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At least 127 records · Page 7Linked to original sources
PubMedDate not supplied

Differentiation of a bipotential glial progenitor cell in a single cell microculture.

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.

Animals↗
PubMed1996

DNA damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay.

The alkaline single-cell gel electrophoresis assay or comet assay is a sensitive and rapid method for DNA strand breaks and detection of alkali labile sites at the single cell level, it further provides information on the presence of damage among individual cells. In this paper we explore the use of this technique utilizing exfoliated buccal mucosa cells from non-smokers (9 donors) and smokers (11 donors). The extent of DNA image length was found to be significantly increased in the smoker group (89.30 +/- 16.18 microns) than in the non-smoker group (52.01 +/- 10.43 microns). Our results indicate that the single-cell gel electrophoresis assay could be applied to human monitoring using exfoliated buccal epithelial cells.

Adult↗
PubMed1989

Enumeration of cytokine-secreting cells at the single-cell level.

A sensitive assay utilizing enzyme-linked immunosorbent assay methodology has been developed for the quantitation of single cells secreting interferon (IFN)-gamma or tumor necrosis factor (TNF). Cloned T cells or cells from lymphoid organs were stimulated with antigen, concanavalin A, or phorbol myristate acetate and ionomycin in microwells coated with antibodies specific for IFN-gamma. Discrete "spots" overlying areas where cells secrete IFN-gamma were then developed by incubation with a second antibody to IFN-gamma, followed by an enzyme-labeled antibody conjugate and substrate. Similarly, using TNF-specific antibody reagents, TNF-secreting cells were detected and quantitated in cell populations obtained from normal lymphoid tissues, bone marrow and peripheral blood, following activation with phorbol myristate acetate and ionomycin. Provided specific antibodies are available, this method has the potential to measure the frequency of cells secreting any cytokine.

Animals↗
PubMedDate not supplied

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.

Biological Assay↗
PubMed2006

In vivo MRI of cancer cell fate at the single-cell level in a mouse model of breast cancer metastasis to the brain.

Metastasis (the spread of cancer from a primary tumor to secondary organs) is responsible for most cancer deaths. The ability to follow the fate of a population of tumor cells over time in an experimental animal would provide a powerful new way to monitor the metastatic process. Here we describe a magnetic resonance imaging (MRI) technique that permits the tracking of breast cancer cells in a mouse model of brain metastasis at the single-cell level. Cancer cells that were injected into the left ventricle of the mouse heart and then delivered to the brain were detectable on MR images. This allowed the visualization of the initial delivery and distribution of cells, as well as the growth of tumors from a subset of these cells within the whole intact brain volume. The ability to follow the metastatic process from the single-cell stage through metastatic growth, and to quantify and monitor the presence of solitary undivided cells will facilitate progress in understanding the mechanisms of brain metastasis and tumor dormancy, and the development of therapeutics to treat this disease.

Animals↗
PubMed2025

Deep learning-based cell-specific gene regulatory networks inferred from single-cell multiome data.

Gene regulatory networks (GRNs) provide a global representation of how genetic/genomic information is transferred in living systems and are a key component in understanding genome regulation. Single-cell multiome data provide unprecedented opportunities to reconstruct GRNs at fine-grained resolution. However, the inference of GRNs is hindered by insufficient single omic profiles due to the characteristic high loss rate of single-cell sequencing data. In this study, we developed scMultiomeGRN, a deep learning framework to infer transcription factor (TF) regulatory networks via unique integration of single-cell genomic (single-cell RNA sequencing) and epigenomic (single-cell ATAC sequencing) data. We create scMultiomeGRN to elucidate these networks by conceptualizing TF network graph structures. Specifically, we build modality-specific neighbor aggregators and cross-modal attention modules to learn latent representations of TFs from single-cell multi-omics. We demonstrate that scMultiomeGRN outperforms state-of-the-art models on multiple benchmark datasets involved in diseases and health. Via scMultiomeGRN, we identified Alzheimer's disease-relevant regulatory network of SPI1 and RUNX1 for microglia. In summary, scMultiomeGRN offers a deep learning framework to identify cell type-specific gene regulatory network from single-cell multiome data.

Deep Learning↗
PubMed2004

Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip.

INTRODUCTION: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. METHODS: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. RESULTS: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. CONCLUSIONS: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.

Apoptosis↗
PubMed2006

Opposing modifications in intrinsic currents and synaptic inputs in post-traumatic mossy cells: evidence for single-cell homeostasis in a hyperexcitable network.

Recent experimental and modeling results demonstrated that surviving mossy cells in the dentate gyrus play key roles in the generation of network hyperexcitability. Here we examined if mossy cells exhibit long-term plasticity in the posttraumatic, hyperexcitable dentate gyrus. Mossy cells 1 wk after fluid percussion head injury did not show alterations in their current-firing frequency (I-F) and current-membrane voltage (I-V) relationships. In spite of the unchanged I-F and I-V curves, mossy cells showed extensive modifications in Na(+), K(+) and h-currents, indicating the coordinated nature of these opposing modifications. Computational experiments in a realistic large-scale model of the dentate gyrus demonstrated that individually, these perturbations could significantly affect network activity. Synaptic inputs also displayed systematic, opposing modifications. Miniature excitatory postsynaptic current (EPSC) amplitudes were decreased, whereas miniature inhibitory postsynaptic current (IPSC) amplitudes were increased as expected from a homeostatic response to network hyperexcitability. In addition, opposing alterations in miniature and spontaneous synaptic event frequencies and amplitudes were observed for both EPSCs and IPSCs. Despite extensive changes in synaptic inputs, cannabinoid-mediated depolarization-induced suppression of inhibition was not altered in posttraumatic mossy cells. These data demonstrate that many intrinsic and synaptic properties of mossy cells undergo highly specific, long-term alterations after traumatic brain injury. The systematic nature of such extensive and opposing alterations suggests that single-cell properties are significantly influenced by homeostatic mechanisms in hyperexcitable circuits.

Animals↗
PubMed2004

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures.

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.

Adolescent↗
PubMed2006

Morphological classification of parvalbumin-containing retinal ganglion cells in mouse: single-cell injection after immunocytochemistry.

PURPOSE: Matching the neuron's morphology with its expression of a particular protein cannot be easily achieved by immunocytochemistry alone, as many proteins are expressed too weakly. In this study, a newly developed method was adopted to match mouse retinal ganglion cell (RGC) morphology with its expression of parvalbumin. METHODS: Parvalbumin-containing ganglion cells were first identified by immunocytochemistry and then were iontophoretically injected with a lipophilic dye DiI. Then confocal microscopy was used to characterize the morphologic classification of the parvalbumin-immunoreactive (IR) ganglion cells on the basis of the dendritic field size, branching pattern, and stratification within the inner plexiform layer. RESULTS: The results indicate that at least eight different types of ganglion cells express parvalbumin in the mouse retina. They were heterogeneous in morphology: monostratified to bistratified, small-to-large dendritic field size, and sparse-to-dense dendritic arbors. CONCLUSIONS: Single-cell injection, after immunocytochemistry, provided the first means to identify the detailed functional anatomy of parvalbumin-containing RGCs in the mouse retina. The combined approach of cell morphology and the selective expression of parvalbumin will not only provide useful data for further correlation of physiological properties of the RGCs, but it will also provide a useful strategy for matching a neuron's morphology with its expression of a particular protein.

Animals↗
PubMedDate not supplied

IL-12 production by human monocyte-derived dendritic cells: looking at the single cell.

Dendritic cells (DCs) are under investigation as immunotherapeutic agents in the treatment of cancer and infectious diseases. One of the important factors in skewing the immune response toward clinically beneficial TH1-type immunity is interleukin-12p70. IL-12p70 is synthesized and secreted in response to inflammatory cytokines, bacterial/viral components, and CD40 ligation. This study investigated the production of IL-12 by DCs at the single-cell level using a sensitive intracellular cytokine flow cytometry-based assay system. The authors observed that immature DCs could be stimulated with several compounds to produce IL-12, but that IL-12 production was a feature of a minority of activated DCs. IL-12+ DCs were characterized as being partially matured (ie, absent or low CD83 expression, with variable expression of CD1a and CD64). Interestingly, activated DCs lacked expression of the CD16 and CD64 Fc gammaR, which may have important implications for exogenous antigen-loading strategies.

Antigens, CD↗
PubMed1984

Mechanism of cell-mediated cytotoxicity at the single cell level. VIII. Kinetics of lysis of target cells bound by more than one cytotoxic T lymphocyte.

We measured the effects of having multiple cytotoxic T lymphocytes (CTL) bound to one target cell by using the single-cell cytotoxicity in agarose assay. We found that even though there is variability in the time at which individual target cells are lysed, we can identify a general trend: the mean rate of lysis increases with the number of CTL bound per target cell, reaching a maximum when the CTL-target cell ratio is three. Combining a quantitative model for the rate of lethal hitting in multicellular conjugates with a multi-event model for the rate of target cell disintegration, we developed a new multistage kinetic model for predicting the rate of target cell lysis in multiple lymphocyte-target cell conjugates. The variability in the time at which target cells are hit and the variability in the time until they disintegrate are incorporated into the model. By analyzing our measured data in the context of the multistage kinetic model, we were able to estimate via nonlinear least squares regression the target cell disintegration rate, but not the lethal hitting rate. Lethal hitting appeared to be too fast, when compared with disintegration, to significantly affect the time of target cell lysis. By using previously determined values of the lethal hitting rate for single lymphocyte-target cell conjugates and by postulating that lymphocytes act independently of each other in delivering lethal hits, we were able to estimate the rate at which target cells are hit in multiple-lymphocyte single target cell conjugates. By using this estimate of the lethal hitting rate and the regression estimate of the disintegration rate, the multistage kinetic model gave a quantitative fit to our data. From this analysis, we found that the rate at which a target cell disintegrates after being lethally hit increases with the number of CTL per conjugate. This result is quite surprising, because once the first hit has been received, a target cell can disintegrate in a killer cell-independent manner. Under the conditions of our experiment, it appears as if target cell disintegration is not killer cell-independent. Furthermore, our analysis of the time course of target cell disintegration suggests that the process is not governed by simple first order kinetics, but rather by a more complex multistep mechanism.

Animals↗
PubMed2026

Identifying fate-determining transcription factors with single-cell omics.

Single-cell sequencing enables the systematic discovery of cell fate-determining transcription factors (TFs), or key TFs, that define cellular identity or drive cell state transitions. A wide range of computational methods have been developed for this goal, but they differ substantially in the input data and the biological questions they address. In this article, we systematically review computational approaches for key TF identification and organize them from three perspectives: whether they identify TFs defining cell state identity or driving state transitions, whether transitions are modeled as discrete or continuous processes, and whether TFs act individually or combinatorially. We summarize key features and application scenarios of relevant methods to guide tool selection and discuss emerging trends in this field toward programmable and active control of cell fate.

Transcription Factors↗
PubMed1992

Stimulation of neuron survival by basic FGF and CNTF is a direct effect and not mediated by non-neuronal cells: evidence from single cell cultures.

The multifunctional proteins, basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF), share a capacity to promote in vitro and in vivo survival of several, partly overlapping neuron populations. Whether they can affect neurons directly or whether their supportive effects are mediated by non-neuronal cells and their growth factor products has been addressed in this study by establishing single neuron cultures from embryonic chick ciliary ganglia. Cultures with one or two neurons and without any non-neuronal cells were obtained by limiting dilution of ganglionic cell suspensions on 96-well microtiter plates. In the presence of bFGF about 80% of the wells that contained 1 or 2 neurons at the time of seeding, had this (these) neuron(s) maintained after 1 and 5 days. Absence of bFGF resulted in the death of neurons in over 80% of the wells screened. Identical results were obtained with CNTF. These data demonstrate the effectiveness of bFGF and CNTF at the single neuron level, but do not rule out that the factors may act indirectly on neurons, particularly in complex in vitro and in vivo situations.

Animals↗
PubMed1989

States of determination of single cells transplanted between 512-cell Xenopus embryos.

Single cells from 512-cell Xenopus embryos, totally labeled with intracellular horseradish peroxidase, were transplanted orthotopically (from either dorsal or lateral marginal zone) or heterotopically (between these two marginal zones) to unlabeled host embryos at the 512-cell stage. At tailbud stage 23 the locations, numbers, and histotypes of labeled cells were recorded. The transplanted cell had divided many times, giving rise to labeled progeny that expressed a wide range of cell types and were located in several different organs. Locations and cell types of progeny derived from orthotopic grafts to the dorsal marginal zone were different from those derived from grafts to the lateral marginal zone. Single cells grafted heterotopically to either dorsal or lateral positions expressed fates that were appropriate to their final grafted positions, and not to their original positions. We conclude that individual cells of the marginal zone at the 512-cell stage have multiple presumptive fates and have not been committed to any single fate.

Animals↗
PubMed2006

Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.

BACKGROUND: Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Several methods for RNA amplification are in general use, but little consideration has been given to the comparative analysis of those methods in terms of the overall validity of the data generated when amplifying from single-cell amounts of RNA, rather than their empirical performance in single studies. RESULTS: We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings. CONCLUSION: Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios.

3T3 Cells↗
PubMed1997

Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis.

Single cell gel electrophoresis (SCGE), or comet assay, appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. A follow-up study of 90 smokers who ceased smoking was undertaken to determine the possible decrease of DNA damage in their leukocytes. Before beginning the trial, volunteers smoked on average 26.1 +/- 8.4 cigarettes/day. Comet length did not correlate with the number of cigarettes/day or with the condensate tar content. At the end of the study, 28 volunteers had abandoned the trial, 40 volunteers relapsed into smoking at different times, but with a reduced number of cigarettes/day, whereas 22 fully succeeded in smoking cessation. Throughout the 5 sampling times, a great variability of comet length at individual level was found. However, after 1 year of follow-up, comet length means were found to be significantly shorter (p < 0.0001) in those volunteers who completely quit smoking compared to those who relapsed into smoking (27.2 +/- 1.6 vs. 31.9 +/- 5.1 microns, respectively), irrespective of the amount of cigarettes previously smoked. No effect of age or sex was found. Six months later, these results were confirmed by a further study carried out on a reduced sample of volunteers. The present data strongly suggest that, in spite of the great variability observed, 1 year of smoking cessation is associated with a significant reduction of DNA damage in circulating leukocytes.

Adult↗
PubMed1997

Survival of human epithelial cells irradiated with cobalt 60 as microcolonies or single cells.

Microcolonies of one to >50 cells were irradiated. They were assayed for survival using the Puck and Marcus clonogenic technique and the distant progeny were tested for expression of lethal mutations. The results show that epithelial cell colonies appear to respond as a unit rather than as individual cells to a radiation dose and the uncorrected initial surviving fraction is relatively constant irrespective of the number of cells present at the time the microcolony was irradiated. Irradiation of colonies or monolayers, which were then dispersed, confirmed this and showed slight sparing of the cells irradiated in contact compared with single cells but no sparing effect when the gap junctions were closed. Measurement of apoptosis 2 h post-irradiation showed higher levels in clones derived from cells irradiated in contact but delayed apoptosis in the progeny and lethal mutations appear to be associated with irradiation of single cells. Lethal mutations occurred in the progeny of cells irradiated as single cells for at least 30 cell generations but if cell microcolonies were irradiated the progeny survival showed a complex relationship with progenitor dose. When gap junction intercellular communication (GJIC) was blocked during and immediately post-irradiation using nitrosamines or TPA, cultures regained the initial survival and lethal mutation frequency seen with single cells. It is concluded that the presence of more than one cell in a microcolony at the time of irradiation does result in an altered and possibly a co-ordinated pattern of survival and lethal mutation expression but that inhibition of GJIC can reverse the effects of contact. The results may have implications for investigations of normal tissue response.

Apoptosis↗