Abstract: Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far. Although single splicing events have been described for ≤200 single cells with statistical confidence, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3' sequencing enables the identification of cellular subtypes, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms.

Abstract: We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Abstract: Solid tumors are composed of mutually interacting cancer cells and tumor microenvironment. Many environmental components, such as extracellular matrix (ECM), mesenchymal stem cells, endothelial and immune cells, and various growth factors and cytokines, provide signals, either stimulatory or inhibitory, to cancer cells and determine their fates. Meanwhile, cancer cells can also educate surrounding cells or tissues to undergo changes that are in favorable of tumor progression. CD44, as a transmembrane receptor for hyaluronic acid (HA) and many other ECM components and a coreceptor for growth factors and cytokines, is a critical cell surface molecule that can sense, integrate, and transduce cellular microenvironmental signals to membrane-associated cytoskeletal proteins or to cell nucleus to regulate a variety of gene expressions that govern cell behaviors. Mounting evidence suggests that CD44, particularly CD44v isoforms, are cancer stem cell (CSC) markers and critical regulators of cancer stemness, including self-renewal, tumor initiation, and metastasis. Thus, CD44 is widely used alone or in combination with other cell surface markers to isolate or enrich CSCs through fluorescence-activated cell sorting of dissociated single cells that originate from the patient, xenograft tumor tissues, or tumor cell cultures. Sorted cells are cultured in a specialized culture medium for spheroid formation or inoculated into immunodeficient mice for the analysis of tumorigenic or metastatic potential. In this chapter, detailed experimental methods regarding CD44+ tumor cell isolation, spheroid culture, and characterization will be described.

Abstract: Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

Abstract: Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.

Abstract: A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP+, and NAD+ to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.

Abstract: Although many reports show that various kinds of stem cells have the ability to recover the function of premature ovarian insufficiency (POI), few studies are associated with the mechanism of stem cell treatment of POI. We designed this experimental study to investigate whether human adipose stem cell-derived exosomes (hADSC-Exos) retain the ability to restore ovarian function and how hADSC-Exos work in this process. A POI mouse model was established and human ovarian granule cells (hGCs) collected from individuals with POI were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing POI. The hematoxylin and eosin assay method was employed to assess the number of follicles. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of sex hormones. The proliferation rate and marker expression levels of hGCs were measured by flow cytometry (fluorescence-activated cell sorting). Real-time PCR and western blot assays were used to determine the mRNA and protein expression levels of SMAD2, SMAD3, and SMAD5. Western blot assays were used to test the protein expression levels of apoptosis genes (Fas, FasL, caspase-3, and caspase-8). After the hADSC-Exos were transplanted into the POI mice model, they exerted better therapeutic activity on mouse ovarian function, improving follicle numbers during four stages. ELISA results showed that hADSC-Exos elevated the hormone levels to the normal levels. In addition, after hADSC-Exos were cocultured with POI hGCs, our results showed that hADSC-Exos significantly promoted the proliferation rate and inhibited the apoptosis rate. Furthermore, hADSC-Exos also increased the marker expression of hGCs to the normal level. Besides, mRNA and protein assays demonstrated that hADSC-Exos downregulated the expression of SMAD2, SMAD3, and SMAD5 in vivo and in vitro. Western blot assay demonstrated that hADSC-Exos inhibited expression of the apoptosis genes in POI hGCs, and SMAD knockdown increased the protein expression of apoptosis genes. These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which exosomes derived from hADSCs improved ovarian function of POI disease via regulation of the SMAD signaling pathway.

Abstract: Isolating and maintaining the appropriate stem cell for large scale cell culture is essential in tissue engineering or food production. For bovine satellite cells an optimized isolation and purification protocol is lacking and there is also no detailed understanding on the factors that maintain stemness of these cells. Here, we set up a fluorescence-activated cell sorting strategy to enrich bovine satellite cells. We found that p38-MAPK signalling is activated and PAX7 expression is gradually lost during satellite cell proliferation. The p38 inhibitor (SB203580) treatment maintained PAX7 expression but inhibited the fusion of satellite cells in a concentration-dependent way in short-term incubation. The mechanism of p38 inhibition was confirmed by inhibiting canonical p38 signalling, i.e. HSP27. Long-term culture with an appropriate concentration of p38i enhanced the proliferation and PAX7 expression, while the differentiation capacity recovered and was enhanced compared to vehicle control. These studies indicate that bovine satellite cells maintenance depends on cell purity and p38 MAPK signalling. Inhibition of p38 MAPK signaling is a promising strategy to facilitate large scale cell expansion of primary cells for tissue engineering and cultured meat purposes.

Abstract: This protocol describes how to isolate endothelial cells and pericytes from adult mouse brain microregions using fluorescence-activated cell sorting without requirement for transgenic mice. The cells may be used for diverse downstream applications. The vasculature is emerging as a key contributor to brain function during neurodevelopment and in mature physiological and pathological states. The brain vasculature itself also exhibits regional heterogeneity, highlighting the need to develop approaches for purifying cells from different microregions. Previous approaches for isolation of endothelial cells and pericytes have predominantly required transgenic mice and large amounts of tissue, and have resulted in impure populations. In addition, the prospective purification of brain pericytes has been complicated by the fact that widely used pericyte markers are also expressed by other cell types in the brain. Here, we describe the detailed procedures for simultaneous isolation of pure populations of endothelial cells and pericytes directly from adult mouse brain microregions using fluorescence-activated cell sorting (FACS) with antibodies against CD31 (endothelial cells) and CD13 (pericytes). This protocol is scalable and takes ∼5 h, including microdissection of the region of interest, enzymatic tissue dissociation, immunostaining, and FACS. This protocol allows the isolation of brain vascular cells from any mouse strain under diverse conditions; these cells can be used for multiple downstream applications, including in vitro and in vivo experiments, and transcriptomic, proteomic, metabolomic, epigenomic, and single-cell analysis.

Abstract: The characterization of specialized cell subpopulations in a heterogeneous tissue is essential for understanding organ function in health and disease. A popular method of cell isolation is fluorescence-activated cell sorting (FACS) based on probes that bind surface or intracellular markers. In this study, we analyze the impact of FACS on the cell metabolome of mouse peritoneal macrophages. Compared with directly pelleted macrophages, FACS-treated cells had an altered content of metabolites related to the plasma membrane, activating a mechanosensory signaling cascade causing inflammation-like stress. The procedure also triggered alterations related to energy consumption and cell damage. The observed changes mostly derive from the physical impact on cells during their passage through the instrument. These findings provide evidence of FACS-induced biochemical changes, which should be taken into account in the design of robust metabolic assays of cells separated by flow cytometry.

Abstract: Intervertebral disc (IVD) degeneration is characterized by an early decrease in cellularity of the nucleus pulposus (NP) region, and associated extracellular matrix changes, reduced hydration, and progressive degeneration. Cell-based IVD therapy has emerged as an area of great interest, with studies reporting regenerative potential for many cell sources, including autologous or allogeneic chondrocytes, primary IVD cells, and stem cells. Few approaches, however, have clear strategies to promote the NP phenotype, in part due to a limited knowledge of the defined markers and differentiation protocols for this lineage. Here, we developed a new protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into NP-like cells in vitro. This differentiation strategy derives from our knowledge of the embryonic notochordal lineage of NP cells as well as strategies used to support healthy NP cell phenotypes for primary cells in vitro. An NP-genic phenotype of hiPSCs was promoted in undifferentiated hiPSCs using a stepwise, directed differentiation toward mesodermal, and subsequently notochordal, lineages via chemically defined medium and growth factor supplementation. Fluorescent cell imaging was used to test for pluripotency markers in undifferentiated cells. RT-PCR was used to test for potential cell lineages at the early stage of differentiation. Cells were checked for NP differentiation using immunohistochemistry and histological staining at the end of differentiation. To enrich notochordal progenitor cells, hiPSCs were transduced using lentivirus containing reporter constructs for transcription factor brachyury (T) promoter and green fluorescent protein (GFP) fluorescence, and then sorted on T expression based on GFP intensity by flow cytometry. Periods of pellet culture following initial induction were shown to promote the vacuolated NP cell morphology and NP surface marker expression, including CD24, LMα5, and Basp1. Enrichment of brachyury (T) positive cells using fluorescence-activated cell sorting was shown to further enhance the differentiation efficiency of NP-like cells. The ability to efficiently differentiate human iPSCs toward NP-like cells may provide insights into the processes of NP cell differentiation and provide a cell source for the development of new therapies for IVD diseases.

Abstract: On antigen encounter, naive CD8+ and CD4+ T cells differentiate into a large number of effector cells that migrate to inflamed tissues to fight infections or tumors. Following elimination of the target, a few cells remain in the long-term, the so-called memory T cells that are capable to reexpand and respond more vigorously on a second encounter with the cognate antigen. While the naive T cell compartment is fairly homogenous, effector and memory T cells are largely diverse, comprising dozens of subsets with diverse functions, molecular characteristics, and localization in the body. In addition, CD4+ and, to some extent, also CD8+ T cells can differentiate into several effector subsets according to their pattern of cytokine expression, including the T helper 1 (Th1), Th2, and Th17 cells. It has become clear that specific subsets of T cells dominate different types of infections and pathological conditions and have different capacities to infiltrate and reject tumors. Their correct phenotypic identification is therefore of foremost importance for their live purification by magnetic or fluorescence-activated cell sorting and subsequent molecular characterization. Here, we present a comprehensive list of the main T cell subpopulations with a major focus on human cells along with their surface phenotypic properties.

Abstract: Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

Abstract: Objective: The mechanisms that lead to endothelial cell (EC) injury and propagate the vasculopathy in Systemic Sclerosis (SSc) are not well understood. Using single cell RNA sequencing (scRNA-seq), our goal was to identify EC markers and signature pathways associated with vascular injury in SSc skin. Methods: We implemented single cell sorting and subsequent RNA sequencing of cells isolated from SSc and healthy control skin. We used t-distributed stochastic neighbor embedding (t-SNE) to identify the various cell types. We performed pathway analysis using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). Finally, we independently verified distinct markers using immunohistochemistry on skin biopsies and qPCR in primary ECs from SSc and healthy skin. Results: By combining the t-SNE analysis with the expression of known EC markers, we positively identified ECs among the sorted cells. Subsequently, we examined the differential expression profile between the ECs from healthy and SSc skin. Using GSEA and IPA analysis, we demonstrated that the SSc endothelial cell expression profile is enriched in processes associated with extracellular matrix generation, negative regulation of angiogenesis and epithelial-to-mesenchymal transition. Two of the top differentially expressed genes, HSPG2 and APLNR, were independently verified using immunohistochemistry staining and real-time qPCR analysis. Conclusion: ScRNA-seq, differential gene expression and pathway analysis revealed that ECs from SSc patients show a discrete pattern of gene expression associated with vascular injury and activation, extracellular matrix generation and negative regulation of angiogenesis. HSPG2 and APLNR were identified as two of the top markers of EC injury in SSc.

Abstract: Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across nine time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells that were validated using electrophysiology. We identified widespread changes in the expression of individual transcript features and their splice variants, gene networks, and global patterns of transcription. We next demonstrated that co-culturing human NPCs with rodent astrocytes resulted in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without potentially disruptive cell sorting. We lastly developed and validated a computational tool to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue, which were maturationally heterogeneous but contained subsets of cells most akin to adult human neurons.

Abstract: Summary Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.

Abstract: Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq.

Abstract: CD146 and STRO-1 are endothelial biomarkers that are co-expressed on the cellular membranes of blood vessels within human dental pulp tissue. This study characterized the percentage of dentin-like structures produced by CD146-positive (CD146+) human dental pulp stem cells (DPSCs), compared with their CD146-negative (CD146−) counterparts. DPSC populations were enriched using magnetic-activated cell sorting (MACS), yielding CD146+ and CD146− cells, as well as mixtures composed of 25% CD146+ cells and 75% CD146− cells (CD146+/−). Cell growth assays indicated that CD146+ cells exhibit an approximate 3–4 h difference in doubling time, compared with CD146− cells. Cell cycle distributions were determined by flow cytometry analysis. The low percentage of CD146+ cells’ DNA content in G0/G1 phase were compared with CD146− and non-separated cells. In contrast to CD146− and non-separated cells, prompt mineralization was observed in CD146+ cells. Subsequently, qRT-PCR revealed high mRNA expression of CD146 and Alkaline phosphatase in mineralization-induced CD146+ cells. CD146+ cells were also observed high adipogenic ability by Oil red O staining. Histological examinations revealed an increased area of dentin/pulp-like structures in transplanted CD146+ cells, compared with CD146− and CD146+/− cells. Immunohistochemical studies detected dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP), as well as human mitochondria, in transplanted DPSCs. Co-expression of CD146 and GFP indicated that CD146 was expressed in transplanted CD146+ cells. CD146+ cells may promote mineralization and generate dentin/pulp-like structures, suggesting a role in self-renewal of stem cells and dental pulp regenerative therapy.

Abstract: Cancers consist of a heterogeneous populations of cells that may respond differently to treatment through drug-resistant sub-populations. The scarcity of these resistant sub-populations makes it challenging to understand how to counter their resistance. We report a label-free microfluidic approach to separate cancer cells treated with chemotherapy into sub-populations enriched in chemoresistant and chemosensitive cells based on the differences in cellular stiffness. The sorting approach enabled analysis of the molecular distinctions between resistant and sensitive cells. Consequently, the role of multiple mechanisms of drug resistance was identified, including decreased sensitivity to apoptosis, enhanced metabolism, and extrusion of drugs, and, for the first time, the role of estrogen receptor in drug resistance of leukemia cells. To validate these findings, several inhibitors for the identified resistance pathways were tested with chemotherapy to increase cytotoxicity sevenfold. Thus, microfluidic sorting can identify molecular mechanisms of drug resistance to examine heterogeneous responses of cancers to therapies.

Abstract: Multicolor flow cytometry and cell sorting are powerful immunologic tools for the study of hepatic mϕ, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of mϕ and endothelial cell reporter mice, flow cytometry, and confocal imaging, we have shown that conventional flow-cytometric strategies for identification of Kupffer cells (KCs) leads to inclusion of a significant proportion of CD31hi endothelial cells. These cells were present regardless of the method used to prepare cells for flow cytometry and represented endothelium tightly adhered to remnants of KC membrane. Antibodies to endothelial markers, such as CD31, were vital for their exclusion. This result brings into focus recently published microarray datasets that identify high expression of endothelial cell-associated genes by KCs compared with other tissue-resident mϕ. Our studies also revealed significant and specific loss of KCs among leukocytes with commonly used isolation methods that led to enrichment of proliferating and monocyte-derived mϕ. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.

Abstract: Background context: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. Ang-1 angiopoetin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle and mouse. These show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration, and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy on the presence and function of these Tie2+-nucleus pulposus cells (NPC), in part due to the difficulty of identification and isolation. Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs. Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2/TEK across species is presented. Results: Our protocols are efficient towards labeling and isolation of Tie2+ cells. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4-16 hours, cell expansion can take up to multiple weeks, dependent on application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species. Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse and human surgical specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. Biological function of identified cells needs to be confirmed by functional assays such as in vitro differentiation. In vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

Abstract: Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14++CD16+) monocytes in whole blood.

Abstract: Recent evidence suggests that the targeting of membrane proteins specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the expression and activity of ion-transport-related molecules in the CSCs of esophageal squamous cell carcinoma. Cells exhibiting strong aldehyde dehydrogenase 1 family member A1 (ALDH1A1) activity were isolated from TE8 cells by fluorescence-activated cell sorting, and CSCs were then generated with the sphere formation assay. The gene expression profiles of CSCs were examined by microarray analysis. Among TE8 cells, ALDH1A1 messenger RNA and protein levels were higher in CSCs than in non-CSCs. The CSCs obtained were resistant to cisplatin and had the ability to redifferentiate. The results of the microarray analysis revealed that the expression of 50 genes encoding plasma membrane proteins was altered in CSCs, whereas that of several genes related to ion channels, including transient receptor potential vanilloid 2 (TRPV2), was upregulated. The TRPV2 inhibitor tranilast was more cytotoxic at a lower concentration in CSCs than in non-CSCs, and effectively decreased the number of tumorspheres. Furthermore, tranilast significantly decreased the cell population that strongly expressed ALDH1A1 among TE8 cells. The results of the present study suggest that TRPV2 is involved in the maintenance of CSCs, and that its specific inhibitor, tranilast, has potential as a targeted therapeutic agent against esophageal squamous cell carcinoma.

Abstract: Although malaria is the world's most life-threatening parasitic disease, there is no clear understanding of how certain biophysical properties of infected cells change during the malaria infection cycle. In this article, we use microfluidic impedance cytometry to measure the dielectric properties of Plasmodium falciparum-infected red blood cells (i-RBCs) at specific time points during the infection cycle. Individual parasites were identified within i-RBCs using green fluorescent protein (GFP) emission. The dielectric properties of cell sub-populations were determined using the multi-shell model. Analysis showed that the membrane capacitance and cytoplasmic conductivity of i-RBCs increased along the infection time course, due to membrane alterations caused by parasite infection. The volume ratio occupied by the parasite was estimated to vary from less than 10% at earlier stages, to approximately 90% at later stages. This knowledge could be used to develop new label-free cell sorting techniques for sample pre-enrichment, improving diagnosis.

Abstract: Brain endothelial cells (BECs) form the integral component of the blood-brain barrier (BBB) which separates the systemic milieu from the brain parenchyma and protects the brain from pathogens and circulating factors. In order to study BEC biology, it was of particular interest to establish a method that enables researchers to investigate and understand the underlying molecular mechanisms regulating their function during homeostasis, aging and disease. Furthermore, due to the heterogeneity of the cerebrovasculature and different vessel types that comprise the BBB, it is of particular interest to isolate primary BECs for single cell analysis from various subregions of the brain, such as the neurogenic and highly vascularized hippocampus and to enrich for specific vessel types. In the past, approaches to isolate endothelial cells were dependent on transgenic mice and often resulted in insufficiently pure cell populations and poor yield. This protocol describes a technique that allows single-cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after perfusion and careful removal of the meninges, and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested resulting in a single cell suspension. Cells are stained with fluorochrome-conjugated antibodies identifying CD31+ brain endothelial cells, as well as CD45+CD11b+ myeloid cells for exclusion. Using flow cytometry, cell populations are separated and CD31+BECs are sorted in bulk into RNA later or as single cells directly into either RNA lysis buffer for single or bulk RNA-Seq analyses. The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 1 × 105 cells isolated from an adult mouse cortex/hippocampus.

Abstract: Endothelial cells (ECs) line the interior surface of blood and lymphatic vessels, and play a key role in a variety of physiological or pathological processes such as thrombosis, inflammation, or vascular wall remodeling. Human-induced pluripotent stem cell (iPSCs)-derived ECs provide a new opportunity for vascular regeneration and serve as a model to study the mechanism and to screen for novel therapies. We use developmental cues in a monolayer differentiation approach to efficiently generate mesoderm cells from iPSCs via small-molecule activation of WNT signaling in chemically defined medium for 4 days, and subsequent EC specification using vascular endothelial growth factor and fibroblast growth factor for another 4 days. After 8 days of differentiation, mature ECs are further purified using magnetic-activated cell sorting for the EC surface marker CD144. These ECs exhibit molecular and cellular characteristics consistent with native ECs, such as expression of specific surface markers, formation of tube-like structures and acetylated low-density lipoprotein uptake. © 2018 by John Wiley & Sons, Inc.

Abstract: Cell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine and adipocytes in white adipose tissue. As this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.

Abstract: Glioblastoma (GBM) is a neoplasm characterized by an extensive blood vessel network. Hypoxic niches of GBM can induce tumorigenic properties of a small cell subpopulation called Glioblastoma stem-like cells (GSCs) and can also increase extracellular adenosine generation which activates the A3 adenosine receptor (A3AR). Moreover, GSCs potentiates the persistent neovascularization in GBM. The aim of this study was to determine if A3AR blockade can reduce the vasculogenesis mediated by the differentiation of GSCs to Endothelial Cells (ECs) under hypoxia. We evaluated the expression of endothelial cell markers (CD31, CD34, CD144, and vWF) by fluorescence-activated cell sorting (FACS), and vascular endothelial growth factor (VEGF) secretion by ELISA using MRS1220 (A3AR antagonist) under hypoxia. We validate our results using U87MG-GSCs A3AR knockout (GSCsA3-KO). The effect of MRS1220 on blood vessel formation was evaluated in vivo using a subcutaneous GSCs-tumor model. GSCs increased extracellular adenosine production and A3AR expression under hypoxia. Hypoxia also increased the percentage of GSCs positive for endothelial cell markers and VEGF secretion, which was in turn prevented when using MRS1220 and in GSCsA3-KO. Finally, in vivo treatment with MRS1220 reduced tumor size and blood vessel formation. Blockade of A3AR decreases the differentiation of GSCs to ECs under hypoxia and in vivo blood vessel formation.