Non-enriched Cells Research Articles

Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers.

Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.

Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia

Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats.

A differential role for CXCR4 in the regulation of normal versus malignant breast stem cell activity

C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis resistant (AR) cells were collected from immortalised (MCF10A, 226L) and malignant (MCF7, T47D, SKBR3) breast cell lines and assessed for stem cell enrichment versus unsorted cells. AR cells had significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR normal cells demonstrated increased formation of 3D structures in Matrigel compared to unsorted cells. In vivo, SKBR3 and T47D AR cells had 7- and 130-fold enrichments for tumour formationrespectively, compared with unsorted cells. AR cells contained significantly elevated CXCR4 transcript and protein levels compared to unsorted cells. Importantly, CXCR4 mRNA was higher in stem cell-enriched CD44+/CD24- patient-derived breast cancer cells compared to non-enriched cells. CXCR4 stimulation by its ligand SDF-1 reduced MFE of the normal breast cells lines but increased the MFE in T47D and patient-derived breast cancer cells. CXCR4 inhibition by AMD3100 increased stem cell activity but reduced the self-renewal capacity of the malignant breast cell line T47D. CXCR4+ FACS sorted MCF7 cells demonstrated a significantly increased MFE compared with CXCR4- cells. This significant increase in MFE was further demonstrated in CXCR4 over-expressing MCF7 cells which also had an increase in self-renewal compared to parental cells. A greater reduction in self-renewal following CXCR4 inhibition in the CXCR4 over-expressing cells compared with parental cells was also observed. Our data establish for the first time that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. Here, we demonstrate that CXCR4 signalling specifically regulates breast cancer stem cell activities and may therefore be important in tumour formation at the sites of metastases.

Catalase enrichment using recombinant adenovirus protects αTN4-1 cells from H 2O 2

Since oxidative stress has been implicated in the development of numerous diseases including cataract, this laboratory has created and investigated the stress response of murine immortal lens epithelial cell lines (αTN4-1) conditioned to withstand lethal peroxide concentrations. Two of a group of antioxidative defense (AOD) enzymes found in such cells to have markedly enhanced activity are catalase (CAT) and GSH S-transferase α2 (GST). In order to determine if enrichment of one or both of these AODs is sufficient to protect αTN4-1 cells from lethal H 2O 2 levels, these cells were infected with adenovirus vectors capable of expressing these AODs at a high level. With this system, gene enrichment and increased enzyme activity were observed with both CAT and GST vectors. The percentage of cells infected ranged from about 50 to 90% depending on the multiplicity of infection (MOI). CAT but not GST protected the cells from H 2O 2 stress. The CAT activity was increased from 15- to 150-fold and even at the lower levels protected the cells from H 2O 2 concentrations as high as 200 μM or more (H 2O 2 levels which rapidly kill nonenriched cells). Even when only about 50% of the cell population is infected as judged by GFP infection, the entire population appeared to be protected based on cell viability. The CAT enrichment appears to protect other intracellular defense systems such as GSH from being depleted in contrast to nonenriched cell populations where GSH is rapidly exhausted. The overall results suggest that enriching the cellular CAT gene level with an appropriate recombinant viral vector may be sufficient to protect in vivo systems from peroxide stress.

Distribution of Betalain Pigments in Red Blood Cells after Consumption of Cactus Pear Fruits and Increased Resistance of the Cells to ex Vivo Induced Oxidative Hemolysis in Humans

Betalain pigments are bioavailable phytochemicals recently acknowledged as natural radical scavengers. This work, which extends previous research on the postabsorbitive fate of dietary betalains, investigated the distribution of betanin and indicaxanthin in red blood cells (RBCs) isolated from healthy volunteers (n = 8), before and during the 1-8 h interval after a cactus pear fruit meal, and the potential antioxidative activity of the pigments in these cells. A peak concentration of indicaxanthin (1.03 +/- 0.2 microM) was observed in RBCs isolated at 3 h after fruit feeding, whereas the concentration at 5 h was about half, and even smaller amounts were measured at 8 h. Indicaxanthin was not detected at 1 h. Betanin (30.0 +/- 5.2 nM) was found only in RBCs isolated at 3 h from fruit feeding. In comparison with homologous RBCs before fruit ingestion, a significant delay (P < 0.05) of the onset of an ex vivo cumene hydroperoxide (cumOOH)-induced hemolysis was evident in the RBCs isolated at 3 h (33.0 +/- 4.5 min) and at 5 h (16.0 +/- 2.0 min). Neither vitamins C and E nor GSH was modified in the RBCs at any time point. Blood collected from the same volunteers after a 12-h fasting was incubated with the purified betalains in the range of 5-25 microM, to enrich the erythrocytes with either betanin or indicaxanthin, and then the cells were exposed to cumOOH. When compared to the relevant nonenriched cells, the betalain-enriched erythrocytes exhibited an enhanced resistance to the cumOOH-induced hemolysis, which was positively correlated (r (2) = 0.99) to the amount of the incorporated compound. On a micromolar basis, betanin and indicaxanthin showed a comparable effectiveness. Taken together, these findings provide evidence that human RBCs incorporate dietary betalains and support the concept that these phytochemicals may offer antioxidative protection to the cells.

The effects of insulin-like growth factor binding protein-3 (IGFBP-3) on T47D breast cancer cells enriched for IGFBP-3 binding sites

To investigate insulin-like growth factor (IGF)-independent effects of IGF binding protein-3 (IGFBP-3), T47D cells were enriched for a population of cells that expressed binding sites for biotinylated-IGFBP-3 by panning on streptavidin-coated plate. Proliferation of cell enriched for IGFBP-3 binding sites was significantly inhibited by IGFBP-3, whereas IGFBP-3 had no significant effect on the non-enriched cell population. Enriched and non-enriched cells were equally responsive to IGF-I, TGF-beta and EGF. Conditioned medium from enriched cells had less IGFBP-3 than that from non-enriched cells. Cross-linking of biotinylated IGFBP-3 to T47D cell membranes identified complexes with Mr of 32, 80 and 100 kDa. All of these complexes were more abundant in enriched cells compared with the non-enriched cell population. These data demonstrate that despite the anti-proliferative effects of IGFBP-3 it is possible to selectively enriched for cell populations with more abundant IGFBP-3 binding sites. These enriched cells are more responsive to IGFBP-3 and secrete less of this binding protein than non-enriched cells, supporting the concept that IGFBP-3 secretion by human breast cancer cells may function as an autocrine or paracrine modulator of cell proliferation.

Evaluation of a Culture System for Enrichment of CD34+ Hematopoietic Progenitor Cells Present in Maternal Blood

Background: Clonogenic expansion of fetal cells in maternal blood is one approach to overcome the very low number of target cells available for prenatal genetic analysis. However, efficient methods of enrichment, culturing conditions and subsequent analysis of fetal cells are lacking. Optimization of this technique requires more detailed evaluation of the composition and distribution of fetal cells that cross the placenta into the maternal circulation. Previous studies by others have shown that fetal blood is rich in CD34+ progenitor cells capable of expansion in cultures supplemented with hematopoeitic growth factors. Moreover, CD34+ fetal cells have been recovered from maternal blood following enrichment. Objective: In this study, we examine the type and frequency of hematopoietic progenitor cells detected in maternal (n = 13) and non-pregnant control (n = 4) peripheral blood specimens. Methods: A methylcellulose-based culture system was used to perform colony assays on CD34+-enriched or non-enriched cells. Overall, a total of 2,249 colonies were scored for colony type among the 17 samples. To determine whether fetal cells were present and expanded, all colonies present in each of the 10 confirmed male-cases (n = 1,525 colonies) were examined either by PCR or FISH. Results: With CD34+-enriched maternal samples, we observed a significantly higher number of burst-forming unit-erythroid (BFU-E) and a reduced number of colony-forming unit-granulocyte, macrophage (CFU-GM) colonies compared to the non-enriched samples. Of 1,067 colonies analyzed by PCR for the amelogenin locus on X and Y, none were found positive for the 250-bp Y-specific sequences. Of 458 colonies tested by FISH for presence of X and Y probe signals, no XY-male cells were detected. Conclusion: We conclude that hematopoiesis is enhanced during pregnancy, but the number of fetal progenitor cells is either very low or fail to expand using the enrichment techniques and culturing conditions described in this study. Further development of methods is warranted before considering this approach for prenatal diagnosis.

Triggering of a phospholipase D pathway upon mitogenic stimulation of human peripheral blood mononuclear cells enriched with 12(S)-hydroxyicosatetraenoic acid.

The influence of 12(S)-hydroxyicosatetraenoic acid (12-HETE), that we have previously shown to decrease the proliferative response of human lymphocytes to mitogens, on diacylglycerol and phosphatidic acid (PtdOH) formation was investigated in stimulated human peripheral blood mononuclear cells (PBMC). When human PBMC were first enriched with 12-HETE, then stimulated by the mitogenic lectin concanavalin A (Con A), the production of PtdOH normally associated with Con A stimulation was markedly increased as compared with non-enriched cells. The Con-A-induced rise in the PtdOH mass was markedly decreased by 1% ethanol in 12-HETE-enriched cells, whereas it was unaffected in control cells stimulated by Con A alone. Furthermore, in [3H]arachidonic-acid-labelled cells previously enriched with 12-HETE, the formation of [3H]arachidonic-acid-labelled phosphatidylalcohol was significantly increased upon Con A stimulation, no phosphatidylalcohol being synthesized in non-enriched cells. Collectively, these results suggest that, in the presence of 12-HETE, Con A stimulates a phospholipase D activity which was not triggered by Con A alone. These data are consistent with the lack of effect of suramin, reported as a phospholipase D inhibitor, which we observed in cells stimulated by Con A alone and with the suramin-induced decrease of PtdOH mass in 12-HETE-plus-Con-A-treated cells. Moreover, 12-[3H]HETE-enriched PBMC produced a significant amount of 12-[3H]HETE-containing PtdOH (0.4% of the total PtdOH) in resting conditions. Upon mitogenic stimulation by Con A, the phorbol ester tetradecanoylphorbol acetate or the anti-CD3 mAb OKT3, this proportion was decreased to 0.1-0.2%, since the total PtdOH mass was more drastically increased than the 12-HETE-containing PtdOH species. Although present in relatively low amount in stimulated cells, 12-HETE-containing PtdOH species might have been generated in strategic compartments of the membrane bilayer so that the following events involved in the transduction of the mitogenic signal could be impaired. GC analyses have pointed out drastic variations in the fatty acid composition of PtdOH in non-enriched and in 12-HETE-enriched stimulated cells. Especially PtdOH synthesized in 12-HETE-enriched cells upon Con A stimulation contained a higher amount of saturated fatty acids and a lower amount of arachidonic acid than that formed in control cells stimulated with Con A alone. Such saturated PtdOH species with a low arachidonic acid content are very likely to have a low mitogenic potential.

Cholesterol content and sterol synthesis in human skin fibroblasts and rat aortic smooth muscle cells exposed to lipoprotein-depleted serum and high density apolipoprotein/phospholipid mixtures

Confluent cultures of human skin fibroblasts and rat aortic smooth muscle cells were shown to lose 15–27% of their cellular cholesterol upon replacement of the fetal calf serum with human high density lipoprotein (50 μg cholesterol/ ml) or lipoprotein-depleted serum at a concentration equivalent to 40% whole serum. Addition to the latter medium of high density apoliproprotein/phospholipid mixtures resulted in further enhancement of cellular cholesterol loss which was evident fey 12 h of incubation. Human skin fibroblasts that had been enriched in cholesterol by previous incubation with low density lipoprotein lost their cholesterol in the presence of a high density apolipoprotein/sphingomyelin mixture as readily as non-enriched cells. Concomitant with the marked cholesterol depletion there was a stimulation of sterol synthesis from acetate. The more pronounced loss of cellular cholesterol induced by the presence of phosphatidylcholine or sphingomyelin resulted in a greater incorporation of acetate into sterol in both smooth muscle cells and skin fibroblasts. The present findings indicate that peripheral cells, in spite of their capacity to synthesize cholesterol, depend on exogenous cholesterol for the maintenance of normal levels. It is suggested that the native cholesterol “acceptor” in the lipoproteindepleted serum is an apolipoprotein which under the experimental conditions can form a complex with phospholipids and might also represent the physiological cholesterol “acceptor” in peripheral lymph.