of magnetic properties after magnetic bead-based cell separation of T cells.

Magnetic‐activated cell sorting (MACS) using magnetic nanoparticles coated with specific antibodies is commonly used in immunology research. For in vitro isolation purposes, it is important to know to what extent the magnetic properties remain present in the isolated cell populations and whether it has consequences for sequential isolations. We hypothesized that only upon cell division, cells will lose their magnetic properties via dilution of the particles in/on their daughter cells. We analysed residual magnetic properties of cells that divided vs cells that did not divide after magnetic bead‐based cell separation. As a model, we isolated T cells using beads targeting the non‐modulating surface molecule CD45RO. Cells were labelled with the cell division tracking dye PKH and cultured under different conditions to induce variable degrees of cell division. We demonstrate that T cells that underwent no, or only minimal, cell divisions after MACS retained magnetic properties for up to at least 2 weeks of in vitro culture. The presence of nanoparticles was detected on their cell surface and intracellularly using Labeling Check reagent. These results have important consequences for procedures requiring repetitive isolation rounds after in vitro culture.