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PubMed1999

Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome.

Methylation of cytosine residues in CpG dinucleotides is generally associated with silencing of gene expression. DNA methylation, as a somatic event, has the potential of diversifying gene expression in individual cells of the same lineage. There is little quantitative data available concerning the extent of methylation heterogeneity in individual cells across the genome. T cells from the peripheral blood can be grown as single-cell-derived clones and can be analyzed with respect to their DNA methylation patterns by restriction landmark genomic scanning. The use of the methylation-sensitive enzyme NotI to cut and end-label DNA fragments before their separation in two dimensions provides a quantitative assessment of methylation at NotI sites that characteristically occur in CpG islands. We have undertaken quantitative analysis of two-dimensional DNA patterns to determine the extent of methylation heterogeneity at NotI sites between peripheral blood single-cell-derived T cell clones from the same individual. A total of 1,068 NotI-tagged fragments were analyzed. A subset of 156 fragments exhibited marked methylation heterogeneity at NotI sites between clones. Their average intensity among clones correlated with their intensity in uncultured, whole-blood-derived T cells, indicating that the methylation heterogeneity observed in clones was largely attributable to methylation heterogeneity between the individual cells from which the clones were derived. We have cloned one fragment that exhibited variable NotI-site methylation between clones. This fragment contained a novel CpG island for a gene that we mapped to chromosome 4. The methylation status of the NotI site of this fragment correlated with expression of the corresponding gene. Our data suggest extensive diversity in vivo in the methylation and expression profiles of individual T cells at multiple unrelated loci across the genome.

Base Composition↗

PubMed2005

Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

Biotechnology↗

PubMed2002

Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of their gene expression profiles.

Circulating CD34(+) cells are used in reparative medicine as a stem cell source, but they contain cells already committed to different lineages. Many think that B-cell progenitors (BCPs) are confined to bone marrow (BM) niches until they differentiate into B cells and that they do not circulate in blood. The prevailing convention is that BCP transit a CD34(+)CD19(-)10(+) early-B-->CD34(+)CD19(+)CD10(+) B-cell progenitor (pro-B)-->CD34(-)CD19(+)CD10(+) B-cell precursor (pre-B) differentiation pathway within BM. However, populations of CD34(+)CD10(+) and CD34(+)CD19(+) cells circulate in adult peripheral blood and neonatal umbilical cord blood (CB) that are operationally taken as BCPs on the basis of their phenotypes, although they have not been submitted to a systematic characterization of their gene expression profiles. Here, conventional CD34(+)CD19(+)CD10(+) and novel CD34(+)CD19(+)CD10(-) BCP populations are characterized in CB by single-cell sorting and multiplex analyses of gene expression patterns. Circulating BCP are Pax-5(+) cells that span the early-B, pro-B, and pre-B developmental stages, defined by the profiles of rearranged V-D-J(H), CD79, VpreB, recombination activating gene (RAG), and terminal deoxynucleotidyl transferase (TdT) expression. Contrary to the expectation, circulating CD34(+)CD19(-)CD10(+) cells are essentially devoid of Pax-5(+) BCP. Interestingly, the novel CD34(+)CD19(+)CD10(-) BCP appears to be the normal counterpart of circulating preleukemic BCPs that undergo chromosomal translocations in utero months or years before their promotion into infant acute lymphoblastic B-cell leukemia after secondary postnatal mutations. The results underscore the power of single-cell analyses to characterize the gene expression profiles in a minor population of rare cells, which has broad implications in biomedicine.

Antigens, CD19↗

PubMed2002

Nrf2 transactivator-independent GSTP1-1 expression in "GSTP1-1 positive" single cells inducible in female mouse liver by DEN: a preneoplastic character of possible initiated cells.

Whether single cells immunohistochemically positive for glutathione S-transferase P1-1 (GSTP1-1) induced in the female mouse liver by DEN (Hatayama et al., Carcinogenesis, 14, 537-538, 1993) are precursor initiated cells of preneoplastic foci, is of importance in chemical hepatocarcinogenesis. Nrf2 transactivates a wide variety of ARE (anti-oxidant response element)-mediated enzymes including GSTP1-1. Quantitative examination revealed that the basal expression of hepatic GSTP1-1 was 60% lower in Nrf2 gene knock-out female mice(-/-) than in wild type females, and that treatment with butyrated hydroxyanisole (BHA) increased by 10-fold GSTP1-1 expression in the liver of wild type female mice but not in knockout female mice(-/-). Despite the lack of Nrf2, GSTP1-1-positive single cells were detected in livers of DEN-treated female(-/-) 3 months after treatment. Subsequent BHA feeding to the positive cell-bearing females for one more week clearly showed that the single cells were detectable with females(-/-) but not with females(+/+,+/-) due to the strong induction of GSTP1-1 in the surrounding hepatocytes. The sensitivity to DEN hepatocarcinogenesis was not significantly different among genotypes. These results demonstrate that Nrf2 is regulatory in normal hepatocytes but not in the single cells positive for GSTP1-1 inducible in the female mouse liver by DEN. The transcriptional distinction observed for the DEN-transformants is suggestive of a preneoplastic character of precursor initiated cells.

Animals↗

PubMed1996

Lhcb Transcription Is Coordinated with Cell Size and Chlorophyll Accumulation (Studies on Fluorescence-Activated, Cell-Sorter-Purified Single Cells from Wild-Type and immutans Arabidopsis thaliana).

To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined [beta]-glucuronidase (GUS) enzyme activities, chlorophyll contents, and cell sizes in fluorescence-activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the green sectors to compensate for a lack of photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis.

Journal Article↗

PubMed1983

Characteristics and requirements of the interaction between human monocytes and tumor cells on the single cell level.

Highly purified (97%-99%) and viable (99%) peripheral blood monocytes obtained by EDTA-reversible adherence to autologous-serum-precoated plastic surfaces could rapidly lyse a variety of tumor cells in a 3-4 hr 51Cr release assay. Using these monocytes as effectors, a short-term agarose/conjugate assay was utilized, permitting us to examine the interaction between fresh human monocytes and neoplastic target cells on a single cell level. That the tumor-bound effector cells were indeed monocytes was confirmed by employing the monocyte-specific monoclonal antibody 61D3, which stained 95%-99% of the mononuclear cells bound to conjugated and killed K562 tumor targets. The binding of monocytes to target cells appeared to be temperature dependent and was extremely rapid, reaching a plateau after 5 min at 30 degrees C. Our findings demonstrated for the first time that only a proportion of human blood monocytes can bind to a particular target cell and that only a fraction of the binding cells have the intrinsic potential to kill those neoplastic targets. The proportion of monocytes capable of binding and killing varies between individuals and also depends on the tumor cell used, indicating heterogeneity in the monocyte and tumor cell populations. The highest proportion of monocytes bind to the human erythromyeloid leukemia K562 cell line (13%-50%). The frequency of monocytes capable of killing K562 tumor cells is relatively low (7%-13%). The system described here should be useful to study the heterogeneity of mononuclear phagocytes and to analyze the molecular basis of the interaction between those effector cells and neoplastic target cells.

Antibodies, Monoclonal↗

PubMed1988

A complex mechanism of facilitation in pituitary ACTH cells: recent single-cell studies.

The transfer of information by chemical signals during complex biological processes can, with increasing frequency, be described in terms of interacting signal pairs. External signalling is rarely monolithic; rather, signal pairs are utilized in processes such as hormone secretion, neurotransmission, cell growth and differentiation. The dualism of external signalling often results in the occurrence of synergy. One signal appears to turn the cell on or off, and its synergistic partner increases cell responsiveness, providing gain control of the cellular response. ACTH release provoked by certain stressors arises from a synergistic interaction between two hypothalamic hormones: corticotropin releasing hormone (CRH) and a modulator such as vasopressin (AVP). The pituitary ACTH cell has been used to unravel the intracellular messenger equivalents of an external signal interaction that generates synergy. Research emphasizes the single cell approach. Direct measurements of intracellular free Ca2+ were performed using the Ca2+-sensitive fluorescent probe fura-2/AM and instrumentation for digital image processing. A reverse haemolytic plaque assay was used to measure cumulative ACTH release from single pituitary cells in culture. What is the physiological role of intracellular Ca2+ as a messenger? What are the feedforward and feedback relationships between major second messengers [cyclic AMP, diacylglycerol (DAG), inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)] and intracellular Ca2+? That is, how are individual messenger circuits "wired' within ACTH cells. Intracellular Ca2+ may act as a common signal into which interacting second messenger signals [cyclic AMP, Ins(1,4,5)P3, DAG] are transduced and integrated to govern ACTH release. A novel circuit of messenger pathways linked by Ca2+ is proposed as the intracellular basis for the synergistic interaction of CRH- and AVP-regulated ACTH release.

Adrenocorticotropic Hormone↗

PubMed1999

Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR).

The determination of the gene expression pattern of single cells has important implications for many areas of cellular and developmental biology including lineage determination, identification of primitive stem cells and temporal gene expression patterns induced by changes in the cellular microenvironment. Global Single Cell Reverse Transcription-Polymerase Chain Reaction (GSC RT-PCR) enables the study of single cell gene expression patterns. Initial observations of significant heterogeneity among single cells derived from a population of cells prompted us to determine how much of this observed heterogeneity was due to the intrinsic variation within the method. In this paper we discuss the sensitivity of GSC RT-PCR for analysis of differences in gene expression between single cells and, in particular, detail the amount of variation generated by the method itself. We found that most of the intrinsic variation in the method occurred in the PCR step. The total variation induced by the method was in the range of 5 fold. While we have determined that there is a five fold methodological variation in GSC RT-PCR, any method which use its components (including generation of cDNAs for microarray analysis) is likely to be affected by such experimental variability, which could limit the interpretation of the resulting data.

Analysis of Variance↗

PubMed2026

ELISA (Embedding-Linked Interactive Single-cell Agent): an interpretable hybrid generative Artificial Intelligence agent for expression-grounded discovery in single-cell genomics.

Translating single-cell RNA sequencing (scRNA-seq) data into mechanistic biological hypotheses remains a critical bottleneck, as agentic AI systems lack direct access to transcriptomic representations while expression foundation models remain opaque to natural language. Here, we introduce ELISA (Embedding-Linked Interactive Single-cell Agent), an interpretable framework that unifies single-cell generative pretrained transformer expression embeddings with biomedical bidirectional encoder representations from transformers-based semantic retrieval and large-language model (LLM)-mediated interpretation for interactive single-cell discovery. An automatic query classifier routes inputs to gene marker scoring, semantic matching, or reciprocal rank fusion pipelines depending on whether the query is a gene signature, natural language concept, or mixture of both. Integrated analytical modules perform pathway activity scoring across 60+ gene sets, ligand-receptor interaction prediction using 280+ curated pairs, condition-aware comparative analysis, and cell-type proportion estimation, all operating directly on embedded data without access to the original count matrix. Benchmarked across six diverse scRNA-seq datasets spanning inflammatory lung disease, pediatric and adult cancers, organoid models, healthy tissue, and neurodevelopment, ELISA significantly outperforms CellWhisperer, a classical lexical retriever (BM25), and a random baseline in cell type retrieval (combined permutation test, $p < 2\times 10^{-5}$ for each), with particularly large gains on gene-signature queries (Cohen's $d = 5.98$ for mean reciprocal rank). ELISA replicates published biological findings (mean composite score 0.88), and generates candidate hypotheses through grounded LLM reasoning, bridging the gap between transcriptomic data exploration and biological discovery.

Generative Artificial Intelligence↗

PubMed2006

Mesenchymal multipotency of adult human periosteal cells demonstrated by single-cell lineage analysis.

OBJECTIVE: To investigate whether periosteal cells from adult humans have features of multipotent mesenchymal stem cells (MSCs) at the single-cell level. METHODS: Cell populations were enzymatically released from the periosteum of the proximal tibia obtained from adult human donors and then expanded in monolayer. Single-cell-derived clonal populations were obtained by limiting dilution. Culture-expanded periosteal cell populations were tested for their growth potential and for expression of conventional markers of MSCs and were subjected to in vitro assays to investigate their multilineage potential. To assess their multipotency in vivo, periosteal cells were injected into a regenerating mouse tibialis anterior muscle for skeletal myogenesis or were either seeded into an osteoinductive matrix and implanted subcutaneously into nude mice for osteogenesis or implanted in a joint surface defect under a periosteal flap into goats for chondrogenesis. Cell phenotypes were analyzed by histochemistry and immunohistochemistry and by reverse transcription-polymerase chain reaction for the expression of lineage-related marker genes. RESULTS: Regardless of donor age, periosteal cells were clonogenic and could be expanded extensively in monolayer, maintaining linear growth curves over at least 30 population doublings. They displayed long telomeres and expressed markers of MSCs. Under specific conditions, both parental and single-cell-derived clonal cell populations differentiated to the chondrocyte, osteoblast, adipocyte, and skeletal myocyte lineages in vitro and in vivo. CONCLUSION: Our study demonstrates that, regardless of donor age, the adult human periosteum contains cells that, upon enzymatic release and culture expansion, are multipotent MSCs at the single-cell level.

Adult↗

PubMed2002

Detection of ochratoxin A-induced DNA damage in MDCK cells by alkaline single cell gel electrophoresis (comet assay).

The mycotoxin ochratoxin A (OTA), a widespread contaminant of food and feedstuffs, is nephrotoxic, immunosuppressive and carcinogenic in domestic and laboratory animals. Additionally, it is suspected as being responsible for urinary tract tumours in patients suffering from Balkan endemic nephropathy. Moreover, evidence has accumulated that OTA is a genotoxic carcinogen, although the mechanism that results in DNA damage has not been fully resolved. In this study, the induction of DNA damage by OTA and the subsequent DNA repair was investigated by alkaline single cell gel electrophoresis (comet assay) in cells originally derived from the kidney, a target organ of OTA. With modifications of the method, the influence of OTA uptake into the cells and of DNA repair on the genotoxic effect of OTA should be investigated. In Madin-Darby canine kidney (MDCK) cells, OTA induced single-strand breaks in a concentration dependent manner. When an external metabolising enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By co-incubation with methotrexate or with the mycotoxin citrinin, a substrate of the organic anion transporter, the adverse effect of OTA was inhibited. When DNA repair was inhibited by addition of cytosine arabinoside and hydroxyurea, the tail length increased dramatically and all treated cells showed single-strand breaks. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the DNA damage within 2 h. Adverse effects on the mechanisms of DNA repair, or exposure to OTA in periods of reduced DNA repair capacity may influence the genotoxic potency of OTA and have to be regarded as a further mechanism by which genotoxic effects of OTA can be performed.

Animals↗

PubMed2003

Modelling the individual cell lag phase. Isolating single cells: protocol development.

AIMS: To develop a protocol to isolate single cells in wells of a microtitre plate, having a high certainty of individual cells, combined with a sufficient yield. METHODS AND RESULTS: Single cells were obtained using 1/2 dilution series in microtitre plates. Seventy-two Lactococcus lactis dilution series were checked by plate counting. When the last five columns of the plates were observed, the chance of having one single cell was 80%, while the yield was 75 wells containing cells. A simulation model confirmed these results. This method was compared with the commonly applied method. CONCLUSIONS: This method makes it possible to combine a higher chance of having one cell in a microtitre well with a slightly higher yield. SIGNIFICANCE AND IMPACT OF THE STUDY: A tool is developed to isolate single cells to provide a suitable base for investigating and modelling the individual cell lag phase.

Cell Division↗

PubMed2001

Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level.

Antigen-presenting cells (APCs) from subcutaneous mouse MOPC315 plasmacytoma phagocytosed immunoglobulin G-coated magnetic beads, enabling efficient isolation within 2 hours by magnetic separation (APC-MB). Cell morphology was heterogeneous, with some of the cells having dendrites. The surface phenotype of purified tumor APCs-MB was CD11b(+), CD11c(+), CD40(+), CD80(+), CD86(+), and MHC class II(+). Tumor APCs-MB expressed messenger RNA for fractalkine and ABCD-1 chemokines, and for CC-type chemokine receptors CCR5 and CCR7, indicating the presence of mature dendritic cells (DCs). Visualized at a single cell level within 4 hours after disruption of the tumor, APCs-MB induced rapid Ca(++) mobilization in MHC class II-restricted tumor idiotype (Id)-specific cloned CD4(+) T cells. In long-term assays, tumor APCs-MB induced proliferation of naive T cells from Id-specific T-cell receptor transgenic mice. The results suggest that tumor APCs-MB represent a heterogeneous cell population that includes myeloid-derived DCs of various stages of maturation. A considerable fraction (> or = 15%) of DCs is spontaneously primed with tumor-specific antigen.

Animals↗

PubMed1996

N-ras genes are not mutated in Hodgkin and Reed-Sternberg cells: results from single cell polymerase chain-reaction examinations.

Ras mutations play an important role in many human tumors. They usually occur at only three codons (12, 13 and 61) of the three ras gene family members and lead to altered proteins resulting in a constitutively activated downstream signal cascade. We have examined the N-ras gene status in Hodgkin's disease (HD). Little is known about the pathogenetic events leading to malignant phenotype in HD. Since Hodgkin and Reed-Sternberg (H and RD) cells comprise only a minority of the cellular infiltrate in HD-lymph nodes, molecular studies concerning the status of oncogenes have been difficult to perform and have yielded conflicting results. We have established a single cell PCR assay for N-ras analysis and have examined H and RS cells from 12 cases of HD by PCR amplification and direct sequencing. None of the single H and RS cells examined carried N-ras mutations at either codons 12/13 or 61. Therefore, N-ras mutations are not involved in the pathogenesis of HD.

Adult↗

PubMed1980

Frequency determination of K cells by a single cell cytotoxic assay.

This report describes a highly reproducible single cell cytotoxic assay which can measure the frequency of K cells in human peripheral blood lymphocytes. This assay is based on the ability of FcR-bearing lymphocytes to bind to antibody sensitized target cells via their FcR. Approximately 30% of lymphocytes which bound antibody sensitized target cells via their FcR. Approximately 30% of lymphocytes which bound antibody sensitized P815 cells (P815-AB) were K cells. Cytolysis of the conjugated P815-AB fulfilled all the criteria for ADCC. Furthermore, an excellent correlation was observed between the frequency of K cells and % cytotoxicity as measured by the ADCC 51Cr-release assay. The mean frequency of human k cells in peripheral blood lymphocytes from 20 volunteers was measured to be 3.2% of the total lymphocyte populations.

Antibody Formation↗

PubMed2002

Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 microM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635 +/- 27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61 +/- 13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.

Antibiotics, Antineoplastic↗

PubMedDate not supplied

Effect of antiepileptic drugs on growth of murine lymphoid tumor cells in single-cell culture.

The effect of three commonly used antiepileptic drugs (AEDs), phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA), on the growth of lymphoid tumor cells was assessed in vitro. A single-cell culture method was used to determine growth rates by direct visualization. The amount of free drug was determined by ultrafiltration to ascertain its correlation to therapeutic drug levels. VPA slowed the growth of B-myeloma (FO) and T-lymphoma (AKR-1) cells significantly within the range of therapeutic drug levels. CBZ and PHT likewise inhibited cell growth in both lineages but at two to four times the therapeutic level of free drug. CBZ was shown to have long-term effects on FO and AKR-1 cells, demonstrated by the reduced growth rates of cloned lines for 2-3 months after drug removal. Cloned sublines of myeloma cells secreting lambda light chain (J558L) treated with CBZ or PHT had a higher frequency of lambda light chain secretion loss mutations than the nontreated parent line.

Animals↗

PubMed1981

Analysis of expression of human mammary epithelial antigens in normal and malignant breast cells at the single cell level by flow cytofluorimetry.

The expression of cell-type specific antigens on the surface of human mammary epithelial cells (HME antigens) is analyzed at the single cell level by flow cytofluorimetry. Methods are described to deal with two technical difficulties that have hampered the quantitation of surface antigens on cells from primary cultures by flow cytofluorimetry, namely, cell clumping and the presence of several cell types. By staining the cells simultaneously with two different fluorescent markers: the cell surface HME antigens with fluorescein-tagged antibodies (green fluorescence) and the cell DNA with propidium iodide (red fluorescence), it was possible to determine the expression of HME antigens per unit DNA, analyze their expression on breast cells in mixed populations, and analyze their expression in different phases of the cell cycle. The HME antigens were diminished in malignant breast epithelial cells as compared to their normal counterpart.

Antigens, Surface↗