Abstract: Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. By using mixtures of fixed, cultured human and mouse cells, we first showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single-cell resolution.

Abstract: Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear. Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis. Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924). ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch’s membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.

Abstract: Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

Abstract: An increase of regulatory T cells, defined as CD25high- and/or FOXP3+-expressing CD4+ T cells, within tumors has been reported in several studies. Tregs promote tumor growth by modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-β. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ. Stomach tissue from gastric cancer patients undergoing gastric resection was analyzed using flow cytometry and cell sorting, followed by RT-PCR. We observed that stomach CD4+ FOXP3+ T cells proliferated to a higher degree than CD4+ FOXP3− T cells, which may contribute to Treg accumulation in the mucosa. By analyzing DNA methylation, we demonstrated that both proliferating and nonproliferating FOXP3+ T cells exhibited complete demethylation of the FOXP3 gene, indicating a stable FOXP3 expression in both cell populations. Furthermore, analysis of T-cell populations isolated directly from the tumor and tumor-free mucosa demonstrated that CD4+ CD25high T cells have a higher IL-10/IFN-γ gene expression ratio but express lower levels of TGF-β than CD4+ CD25low/− T cells. We demonstrate strong proliferation among regulatory CD4+ FOXP3+ CD25high T cells in the gastric cancer mucosa. These local Treg express a suppressive cytokine profile characterized by high IL-10 and low TGF-β and IFN-γ production.

Abstract: A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

Abstract: Although researchers have proposed various methods of on-chip cell sorting, high-throughput sorting of large cells remains hampered by the difficulty of controlling high-speed flow over a wide sorting area. To overcome this problem, we proposed high-speed local-flow control using dual membrane pumps driven by piezoelectric actuators placed on the outside of a microfluidic chip in this paper. We evaluated the controllability of shifting the flow profile by the local-flow. The results indicated that we could sort large cells up to approximately 150 μm in size with an equivalent throughput of 31 kHz. Because our method can control the flow profiles, it is applicable not only to large cells but also to small cells. The cell-sorting efficacy of the proposed method was experimentally evaluated on Euglena gracilis NIES-48 (E. gracilis) cells as large target cells and GCIY-EGFP (GCIY) cells derived from a gastric cancer cell line as small target cells. In E. gracilis cells sorting, the throughput is 23 kHz with a 92.8% success rate, 95.8% purity, and 90.8% cell viability. In GCIY sorting, the throughput is 11 kHz with a 97.8% success rate, 98.9% purity, and 90.7% cell viability. These results confirm that the proposed method sorts differently sized cells with high throughput and hence, overcomes the throughput–size trade-off that exists in conventional on-chip cell sorters.

Abstract: ACTA2 expression identifies pulmonary airway and vascular smooth muscle cells (SMCs) as well as alveolar myofibroblasts (MYF). Mesenchymal progenitors expressing fibroblast growth factor 10 (Fgf10), Wilms tumor 1 (Wt1), or glioma-associated oncogene 1 (Gli1) contribute to SMC formation from early stages of lung development. However, their respective contribution and specificity to the SMC and/or alveolar MYF lineages remain controversial. In addition, the contribution of mesenchymal cells undergoing active WNT signaling remains unknown. Using Fgf10CreERT2 , Wt1CreERT2 , Gli1CreERT2 , and Axin2CreERT2 inducible driver lines in combination with a tdTomatoflox reporter line, the respective differentiation of each pool of labeled progenitor cells along the SMC and alveolar MYF lineages was quantified. The results revealed that while FGF10+ and WT1+ cells show a minor contribution to the SMC lineage, GLI1+ and AXIN2+ cells significantly contribute to both the SMC and alveolar MYF lineages, but with limited specificity. Lineage tracing using the Acta2-CreERT2 transgenic line showed that ACTA2+ cells labeled at embryonic day (E)11.5 do not expand significantly to give rise to new SMCs at E18.5. However, ACTA2+ cells labeled at E15.5 give rise to the majority (85%-97%) of the SMCs in the lung at E18.5 as well as alveolar MYF progenitors in the lung parenchyma. Fluorescence-activated cell sorting-based isolation of different subpopulations of ACTA2+ lineage-traced cells followed by gene arrays, identified transcriptomic signatures for alveolar MYF progenitors versus airway and vascular SMCs at E18.5. Our results establish a new transcriptional landscape for further experiments addressing the function of signaling pathways in the formation of different subpopulations of ACTA2+ cells. Stem Cells 2017;35:1566-1578.

Abstract: Sorting cells by their type is an important capability in biological research and medical diagnostics. However, most cell sorting techniques rely on labels or tags, which may have limited availability and specificity. Sorting different cell types by their different physical properties is an attractive alternative to labels because all cells intrinsically have these physical properties. But some physical properties, like cell size, vary significantly from cell to cell within a cell type; this makes it difficult to identify and sort cells based on their sizes alone. In this work we continuously sort different cells types by their density, a physical property with much lower cell-to-cell variation within a cell type (and therefore greater potential to discriminate different cell types) than other physical properties. We accomplish this using a 3D-printed microfluidic chip containing a horizontal flowing micron-scale density gradient. As cells flow through the chip, Earth’s gravity makes each cell move vertically to the point where the cell’s density matches the surrounding fluid’s density. When the horizontal channel then splits, cells with different densities are routed to different outlets. As a proof of concept, we use our density sorter chip to sort polymer microbeads by their material (polyethylene and polystyrene) and blood cells by their type (white blood cells and red blood cells). The chip enriches the fraction of white blood cells in a blood sample from 0.1% (in whole blood) to nearly 98% (in the output of the chip), a 1000x enrichment. Any researcher with access to a 3D printer can easily replicate our density sorter chip and use it in their own research using the design files provided as online Supporting Information. Additionally, researchers can simulate the performance of a density sorter chip in their own applications using the Python-based simulation software that accompanies this work. The simplicity, resolution, and throughput of this technique make it suitable for isolating even rare cell types in complex biological samples, in a wide variety of different research and clinical applications.

Abstract: Extracellular vesicles (EVs) are nanoscale membrane-derived vesicles that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived EVs, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here we demonstrate for the first time that squamous cell carcinoma (SCC) EVs were enriched with the C-terminal fragment of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in malignancies. Overexpression of Dsg2 increased EV release and mitogenic content including epidermal growth factor receptor and c-Src. Inhibiting ectodomain shedding of Dsg2 with the matrix metalloproteinase inhibitor GM6001 resulted in accumulation of full-length Dsg2 in EVs and reduced EV release. When cocultured with Dsg2/green fluorescence protein-expressing SCC cells, green fluorescence protein signal was detected by fluorescence-activated cell sorting analysis in the CD90+ fibroblasts. Furthermore, SCC EVs activated Erk1/2 and Akt signaling and enhanced fibroblast cell proliferation. In vivo, Dsg2 was highly up-regulated in the head and neck SCCs, and EVs isolated from sera of patients with SCC were enriched in Dsg2 C-terminal fragment and epidermal growth factor receptor. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.-Overmiller, A. M., Pierluissi, J. A., Wermuth, P. J., Sauma, S., Martinez-Outschoorn, U., Tuluc, M., Luginbuhl, A., Curry, J., Harshyne, L. A., Wahl, J. K. III, South, A. P., Mahoney, M. G. Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

Abstract: The enrichment of viable cells is an essential step to obtain effective products for cell therapy. While procedures exist to characterize the viability of cells, most methods to exclude nonviable cells require the use of density gradient centrifugation or antibody-based cell sorting with molecular labels of cell viability. We report a label-free microfluidic technique to separate live and dead cells that exploits differences in cellular stiffness. The device uses a channel with repeated ridges that are diagonal with respect to the direction of cell flow. Stiff nonviable cells directed through the channel are compressed and translated orthogonally to the channel length, while soft live cells follow hydrodynamic flow. As a proof of concept, Jurkat cells are enriched to high purity of viable cells by a factor of 185-fold. Cell stiffness was validated as a sorting parameter as nonviable cells were substantially stiffer than live cells. To highlight the utility for hematopoietic stem cell transplantation, frozen samples of cord blood were thawed and the purity of viable nucleated cells was increased from 65% to over 94% with a recovery of 73% of the viable cells. Thus, the microfluidic stiffness sorting can simply and efficiently obtain highly pure populations of viable cells.

Abstract: Background: Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome. Methods and Results: Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecule inhibitors. Fluorescence-activated cell sorting (FACS) analysis confirmed ∼85% and ∼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable to the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective. Conclusions: We demonstrate for the first time that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. This article is protected by copyright. All rights reserved.

Abstract: Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

Abstract: During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDHhi) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDHl° and ALDHhi MSC subsets demonstrated similar expression of stromal cell (>95% CD73+, CD90+, CD105+) and pericyte (>95% CD146+) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDHhi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDHhi MSC or CDM produced by ALDHhi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDHl° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDHhi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDHhi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542–1553

Abstract: Separation of pure cell populations from the liver is a prerequisite to study the role of hepatic parenchymal and non-parenchymal cells in liver physiology, pathophysiology, and immunology. Traditional methods for hepatic cell separation usually purify only single cell types from liver specimens. Here, we describe an efficient method that can simultaneously purify populations of hepatocytes (HCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs) from a single mouse liver specimen. A liberase-based perfusion technique in combination with a low-speed centrifugation and magnetic-activated cell sorting (MACS) led to the isolation and purification of HCs, KCs, and LSECs with high yields and purity.

Abstract: A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.

Abstract: Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. We hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression of five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.

Abstract: B cells reactive with a specific antigen usually occur at a frequency of <0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

Abstract: Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1eGFP/+ knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1+ cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations.

Abstract: The capacity of bone and cartilage to regenerate can be attributed to skeletal stem cells (SSCs) that reside within the bone marrow (BM). Given SSCs are rare and lack specific surface markers, antibody-based sorting has failed to deliver the cell purity required for clinical translation. Microfluidics offers new methods of isolating cells based on biophysical features including, but not limited to, size, electrical properties and stiffness. Here we report the characterization of the dielectric properties of unexpanded SSCs using single-cell microfluidic impedance cytometry (MIC). Unexpanded SSCs had a mean size of 9.0 µm; larger than the majority of BM cells. During expansion, often used to purify and increase the number of SSCs, cell size and membrane capacitance increased significantly, highlighting the importance of characterizing unaltered SSCs. In addition, MIC was used to track the osteogenic differentiation of SSCs and showed an increased membrane capacitance with differentiation. The electrical properties of primary SSCs were indistinct from other BM cells precluding its use as an isolation method. However, the current studies indicate that cell size in combination with another biophysical parameter, such as stiffness, could be used to design label-free devices for sorting SSCs with significant clinical impact.

Abstract: Efficient methods to isolate highly purified Arabidopsis thaliana pollen and the subcellular components of the male gametophyte (the vegetative nucleus and two sperm cells) have enabled genome-scale studies revealing a highly dynamic reprogramming of the transcriptome and epigenome during pollen development. Here, we describe the isolation of uninucleate microspores, mature pollen, as well as sperm cells and vegetative nuclei by Fluorescence-Activated Cell Sorting.

Abstract: Mitotically quiescent cancer stem cells (CSCs) possess higher malignant potential than other CSCs, indicating their higher contribution to therapeutic resistance than that of other CSCs. In esophageal squamous cell carcinoma (ESCC), p75 neurotrophin receptor (p75NTR) is expressed in a candidate CSC population showing high tumorigenicity and chemoresistance. In the present study, we isolated and characterized quiescent CSCs population in ESCC based on p75NTR expression and cell cycle status. Expression of p75NTR and Ki-67 in ESCC cell lines (KYSE cells) and surgically resected ESCC specimens was detected by performing immunocytochemical analysis. p75NTR-positive KYSE cells were fractionated into quiescent and proliferating cells by performing flow cytometry with a fluorescent DNA-staining dye to determine their CSC phenotype. Immunocytochemical analysis showed that 21.8 and 36.5% of the p75NTR-positive cells were Ki-67-negative (G0), which accounted for 11.4 and 15.7% of cells in KYSE-30 and KYSE-140 cell lines, respectively. Flow cytometric cell sorting showed that p75NTR-positive cells in the G0-G1 phase (p75NTR-positive/G0-1 cells) but not in the S-G2-M phase (p75NTR-positive/S-G2-M cells) showed strong expression of stem cell-related genes Nanog, BMI-1, and p63; high colony formation ability; high tumorigenicity in a mouse xenograft model; and strong chemoresistance against cisplatin because of the expression of drug resistance genes ABCG2 and ERCC1. Label-retention assay showed that 3.4% p75NTR-positive cells retained fluorescent cell-tracing dye, but p75NTR-negative cells did not. Immunohistochemical analysis of ESCC specimens showed p75NTR expression in 39 of 95 (41.1%) patients, with a median of 13.2% (range, 3.0-80.1%) p75NTR-positive/Ki-67-negative cells, which were found to be associated with poorly differentiated histology. Our results suggest that p75NTR-positive/G0-1 cells represent quiescent CSCs in ESCC and indicate that these cells can be used as targets to investigate molecular processes regulating CSC phenotype and to develop novel therapeutic strategies.

Abstract: A number of diverse studies have reported the anticancer properties of Cnidium officinale Makino (CO). However, the apoptotic effect of this traditional medicinal herb in human hepatocellular carcinoma cells (HepG2) remains to be elucidated. Therefore, the present study investigated the ability of CO to reduce cell viability through apoptotic pathways. Cell viability was determined using the 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay. CO extract-induced apoptosis in HepG2 cells was assessed by Hoechst 33258 staining. The cell cycle was monitored using fluorescence-activated cell sorting analysis with propidium iodide staining. Furthermore, the present study explored whether various signaling molecules associated with HepG2 cell death were affected by CO treatment, including caspase-3, B-cell lymphoma 2 (Bcl-2), tumor protein p53 (p53), cyclin-dependent kinase 4 (CDK4) and cyclin D. The expression levels of these genes were examined by reverse-transcription polymerase chain reaction and western blotting. The expression levels of caspase-3 and p53 were upregulated with CO extract treatment, whereas those of Bcl-2, CDK4 and cyclin D were significantly downregulated. Cleaved caspase-3 expression was upregulated following treatment with CO extract in a dose-dependent manner. Collectively, the data suggest that CO extract has the potential to induce apoptosis of HepG2 cells and may act by suppressing the cell cycle, which leads to caspase-3 cleavage and p53 signaling.