Bulk Cell Populations Research Articles

A subpopulation of CD24+ cells in colon cancer cell lines possess stem cell characteristics

Cancer stem cells (CSCs) have been shown to contribute to the resistance and relapse in a range of cancer types such as breast cancer and glioma. However, colon cancer stem cells remain poorly characterized. Here we reported that CD24+ subpopulation in colon cancer cell lines HCT116 and SW480 exhibited cancer stem cell-like characteristics. Using flow cytometry candidate CSCs markers were selected after initial screening of known CSCs markers from other types of cancer on colon cancer cell lines HCT116, SW480 and HT29. CD24 was expressed in the minority of bulk cell population of HCT116 and SW480 cell lines. Moreover, functional tests demonstrated that CD24+ cells exhibited enhanced chemotherapy-resistance, self-renewal and tumorigenic capacity both in vitro and in vivo, compared to CD24- subpopulations. These results suggest that CD24+ subpopulation in colon cancer cell lines HCT116 and SW480 exhibits CSCs like characteristics, and represents a nice model to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

GATA1 mutations Outside the Blast Cell Population in Transient Leukemia of Down Syndrome

Abstract 2562 Background.Transient leukemia (TL), a disorder of fetal hematopoiesis, occurs in approximately 10% of infants with Down syndrome and in one fifth of cases is followed by the development of acute myeloid leukemia. The diagnosis of TL is based on the detection of a spontaneously resolving population of blasts in the peripheral blood of newborns with DS and, more recently, the detection of clone-specific somatic mutations of the hematopoietic transcription factor GATA1. The alternative terms for this condition, transient myeloproliferative disorder (TMD) and transient abnormal myelopoiesis (TAM), reflect the observation that the peripheral blood of affected infants frequently shows an expansion of immature granulocytic precursor cells at various stages of development as well as nucleated erythroid precursors in addition to a population of blast cells. We hypothesized that the immature myeloid precursor cells observed in TL arise from the same hematopoietic cell clone that is defined by the specific GATA1 mutation found in TL blasts. Methods.Cryopreserved mononuclear cells from peripheral blood samples of three male infants with TL of Down syndrome were stained at a density of 106 cells/100ul with the following antibodies: CD45-Alexa Flour 700, CD61-PE, CD11b-PE-Cy5, CD235a-PE-Cy7, CD3-APC, CD34-APC-Cy7, CD15-Pacific Blue. In addition, aliquots of these cell populations were fixed, permeabilized and stained with for intracellular myeloperoxidase (MPO-FITC). Fractions of cells corresponding to different hematopoietic lineages were prepared by fluorescence activated cell sorting (FACSAriaII, BD Biosciences, San Jose, CA) both as bulk populations and single cells (sorted into 96-well plates). Morphology of sorted cell populations was determined by Giemsa staining. Lineage-specific hematopoietic colonies were cultured from primary mononuclear cell samples in a standard CFC assay. DNA of sorted bulk and single cell populations as well as individual lineage-specific hematopoietic colonies was used to detect GATA1 mutations in exon 2 by PCR, restriction enzyme analysis or sequencing of PCR products, or nested PCR. The location of the GATA1 gene on the X chromosome and the use of samples derived from males meant that wild type GATA1 alleles were not derived from TL cells. Results.Primary TL blood samples could be fractionated in a blast cell (CD45+CD34+and/or CD61+), erythroid (CD45-CD235a+), immature myeloid (CD45+CD15+MPO+) and lymphoid (CD45+CD3+) cell population. Cell morphology was consistent with this sorting strategy. The cell fraction designed to enrich TL blasts in all cases showed the presence of cells harboring only the mutant GATA1 allele indicating a high degree of purity of the blast fraction. As expected, the lymphoid cell populations contained in the CD3+ fractions exclusively harbored wild type alleles. Similarly, both sorted erythroid cell populations and individual erythroid colonies (Bfu-E) derived from unsorted input cells harbored only wild type GATA1 alleles. In contrast, CD15+ and/or MPO+ myeloid cell fractions consistently showed the presence of both mutant and wild type GATA1 alleles. Quantification by single cell PCR is in progress. Conclusion.Somatic GATA1 mutations which result in the translation of a N-terminally truncated mutant protein (GATA1s), are pathognomonic for TL and myeloid leukemia of Down syndrome. Published data do not determine if the cell clone harboring GATA1 mutations in TL extends beyond the population of blast cells and megakaryocytic lineage. We confirmed the presence of GATA1 mutations in TL blasts and their expected absence from lymphoid cell populations. The absence of mutations from cells of erythroid lineage suggests that the altered GATA1 function induced by mutations found in TL is incompatible with erythroid differentiation. The detection of clone-specific GATA1 mutations in myeloid cells expressing MPO was unexpected and raises the possibility that either the progenitor cells giving rise to TL blasts or the blasts themselves retain myeloid lineage differentiation potential. The finding lends support to definitions of TL and myeloid leukemia in Down syndrome that are based on the specific mutational and cellular mechanism and transcend a megakaryoblastic blast phenotype. Disclosures:No relevant conflicts of interest to declare.

Oxaliplatin-incorporated micelles eliminate both cancer stem-like and bulk cell populations in colorectal cancer

PurposeThe failure of cancer treatments is partly due to the enrichment of cancer stem-like cells (CSLCs) that are resistant to conventional chemotherapy. A novel micelle formulation of oxaliplatin (OXA) encapsulated in chitosan vesicle was developed. The authors postulate that micelle encapsulation of OXA would eliminate both CSLCs and bulk cancer cells in colorectal cancer (CRC).Experimental designIn this study, using stearic acid-g-chitosan oligosaccharide (CSO-SA) polymeric micelles as a drug-delivery system, OXA-loaded CSO-SA micelles (CSO-SA/OXA) were prepared. Intracellular uptake of CSO-SA/OXA micelles was assessed by confocal microscope. The effects of free OXA, the empty carrier, and CSO-SA/OXA micelles were tested using human CRC cell lines in vitro and in vivo.ResultsThe micelles showed excellent internalization ability that increased OXA accumulation both in CRC cells and tissues. Furthermore, CSO-SA/OXA micelles could either increase the cytotoxicity of OXA against the bulk cancer cells or reverse chemoresistance of CSLC subpopulations in vitro. Intravenous administration of CSO-SA/OXA micelles effectively suppressed the tumor growth and reduced CD133+/CD24+ cell (putative CRC CSLC markers) compared with free OXA treatment, which caused CSLC enrichment in xenograft tumors (P < 0.05).ConclusionThe results of this study indicate that CSO-SA micelle as a drug-delivery carrier is effective for eradicating CSLCs and may act as a new option for CRC therapy.

Mobilization of CD34 + Progenitor Cells in Association with Decreased Proliferation in the Bone Marrow of Macaques after Administration of the Fms-Like Tyrosine Kinase 3 Ligand

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34+ progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo. However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34+ progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34+ progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34+ cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34+ progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34+ cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34+ cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.

Prognostic Significance of Tumorigenic Cells With Mesenchymal Features in Pancreatic Adenocarcinoma

Specific populations of highly tumorigenic cells are thought to exist in many human tumors, including pancreatic adenocarcinoma. However, the clinical significance of these tumor-initiating (ie, cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma. ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays, respectively. All statistical tests were two-sided. ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens, and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months, hazard ratio of death = 1.28, 95% confidence interval = 1.02 to 1.68, P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors, and in four (67%) of these six patients, the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells, expressed genes consistent with a mesenchymal state, and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells. ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma.

Single amino acid resolution of proteolytic fragments generated in individual cells

The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single-cell capillary electrophoresis (CE) coupled with laser-induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single-cell analysis applications. Thus, it follows that by determining the relative amounts of reaction products generated in single cells using CE and by producing larger quantities of these products using bulk cell populations to identify them using MS, it is possible to determine enzyme activity profiles in single cells. In this study, the applicability of this approach was demonstrated by examining the intracellular fate of a protease substrate derived from the beta-amyloid precursor protein (beta-APP). In single live TF-1 cells, three distinct fragments were generated from the beta-APP peptide, which differed by a single uncharged amino acid. The CE measurements indicated that the proteolytic fragment profiles (i.e., the relative amounts of each fragment) were consistent from cell to cell but that they were different from those obtained in cell lysates. Furthermore, measurements obtained at the single cell level made it possible to observe a modest but statistically significant negative correlation between the total amount of beta-APP peptide loaded in cells and the fraction of peptide that remained intact. This study demonstrates how single-cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells.

Modulation of T-Cell Activation by Malignant Melanoma Initiating Cells

Highly immunogenic cancers such as malignant melanoma are capable of inexorable tumor growth despite the presence of antitumor immunity. Thus, only a restricted minority of tumorigenic malignant cells may possess the phenotypic and functional characteristics needed to modulate tumor-directed immune activation. Here we provide evidence supporting this hypothesis. Tumorigenic ABCB5(+) malignant melanoma initiating cells (MMICs) possessed the capacity to preferentially inhibit IL-2-dependent T-cell activation and to support, in a B7.2-dependent manner, induction of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Compared with melanoma bulk cell populations, ABCB5(+) MMICs displayed lower levels of MHC class I, aberrant positivity for MHC class II, and lower expression levels of the melanoma-associated antigens MART-1, ML-IAP, NY-ESO-1, and MAGE-A. Additionally, these tumorigenic ABCB5(+) subpopulations preferentially expressed the costimulatory molecules B7.2 and PD-1, both in established melanoma xenografts and in clinical tumor specimens. In immune activation assays, MMICs inhibited mitogen-dependent human peripheral blood mononuclear cell (PBMC) proliferation and IL-2 production more efficiently than ABCB5(-) melanoma cell populations. Moreover, coculture with ABCB5(+) MMICs increased the abundance of Tregs, in a B7.2 signaling-dependent manner, along with IL-10 production by mitogen-activated PBMCs. Consistent with these findings, MMICs also preferentially inhibited IL-2 production and induced IL-10 secretion by cocultured patient-derived, syngeneic PBMCs. Our findings identify novel T-cell modulatory functions of ABCB5(+) melanoma subpopulations and suggest specific roles for these MMICs in the evasion of antitumor immunity and in cancer immunotherapeutic resistance.

Measurement of Human Immunodeficiency Virus Type 1 Preintegration Transcription by Using Rev-Dependent Rev-CEM Cells Reveals a Sizable Transcribing DNA Population Comparable to That from Proviral Templates

Preintegration transcription is an early process in human immunodeficiency virus type 1 infection and has been suggested to occur at a low level. The templates have also been suggested to represent a small population of nonintegrated viral DNA, particularly the two-long-terminal-repeat (2-LTR) circles. However, these determinations were made by either using PCR amplification of viral transcripts in bulk cell populations or utilizing the LTR-driving reporter cells that measure the synthesis of Tat. The intrinsic leakiness of LTR often makes the measurement of low-level viral transcription inaccurate. Since preintegration transcription also generates Rev, to eliminate the nonspecificity associated with the use of LTR alone we have developed a novel Rev-dependent indicator cell, Rev-CEM, to measure preintegration transcription based on the amount of Rev generated. In this report, using Rev-CEM cells, we demonstrate that preintegration transcription occurs on a much larger scale than expected. The transcribing population derived from nonintegrated viral DNA was comparable (at approximately 70%) to that derived from provirus in a productive viral replication cycle. Nevertheless, each nonintegrated viral DNA template exhibited a significant reduction in the level of transcriptional activity in the absence of integration. We also performed flow cytometry sorting of infected cells to identify viral templates. Surprisingly, our results suggest that the majority of 2-LTR circles are not active in directing transcription. It is likely that the nonintegrated templates are from the predominant DNA species, such as the full-length, linear DNA. Our results also suggest that a nonintegrating lentiviral vector can be as effective as an integrating vector in directing gene expression in nondividing cells, with the proper choice of an internal promoter.

Tumor-Initiating Cells of HER2-Positive Carcinoma Cell Lines Express the Highest Oncoprotein Levels and Are Sensitive to Trastuzumab

The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer. Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants. Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.

Effects of Steroid Treatment on Childhood ALL Stem Cells.

Several lines of evidence indicate a central role for stem cells in the pathogenesis of human leukaemias and exemplify the need to develop strategies that target this sub-population of cells. It is proposed that these cells may exhibit different chemo-sensitivity and consequently may be resistant to drug regimens designed to kill the bulk leukaemia population. Inherently resistant leukaemia stem cells may contribute to subsequent disease relapse. Clearly, there is a need to assess the relative efficacy of therapeutic agents on the sub-populations of cells in addition to the bulk leukaemia. We have previously demonstrated that the sub-population of childhood acute lymphoblastic leukaemia (ALL) cells, capable of serially engrafting NOD/SCID mice, have a CD34+/CD19− or CD34+/CD7− phenotype in B cell precursor (BCP) ALL and T-ALL, respectively. In this investigation we have compared the efficacy of a current glucocorticoid therapeutic agent on these putative ALL stem cells with their effects on the bulk leukaemia population. The effect of dexamethasone (dex), a key component of the treatment of childhood ALL, on primary ALL cells from 13 paediatric cases was examined. Unsorted ALL cells were co-cultured with and without dex for 48 hours. Subsequently, cell viability and apoptosis were evaluated by flow cytometry using propidium iodide and annexin V staining, with Flow-Count fluorospheres to directly determine absolute cell counts. Primary cells from 11 patients with BCP ALL were sorted for expression of CD34/CD19 and cells from 2 T-ALL cases were sorted for expression of CD34/CD7. The unsorted cells and the sorted sub-fractions were co-cultured with increasing concentrations of dex (0.05 to 500 μM) to compare the relative chemosensitivity of the bulk and putative leukaemia stem cell populations. The unsorted leukaemia populations were completely refractory to dex with no significant difference in the levels of apoptosis observed or the absolute number of viable cells in the treated samples and in the untreated controls. Interestingly, when the sorted populations were assessed, an increase in the absolute numbers of viable CD34+/CD19− (1.2–9.7 fold, P<0.02) and CD34+/CD7− (2.6–5 fold, P<0.04) leukaemia cells were observed even at the highest steroid dose, compared to the respective untreated sub-fractions. The other leukaemic sub-fractions did not show a significant increase in the number of viable cells following dex exposure. These data show that 10 out of 11 drug treated primary leukaemia cells were resistant to dex. The putative CD34+/CD19− BCP ALL cells and CD34+/CD7− T-ALL cells showed a significantly enhanced proliferative potential when exposed to the drug, suggesting that it is these cells that may be responsible for disease relapse.

Effect of low intensity laser interaction with human skin fibroblast cells using fiber-optic nano-probes

Over the past forty years, many efforts have been devoted to study low power laser light interactions with biological systems. Some of the investigations were performed in-vitro, on bulk cell populations. Our present work was undertaken to apply specially engineered fiber-optic based nano-probes for the precise delivery of laser light on to a single cell and to observe production of low power laser light induced reactive oxygen species (ROS). A normal human skin fibroblast (NHF) cell line was utilized in this investigation and the cells were irradiated under two different schemes of exposure: (1) an entire NHF cell population within a Petri dish using a fan beam methodology, and (2) through the precise delivery of laser energy on to a single NHF cell using fiber-optic nano-probe. Photobiostimulative studies were conducted through variation of laser intensity, exposure time, and the energy dose of exposure. Laser irradiation induced enhancement in the rate of cell proliferation was observed to be dependent on laser exposure parameters and the method of laser delivery. The total energy dose (fluence) had a greater influence on the enhancement in the rate of cellular proliferation than compared to laser intensity. The enhancement in the growth rate was observed to have a finite life-time of several days after the initial laser exposure. Fluorescent life-time imaging of ROS was performed during the nano-based single cell exposure method. The kinetics of ROS generation was found to depend strongly on the laser fluence and not on the laser intensity.

Quantitative Real Time PCR Analysis of MDR1 Transduced Rhesus Macaque Hematopoietic Stem Cells Shows No Indication for Proliferative Advantage after Long-Term Follow-Up.

Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking.Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR.Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively.Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: No Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model.

It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking.One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites.In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive.By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector.We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

634. The Role of Ink4a-Arf Locus in Control of Hematopoietic Stem Cell Proliferation

The mechanisms that control hematopoietic stem cell (HSC) proliferation and self-renewal are incompletely understood, but are relevant for developing gene therapy strategies that target HSCs. Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of the Ink4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis. The Ink4a-Arf locus encodes two tumor suppressor proteins, p16Ink4a and p19Arf. Induced transcription of these genes results in cell cycle arrest in several model systems. Based on these facts, we have tested the hypothesis that these Ink4a-Arf gene products may play a role in limiting HSC self renewal during conditions of enhanced proliferation. We first tested whether p19Arf was induced in murine bone marrow cells that were cultured for 10–12 days in IL3, IL6, SCF, and 15% fetal calf serum, hypothesizing that the well described loss of HSCs under these conditions could be due to induction of p19Arf. Using cells from mice that had a GFP reporter gene inserted into the first exon of the p19Arf gene, we found that the p19Arf/GFP allele was significantly induced in the bulk cell population after culture, but not expressed in lineage negative, c-kit+, Sca1+ cells derived from uncultured bone marrow. Competitive repopulation studies were then performed using bone marrow from either p19Arf null mice, or from mice with a homozygous deletion that disrupt both p19Arf and p16Ink4a (Ink4a-Arf–/–). In both cases, there were no defects in repopulating activity when using either freshly isolated bone marrow cells, or cells grown in our 12 day cultures. These data indicate that the loss of HSCs with in vitro culture does not require either p19Arf or p16Ink4a. We next asked whether these genes play a role in the self-renewal of HSCs in vivo using serial bone marrow transplantation assay. Bone marrow cells from either strain or from wildtype controls were serially transplanted into lethally irradiated recipients in doses ranging from 1×105 to 1×106 cells per mouse. We are currently following the fourth group of transplant recipients. In the mice transplanted with bone marrow cells from Ink4a-Arf–/– donors, a survival advantage was noted relative to mice transplanted with strain-matched, wildtype controls. For instance, in secondary recipients receiving 1×105 bone marrow cells, 100% survival was noted in mice transplanted with Ink4a-Arf–/– cells after 10 weeks compared to 20% survival in mice receiving the same dose of wildtype cells. A similar survival advantage was also noted in groups given the lowest cell doses within the 3rd and 4th transplants. In parallel experiments using p19Arf null mice, there was no difference in survival relative to wildtype controls. Measurement of blood counts from recipients in all cohorts clearly showed that the deaths were due to hematopoietic failure. These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16Ink4a is necessary. Because animals transplanted with Ink4a-Arf–/– cells do die with hematopoietic failure, albeit later than wildtype controls, we conclude that mechanisms not associated with the Ink4a genes also play a role in limiting HSC self-renewal in vivo.

A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-microm widths were readily traversed by the 8 microm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-microm channels; some that passed through the 4-microm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-microm channels, trophozoites mimicked "pitting," a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-microm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-microm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.

Antigen loss variants of a murine renal cell carcinoma: implications for tumor vaccination.

Vaccination with tumour cells genetically modified to support induction of an immune response either by production of cytokines or expression of co-stimulatory molecules provides a promising therapeutic approach. We have evaluated the efficiency of tumour vaccination using RENCA cells, a renal cell carcinoma of the BALB/c strain, which were stably transfected with MHC class II, B7.1 or both. Tumour growth after vaccination with MHC class II and/or B7.1 transfected RENCA cells was extremely variable, with protection close to 100% after vaccination with some clones and no effect of vaccination with others. To unravel the underlying mechanism, untransfected RENCA cells were cloned, and individual clones were tested for immunogenicity; that cloned RENCA cells varied considerably in immunogenicity. Whereas all clones displayed comparable growth rates in nude mice, some grew very slowly in immunocompetent syngenetic hosts. Vaccination with rapidly growing clones was ineffective and, importantly, this feature remained unaltered by vaccination with MHC class II and/or B7.1 transfected clones. Instead, 8 of 10 mice rejected the parental line after immunisation with a pool of MHC class II and B7.1 transfected clones. Finally, by cloning RENCA cells, we obtained one highly immunogenic clone (P2). Vaccination with this clone led to an individual-specific response, which indicates that during the cloning procedure a new strongly immunogenic entity must have arisen. Taken together, our results indicate that vaccination with MHC II and/or B7.1 transfected tumour cells induces an efficient immune response, but only if the tumour is weakly immunogenic. Since tumours may be composed of clones displaying different antigenicities, it is mandatory to use bulk cell populations for transfection and vaccination.

Host-cell reactivation of reporter genes introduced into cells by adenovirus as a convenient way to measure cellular DNA repair

In order to conveniently measure cellular DNA repair in immortalized and primary human cells we have combined the features of high cellular infectivity of adenovirus (Ad) with that of host-cell reactivation (HCR) of ultraviolet light (UV)-damaged reporter genes. We show that Ads having either the cat (chloramphenicol acetyltransferase) or seap (secreted alkaline phosphatase) reporter gene under control of a strong constitutive promoter can be used to measure relative levels of DNA repair by HCR. Most importantly, the SEAP assay allows for a convenient, inexpensive, and sensitive colorimetric microtiter assay. Only a few steps are involved and it is possible to process many samples simultaneously in a relatively short time, which is not as easily done with other reporter gene assays. Furthermore, we show that co-infection of UV-damaged SEAP Ad with an Ad carrying a prokaryotic repair gene significantly increased the HCR levels in xeroderma pigmentosum cells. The Ad gene delivery system, and the SEAP assay in particular, should simplify existing HCR assays considerably. By using non-lytic Ad as a vehicle it should be possible to quantitatively introduce normal or dominant negative mutant DNA repair genes into bulk cell populations for DNA repair studies.

Changes in Friend murine erythroleukaemia cell membranes during induced differentiation determined by electrorotation

We used electrorotation measurements to investigate alterations in the plasma membranes of DS19 murine erythroleukaemia cells that accompanied erythropoietic differentiation induced by hexamethylene bisacetamide (HMBA). Following 3 days of HMBA treatment, the mean cell membrane specific capacitance determined from electrorotation spectra of individual, viable cells at physiological tonicity (300 mosmol/kg) fell from 1.74 to 1.53 μF/cm 2, in agreement with trends observed earlier by dielectrophoretic measurements on bulk cell populations. Scanning and transmission electron microscopy revealed that the relatively high values found for cell membrane capacitance ( > 1 μ F/cm 2 ) reflected the large area of plasma membrane associated with complex surface morphology including numerous microvilli. Furthermore, it demonstrated that the fall in membrane capacitance during HMBA treatment correlated with a reduction in the density of these complex surface features. Differences in the mechanical characteristics of the cell membranes of untreated and treated cells were then examined by exposing cells to osmotic stress. The intricacy of membrane morphology intensified with increasing osmolality of the suspending medium and this was reflected in higher specific capacitance values. When the osmolality was increased from 210 to 450 mosmol/kg, the mean membrane capacitance of untreated DS19 cells changed from 1.58 to 2.05 μF/cm 2 while that for HMBA-treated cells changed from 1.47 to 1.72 μF/cm 2, a significantly smaller response. This demonstrated that cells exposed to 72 h of differentiation treatment had an enhanced mechanical resilience as compared with their untreated counterparts, evidencing the early stages of the development of the membrane skeleton which becomes fully developed in mature erythrocytes. Our findings demonstrate the value of electrorotation measurements as a method for the non-invasive characterisation of viable leukaemic cells and their responses to stimuli and show that the membrane capacitance values so derived reflect membrane morphology.

Analysis of cell structural and functional diversity by combination of micromanipulation and microfluorimetry

Fluorescent molecules are widely used to study quantitative cell properties, such as density of different antigenic markers or membrane responses to various stimuli. In most cases, studies are done on bulk cell populations with a spectrofluorimeter or at the single cell level with a cytofluorograph. However, only microspectrofluorimetric techniques allow continuous recording of dynamic events undergone by individual cells. The aim of the present report was twofold: first, to describe a methodology easily accessible to cell biologists that allows simultaneous manipulation of single cells and measurements of their fluorescence properties; and second, through this methodology to study quantitative aspects of cell structure and function such as binding of a fluorescein-labeled lectin, transfer of fluorescent molecules between labeled and unlabeled cells brought in close contact, or fluorescence response of individual cells stimulated after being loaded with a potential-sensitive dye. We conclude that the understanding of many aspects of cell structure and behavior requires that individual cells be studied under dynamic conditions and for prolonged periods of time.