Limiting Dilution Cultures Research Articles

Activation of mir-30a-wnt/β-catenin signaling pathway upregulates cathepsin K expression to promote cementogenic differentiation of periodontal ligament stem cells

To explore the role of cathepsin K (CTSK) regulated by mir-30a-wnt/β-catenin signaling pathway in cementogenic differentiation of periodontal ligament stem cells (PDLSCs). Human PDLSCs isolated by limiting dilution culture were induced by enamel matrix protein derivative (EMD) for differentiation into cementoblast-like cells. MicroRNA chip technique was employed to screen the differentially expressed microRNAs in the cells during induced differentiation. The effect of inhibiting miR-30a on CTSK expression in the induced cells was examined using RT-PCR and Western blotting. Ceramic scaffolds coated with PDLSCs treated with EMD and transfected with the miR-30a inhibitor or a lentiviral vector for CTSK overexpression were prepared and implanted subcutaneously in nude mice, and 8 weeks later the cellular expressions of cementoblast markers CAP and CEMP-1 were detected with immunohistochemistry to verify whether CTSK participate in cementogenic differentiation of PDLSCs. The role of wnt signaling pathway in miR-30a-mediated regulation of CTSK expression was explored by examining CTSK protein expressions after blocking wnt signaling in PDLSCs. In PDLSCs with EMD-induced differentiation into cementoblast-like cells, multiple microRNAs exhibited differential expressions; and among them, miR-30a was specifically and significantly up-regulated (P < 0.05). Up-regulation of miR-30a obviously increased the expression of CTSK (P < 0.05) and promoted PDLSCs to form cementum-like tissues with high expressions of CAP and CEMP-1. The regulatory effect of miR-30a on CTSK expression was obviously attenuated after inhibiting wnt/β-catenin signaling pathway. EMD induces cementogenic differentiation of PDLSCs possibly by up-regulating the expression of miR-30a, which further activates the wnt/β-catenin signaling pathway to enhance the expression of CTSK.

TGF-\u03b2 signaling promotes tube-structure-forming growth in pancreatic duct adenocarcinoma

Tube-forming growth is an essential histological feature of pancreatic duct adenocarcinoma (PDAC) and of the pancreatic duct epithelium; nevertheless, the nature of the signals that start to form the tubular structures remains unknown. Here, we showed the clonal growth of PDAC cell lines in a three-dimensional (3D) culture experiment that modeled the clonal growth of PDAC. At the beginning of this study, we isolated the sphere- and tube-forming clones from established mouse pancreatic cancer cell lines via limiting dilution culture using collagen gel. Compared with cells in spherical structures, the cells in the formed tubes exhibited a lower CK19 expression in 3D culture and in the tumor that grew in the abdominal cavity of nude mice. Conversely, the expression of the transforming growth factor β (TGF-β)-signaling target mRNAs was higher in the formed tube vs the spherical structures, suggesting that TGF-β signaling is more active in the tube-forming process than the sphere-forming process. Treatment of sphere-forming clones with TGF-β1 induced tube-forming growth, upregulated the TGF-β-signaling target mRNAs, and yielded electron microscopic findings of a fading epithelial phenotype. In contrast, the elimination of TGF-β-signaling activation by treatment with inhibitors diminished the tube-forming growth and suppressed the expression of the TGF-β-signaling target mRNAs. Moreover, upregulation of the Fn1, Mmp2, and Snai1 mRNAs, which are hallmarks of tube-forming growth in PDAC, was demonstrated in a mouse model of carcinogenesis showing rapid progression because of the aggressive invasion of tube-forming cancer. Our study suggests that the tube-forming growth of PDAC relies on the activation of TGF-β signaling and highlights the importance of the formation of tube structures.

Genetic complexity in the replication-competent latent HIV reservoir increases with untreated infection duration in infected youth.

Timely initiation of combination antiretroviral therapy (ART) limits latent HIV reservoir size and should also limit reservoir genetic complexity. However, the relationship between these two factors remains unclear, particularly among HIV-infected youth. Retrospective analysis of replication-competent latent HIV clones serially isolated by limiting-dilution culture from resting CD4 T-cell reservoirs from ART-suppressed, young adult participants of a historic phase I therapeutic vaccine trial (PACTG/IMPAACT-P1059). Replication-competent latent HIV clones isolated from resting CD4 T cells of four perinatally and 10 nonperinatally infected young adults (average 22 versus 6 years uncontrolled infection, respectively) were sequenced in Pol and Nef. Within-host HIV sequence datasets were characterized with respect to their genetic diversity and inferred immune escape mutation burden. Although participants were comparable in terms of sociodemographic and HIV sampling characteristics (e.g. on average, a mean 17 Pol sequences were recovered at five timepoints over up to 70 weeks) and the length of ART suppression at study entry (average 3 years), replication-competent HIV reservoir size, genetic diversity, immune escape mutation burden and variant complexity were significantly higher among the perinatally infected participants who experienced longer durations of uncontrolled viremia. Nevertheless, viral sequences inferred to retain susceptibility to host cellular immune responses were detected in all participants, irrespective of uncontrolled viremia duration. HIV elimination in late-suppressed youth may be doubly challenged by larger and more genetically complex reservoirs. Strategies that integrate host and viral genetic complexity to achieve HIV remission or cure may merit consideration in such cases.

Establishment and characterization of cell clones from the Papilio cell line RIRI-PaDe-3 by a high-efficiency clonal method.

Cell cloning is of great importance in keeping particular properties of cultured cells, and interesting cells can be selected by cloning from heterogeneous cell populations. In addition, continuous cell lines usually from primary culture are prone to heterologous constitution and genetic instability, so that supplementary cloning steps are necessary for achieving a homogenous cell population. In this study, limiting dilution culture and feeder layer culture were originally used for cloning RIRI-PaDe-3 cell line, but both failed. Afterward, we designed a cloning protocol which was composed of two steps: cells in semisolid medium with seeding density in the range of 3.05 × 105-6.10 × 105cells/mL formed colonies from monodispersed cell suspensions; 40 well-dispersed colonies were removed from the suspended state by using micromanipulator system and finally scaled up. To determine whether this method can isolate cell lines possessing characteristics different from the parent population, we made an evaluation of cells monoclonal in biological characteristics. Significant differences have been found among clones isolated from the RIRI-PaDe-3 insect cell line in cell morphology, chromosome numbers, and genetic background. Thus the indicated modified semisolid medium cloning protocol was advantageous to the convenient and genuine cloning from the previously heterogeneous population.

Meso-Endothelial Bipotent Progenitors from Human Placenta Display Distinct Molecular and Cellular Identity.

SummaryThe existence of bipotential precursors for both mesenchymal and endothelial stem/progenitor cells in human postnatal life is debated. Here, we hypothesized that such progenitors are present within the human term placenta. From a heterogeneous placental single-cell suspension, a directly flow-sorted CD45−CD34+CD144+CD31Lo population uniquely differentiated into both endothelial and mesenchymal colonies in limiting dilution culture assays. Of interest, these bipotent cells were in vessel walls but not in contact with the circulation. RNA sequencing and functional analysis demonstrated that Notch signaling was a key driver for endothelial and bipotential progenitor function. In contrast, the formation of mesenchymal cells from the bipotential population was not affected by TGFβ receptor inhibition, a classical pathway for endothelial-mesenchymal transition. This study reveals a bipotent progenitor phenotype in the human placenta at the cellular and molecular levels, giving rise to endothelial and mesenchymal cells ex vivo.

Interaction of Bovine Peripheral Blood Polymorphonuclear Cells and Leptospira Species; Innate Responses in the Natural Bovine Reservoir Host.

Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and Leptospira interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia, and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs) and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2) was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of Leptospira strains with bovine PMNs did not affect Leptospira viability as measured by limiting dilution culture. This is in contrast to previously reported results of innate inflammatory activation by Leptospira in human and other animal models, or the activation and interaction of bovine PMNs with Escherichia coli and other bacterial pathogens. While it could be hypothesized that variations in innate receptor recognition, specifically variance in toll-like receptor 2, could underlie the observed reduction of activation in bovine PMNs, additional studies would be needed to explore this possibility. Reduction in neutrophil responses may help to establish nearly asymptomatic chronic Leptospira infection of cattle. This study emphasizes the importance of studying host-pathogen relationships in the appropriate species as extrapolation from other animal models may be incorrect and confounded by differences in the host responses.

Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

PurposeHuman and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs).MethodsSGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis.ResultsSG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype.ConclusionsEpithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.

Inflammatory Signaling Regulates Embryonic Hematopoietic Stem and Lymphoid Progenitor Cell Formation

Hematopoietic stem cells (HSCs) are defined as cells that can self-renew and provide robust, long-term multilineage engraftment of immunocompromised adults. Only one HSC is sufficient to reconstitute the entire hematopoietic system of an adult mouse, thus blood should theoretically be a simple organ system to generate from embryonic stem cells or induced pluripotent stem cells. Yet, despite two decades of effort, no one has been able to produce HSCs ex vivo from these sources. In the vertebrate embryo, transient waves of hematopoietic cells are produced prior to the formation of HSCs. While the role of erythrocyte production is self-evident, the necessity of myeloid production in the midgestation embryo is less clear. HSCs differentiate from hemogenic endothelium, and in mammalian embryos briefly accumulate in the aorta/gonad/mesonephros (AGM) region within intra-aortic hematopoietic clusters, although the vast majority of cluster cells are not functional HSCs. Approximately 20% of cells in hematopoietic clusters express a Ly6a-GFP transgene (Ly6a encodes Sca-1), and all lymphoid progenitors, pre-HSCs, and HSCs at embryonic day (E)11.5 are found within the Ly6a-GFP+ population. Genetic profiling of Ly6a-GFP+ and Ly6a-GFP- hematopoietic cluster cells from the AGM region revealed enrichment in gene ontology (GO) terms associated with innate immunity and inflammatory response in the Ly6a-GFP+ population, suggesting inflammatory signaling may regulate the formation of lymphoid progenitors and HSCs. To identify inflammatory cytokines that may be involved in lymphoid progenitor/HSC formation, we performed explant cultures of E9.5 embryos and added inflammatory factors known to induce Sca-1 expression in the adult mouse. Both type I and type II interferons (IFN-α4 and IFN-γ), and to a lesser extent TNFα, induced Ly6a-GFP expression in hematopoietic cells of explanted embryos. IFN-γ receptor 1 (IFN-γR1) was expressed on >80% of Ly6a-GFP+ hematopoietic cluster cells in the AGM, and Stat1 was phosphorylated in response to the addition of either IFN-γ or IFN-α4 to AGM explant cultures, indicating that embryonic hematopoietic stem and progenitor cells (HSPCs) are wired to respond to interferon stimulation. Mouse embryos deficient for IFN-g, the IFN-gR1, or the IFN-a4 receptor had decreased numbers of progenitors with lymphoid potential in the AGM region, as measured by limiting dilution culture on OP9 stromal cells, and IFN-gR1 deficient embryos had fewer HSCs, as determined by transplantation into adult mice. The role of IFN-γ signaling in HSC production in the AGM is evolutionarily conserved, as morpholino (MO) knockdown of IFN-g and its receptor likewise reduced HSPC numbers in the dorsal aorta and caudal hematopoietic tissue of zebrafish embryos, and the subsequent population of rag2 expressing lymphoid cells in the thymus. ChIP-Seq demonstrated that interferon regulatory factor 2 (IRF2) occupied genes in human fetal liver CD34+ HSPCs, with GO analysis indicating innate immunity and interferon signaling as significantly enriched processes, further demonstrating that interferon signaling is active during vertebrate development. IRF2 is a negative regulator of interferon signaling in the adult, and consistent with this, MO-knockdown of IRF2 in the zebrafish embryo enhanced the production of HSPCs, whereas knockdown of the positive effector IRF1 mimicked the phenotype of IFN-g loss. Myeloid cells have previously been shown to produce IFNs, and primitive F4/80+ macrophages were observed in close proximity to hematopoietic cluster cells in the murine AGM region. To determine if the presence of primitive myeloid cells impacted HSPC formation, macrophage specific as well as pan-myeloid knockdown was employed in zebrafish; macrophage depletion by IRF8-MO significantly reduced runx1 expression in the AGM and HSPC production. Significantly, MO-mediated inhibition of PU.1 function, which depletes the majority of myeloid cells, caused an even more striking loss, suggesting IFN-γ is not the only inflammatory mediator, nor primitive macrophages the only myeloid cells, involved in controlling embryonic HSPC production. Together these data demonstrate that myeloid-mediated inflammatory signaling is active in the developing vertebrate embryo, and in the absence of pathogenic challenge function to regulate the production of lymphoid progenitors and definitive HSCs. DisclosuresNo relevant conflicts of interest to declare.

Functional Analysis of the Subclonal Architecture of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) at a Single Cell Level

Introduction: Transforming events in B-lineage (ALL) occur primarily at the level of committed progenitor cells, but the phenotype, frequency and hierarchical organization of leukemia-initiating cells (LICs) are controversial. Pronounced clonal heterogeneity in ALL and the lack of preclinical models impede functional analysis of LICs and dissecting the clonal architecture of B-cell precursor acute lymphoblastic leukemia (BCP-ALL).Aims and methods: To identify cellular features associated with cell fate and decisions, engraftment potential, growth kinetics and resistance to therapy of BCP-ALL LICs at the clonal level, we generated multiple single-cell-derived clones from patient derived-long-term cultures (PD-LTCs) of human BCP-ALL. This in vitro ALL-LTC model allows prolonged serum-, cytokine-, and stroma-free culture of patient-derived BCP-ALL cells that are largely stable in terms of cytogenetic and immunophenotypic features for up to 6 months [Nijmeijer B et. al, Exp Hematol. 2009;37:376; Badura S et al., PLoS One. 2013; 8:e80070] Three ALL-LTCs were selected on the basis of different patterns of surface marker expression and cytogenetics, i.e. Ph+ ALL (LTCs PH and BV; common-ALL) and BCR-ABL negative ALL (LTC CR; pre B-ALL), respectively.Results: Clonogenic growth in semisolid medium ranged from 0.25% to 8% for these ALL-LTCs (CR 8%, PH 0.45% and BV 0.25%), consistent with functional diversity within the bulk as well as the immunophenotypically defined subsets. To analyze these subpopulations at the clonal level, we isolated leukemic cell subsets by cell sorting on the basis of CD20, CD34 and CD38 expression, and then generated hundreds of clones by expanding single-cell sorted subpopulations. All sorted cell fractions from all three ALL-LTCs were capable of initiating long-term growth in vitro. In limiting dilution cultures, 1% to 5% of single sorted cells derived from these cell fractions gave rise to prolonged leukemic cell growth. To evaluate the leukemia-initiating capacity of the clonal ALL subpopulations in vivo, we examined engraftment and outgrowth kinetics of different ALL clones in a xenograft model with sublethally irradiated NSG mice. Individual clones derived from different sorted subpopulations displayed pronounced differences in engraftment potential and aggressiveness based on analyses of blood, bone marrow and spleen. This ranged from rapid appearance of ALL and death within 70-80 days (clone PH 20), to complete lack of leukemic outgrowth 155 days after transplantation (clone PH clone 14). The other four clones derived from this ALL (PH) displayed intermediate engraftment and outgrowth kinetics. Notably, leukemogenic properties were not associated with a distinct surface marker profile based in CD20, CD34 and CD38 expression. A similar heterogeneity of leukemogenic potential was determined with clonal subpopulations from the LTC BV.To assess the hierarchical relationship between different clones and the association with LIC capacity, we examined the pattern of immunoglobulin VDJ-rearrangement of different clones. The Ig rearrangement patterns confirm that the clones represent distinct clonal populations derived from the original polyclonal LTC, but we observed no hierarchical relationship with respect to the biological properties of different clones. Further functional evaluation of these subclones was performed by videomicroscopy-based single cell tracking that allows analysis of individual cells and their progeny over many cell divisions. We observed concordance between cell cycle length and cell death events as determined by single cell tracking and the engraftment potential and aggressiveness of LTCs derived single cell clones in vivo.To elucidate the molecular basis for the biologic differences between the clonal ALL populations, we compared highly and poorly leukemogenic clones by SILAC based proteomics. Only 5% of 6500 proteins measured in clones differing in their leukemia-initiating potential were differentially regulated, and these proteins could be assigned to a limited number of pathways.In conclusion, comparative evaluation of clonal lymphoblast populations generated from patient-derived ALL long-term cultures by combined in vitro analysis, single cell videomicroscopy, xenotransplantation and proteomics is a promising approach to identify specific markers and therapeutic strategies that target LIC in ALL. DisclosuresWojcik:Novartis: Research Funding, Travel, Accommodation Other; SAnofi Aventis: Consultancy, Travel, Accommodation, Travel, Accommodation Other. Lang:Novartis: Research Funding, Travel, Accommodation Other. Brüggemann:Amgen Inc.: Consultancy, Research Funding.

A scalable RNA RT-PCR based assay to quantify latency disruption in HIV-1 infected cells from patients on antiretroviral therapy (VIR1P.1001)

Abstract Antiretroviral therapy suppresses HIV-1 replication but virus persists in stable reservoirs. A facile way to quantify cells in such reservoirs is HIV DNA PCR, however most of these cells are unlikely to ever produce replication-competent virus. On the other hand, viral outgrowth assays provide virologic realism, but are resource intensive. Detection of RNA by RT-PCR provides some virologic realism while allowing for high throughput. We present a method in which flow cytometry purified CD4+ T cells from patients on antiretroviral therapy are placed in limiting dilution culture for 4 days. The cells are stimulated with antibodies to CD2, CD3, and CD28 and viral replication is inhibited with efavirenz. Viral RNA is purified from the culture media using a paramagnetic nanoparticle based method that is scalable for robotics. Real time quantitative RT-PCR is performed with an HIV gag primer and probe set. This assay detects as few as 47 copies of HIV RNA induced from a single cell from the ACH-2 cell line. Using resting memory CD4+ T cells from patients on antiretroviral therapy, the frequency of induced HIV RNA production varied from 1 in 40,000 cells to fewer than 1 in 1,000,000. There was no correlation between frequency of HIV RNA producing cells and frequency of proviral DNA. This assay represents a scalable method for quantifying the frequency of HIV producing cells from patients with viral suppression and for evaluating reservoir elimination strategies for such patients.

Generation of a Platform for Identification of CLL Specific Cell Surface Proteins Targeted by Anti-Tumor Antibodies in Patient Sera After Allogeneic Hematopoietic Cell Transplantion

AbstractAbstract 1349 Background:Clinical and translational studies suggest that allogeneic hematopoietic cell transplantation (alloHCT) may induce production of anti-tumor antibodies in the recipient after transplantation. We hypothesized that this phenomenon can serve as a platform for the generation of novel therapeutic monoclonal antibodies (mAbs). An important step to this goal is the identification of targeted antigens with defined ability to support antibody induced cell death. To address this challenge, we developed a chronic lymphocytic leukemia (CLL) membrane protein display system capable of discovering cell surface proteins targeted by serum antibodies and applied this tool to post-alloHCT patient samples. Methods:Total and mRNA was purified from peripheral blood mononuclear cells from six untreated CLL patients and used to generate cDNA. The patient cDNA was pooled, cloned into the retroviral vector pBMN, and expressed in the murine T-cell line Bw 5147 (Bw-CLL-Lib). Enrichment of Bw-CLL-Lib cells displaying membrane proteins of interest was performed via fluorescence activated cell sorting. The unselected Bw-CLL-Lib pool was blocked with recombinant human Fc followed by staining with 1:200 diluted post-alloHCT patient sera and by secondary staining with pooled Alexa Fluor 647 labeled goat-anti-human-lambda and -anti-kappa light chain specific antibodies. Bw-CLL-Lib cells positively binding to serum antibodies were collected and re-grown in culture to 1×106 cells. A second round of enrichment was performed, after which the Bw-CLL-Lib cells were placed in limiting dilution culture. Individual clones were screened for serum reactivity and the retroviral inserts in reactive Bw-CLL-Lib clones were rescued via PCR and sequenced. Proteins of interest were re-expressed in Bw 5147 cells and used to confirm reactivity of the patient serum with specific cell surface proteins. Results:The Bw-CLL-Lib cell pool was screened separately with serum from ten patients with CLL post unrelated donor alloHCT. Serum from three patients enriched positively binding Bw-CLL-Lib clones. Thus far, we have successfully identified serum antibodies to a membrane proximal epitope on a therapeutically relevant CLL cell surface protein. Conclusions:Here we demonstrate a methodology for identifying targets of anti-tumor antibodies in serum from patients after alloHCT. This technique yields a high degree of successful identification of antibody reactivity with cell surface proteins in post alloHCT serum samples. When combined with post-alloHCT antibody Fab phage display (Baskar et al., Blood 114, 4494–4502, 2009) this methodology forms a complete drug and target discovery platform for the generation of tumor specific mAbs derived from alloHCT patients. Disclosures:No relevant conflicts of interest to declare.

Real-Time PCR to QuantifyLeishmania donovaniin Hamsters

Visceral leishmaniasis, a vector-borne disease caused by Leishmania donovani and Leishmania infantum , currently affects 12 million individuals in 88 countries. In the present study, a real-time PCR (rt-PCR) assay has been optimized and validated against 2 other routine methods, i.e., microscopy and limiting dilution culture assay, to estimate parasite load in the liver of infected Syrian hamsters (Mesocricetus auratus). A set of specific primers amplified a 116-bp target template of the kinetoplastid DNA of L. donovani in a SYBR® Green-based rt-PCR assay. To assess the methods, we tested 2 anti-leishmanial compounds belonging to the class of arylimidamides, DB745 (2,5-bis[2-ethoxy-4-(2-pyridylimino)aminophenyl]furan) and DB766 (2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan) for efficacy in vivo in Syrian hamsters infected with L. donovani promastigotes. Parasite load was quantified in liver by all 3 methods and was found comparable. Of the 3 methods, rt-PCR was the fastest and most convenient, sensitive, and reproducible method.

Differential sensitivity to paclitaxel-induced apoptosis and growth suppression in paclitaxel-resistant cell lines established from HEC-1 human endometrial adenocarcinoma cells

To investigate acquired paclitaxel (PTX) resistance in cancer cells, we established five monoclonal PTX-resistant cell lines from HEC-1 human endometrial adenocarcinoma cells by means of long-term PTX-exposed cultures and limiting dilution cultures. The established PTX-resistant subclones showed apparent resistance to PTX-induced DNA fragmentation but not to PTX-induced growth suppression. None of the five PTX-resistant subclones showed apparent resistance to other anticancer drugs such as cisplatin, etoposide, 5-fluorouracil, pirarubicin-HCl, 4-hydroxy-cyclophosphamide or mitomycin C. Semiquantitative flow cytometric analysis revealed no apparent differential expression of 17 molecules that were previously reported to regulate apoptosis or drug resistance, between the five PTX-resistant subclones and the parent cells. Karyotyping analysis revealed common changes in chromosomes 4 and 18 in the five PTX-resistant subclones but not in the HEC-1 parent cells. These results indicate that PTX-induced growth suppression is regulated by different mechanisms from those involved in PTX-induced apoptosis. It was concluded that these established PTX-resistant subclones can be useful models in studies related to the prevention or treatment of recurrent cancers after PTX chemotherapy.

Plasma apolipoprotein O level increased in the patients with acute coronary syndrome

Apolipoprotein (apo) O is a novel apolipoprotein that is present predominantly in high density lipoprotein (HDL). However, overexpression of apoO does not impact on plasma HDL levels or functionality in human apoA-I transgenic mice. Thus, the physiological function of apoO is not yet known. In the present study, we investigated relationships between plasma apoO levels and high-sensitive C-reactive protein (hs-CRP) levels, as well as other lipid parameters in healthy subjects (n = 111) and patients with established acute coronary syndrome (ACS) (n = 50). ApoO was measured by the sandwich dot-blot technique with recombinant apoO as a protein standard. Mean apoO level in healthy subjects was 2.21 ± 0.83 µg/ml whereas it was 4.94 ± 1.59 µg/ml in ACS patients. There were significant differences in plasma level of apoO between two groups (P < 0.001). In univariate analysis, apoO correlated significantly with lg(hsCRP) (r = 0.48, P < 0.001) in ACS patients. Notably, no significant correlation between apoO and other lipid parameters was observed. Logistic regression analysis showed that plasma apoO level was an independent predictor of ACS (OR = 5.61, 95% CI 2.16–14.60, P < 0.001). In conclusion, apoO increased in ACS patients, and may be regarded as an independent inflammatory predictor of ACS patients.

A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

BackgroundMolecular and cellular studies of Plasmodium falciparum require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported.MethodsIn vitro parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable pKa value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites.Results5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 × Dd2 genetic cross.ConclusionsThis fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.

Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells

We previously reported that transfection of Pax6 gene which regulated early events in eye development into mouse ES cells brought about their differentiation into retinal progenitors. Here, we attempted to establish cloned retinal progenitors which had ability to further differentiate into photoreceptor like cells by transfecting mouse induced pluripotent stem (iPS) cells with Pax6 gene. Undifferentiated iPS cells were transfected with Pax6 cDNA, followed by selection with G418. After limiting dilution culture, we selected cloned Pax6-transfected cells, which simultaneously expressed mRNAs of Nestin, Musashi1, Six3 and Chx10 for further characterization. We obtained totally 8 clonally expanding Pax6-transfected cells. They started to express mRNAs of Brn3b, Cone-rod homeobox (Crx), pkc, CD73, rhodopsin and the γ-subunit of rod cGMP phosphodiesterase (PDEγ). Flow cytometric analysis revealed that almost half of the cells were CD73+, a marker of photoreceptor precursors. Western blotting confirmed cytoplasmic protein expression of rhodopsin. High KCl stimulation increased free Ca influx into the cells on Ca2+ imaging. iPS cells transfected with Pax6 gene, followed by subsequent limiting dilution culture became retinal progenitors including photoreceptor like cells. The cloned cell lines may be useful for analyzing differentiation requirement of retinal progenitors.

Establishment and characterization of monoclonal 5-fluorouracil-resistant cell lines derived from human endometrial adenocarcinoma

To investigate acquired 5-fluorouracil (5FU)-resistance in cancer cells, we established four monoclonal 5FU-resistant cell lines from human endometrial adenocarcinoma cells by long-term 5FU-exposure cultures and limiting dilution cultures. The established subclones exhibited 5-25 times greater 5FU-resistance than the parent cells, and showed suppression of 5FU-induced DNA fragmentation. All four 5FU-resistant subclones were 25-125 times more resistant to SN38, 4-hydroxy-cyclophosphamide, paclitaxel and etoposide than the parent cells while none of the four subclones showed resistance to mitomycin. Two of the four subclones showed no resistance to pirarubicin and bleomycin. Although all four 5FU-resistant subclones were 5-25 times more resistant to anti-Fas IgM than the parent cells, the resistance levels to anti-Fas IgM of the individual subclones did not coincide with the strengths of their multidrug resistance. Karyotyping analyses revealed that the parent cells and the three 5FU-resistant subclones examined had normal 46XX karyotypes. These results indicate that the cell death signals induced by mitomycin, pirarubicin and bleomycin are distinctly different from the Fas-mediated apoptotic signals, that acquisition of 5FU-resistance can occur without any large chromosomal deletions or rearrangements, and that there are several possible molecular changes during the acquisition of 5FU-resistance. The established cell lines represent useful tools for investigating the mechanisms and treatments of acquired 5FU-resistance.

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

BackgroundWe have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells.ResultsSixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro.ConclusionsThis is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

Cytokine production in varicella zoster virus-stimulated limiting dilution lymphocyte cultures.

Human blood lymphocytes were stimulated with varicella zoster virus (VZV) antigen in limiting dilution cultures and the amounts of interferon-gamma (IFN-gamma) and IL-4 measured in the supernatants. The results indicate that up to 85% of proliferating cells of young adults produce IFN-gamma and up to 10% make IL-4. At limiting dilution, few if any wells were positive for both IFN-gamma and IL-4. The amount of IFN-gamma per well increased in the presence of antibody to IL-4, but anti-IFN-gamma not increase IL-4 production. The frequency of wells containing IFN-gamma was lower in subjects < 19 or > 55 years of age, and the amounts of IFN-gamma in positive wells was significantly lower in cultures of the older subjects' lymphocytes. The frequency of IL-4-making cells did not fall significantly with age. The data suggest that the age-related decline in the frequency of blood T cells which responds to VZV affects mainly the cells with a Th1-like cytokine phenotype.

Characterization of an HLA-DR4-restricted T cell clone recognizing a protein moiety of small nuclear ribonucleoproteins (UsnRNP).

In sera of patients with mixed connective tissue disease (MCTD, Sharp Syndrome) high titres of IgG autoantibodies to U1snRNP-specific proteins are found. The isolated occurrence of these autoantibodies is highly associated with the HLA-DR4 haplotype. snRNP-specific T cells are supposed to be involved in this autoantibody production. To address this question we cultured mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells using bulk or limiting dilution cultures. Secondary responses to snRNP were detected only rarely with T cell lines from two patients and two controls, and turned out to be unstable during further expansion. One T cell line derived from a healthy individual retained its snRNP reactivity upon limiting dilution cloning and could be characterized in detail. The CD4+ T cell clone recognized native snRNP particles presented by monocytes in an HLA-DR4 (B1*0401)-restricted manner. Separation of the protein and RNA moieties of snRNP particles revealed that the T cell clone responded specifically to the protein fraction, but not to RNA and diverse control antigens. Sequencing of the T cell receptor alpha and beta chain cDNAs revealed that the clone used the V alpha 14.2 and V beta 14 elements. Upon antigen-specific and mitogenic stimulation the T cell clone showed a Th1-specific cytokine pattern, and did not provide helper activity for in vitro immunoglobulin production. This study demonstrate the presence of self-reactive snRNP-specific T cells in a healthy donor. The T cell clone may not represent a helper T cell for the formation of U1snRNP-specific autoantibodies.