LSC Cells Research Articles

Changes of the biophysical properties of voltage-gated Na+ currents during maturation of the sodium-taste cells in rat fungiform papillae.

Taste cells are a heterogeneous population of sensory receptors that undergo continuous turnover. Different chemo-sensitive cell lines rely on action potentials to release the neurotransmitter onto nerve endings. The electrical excitability is due to the presence of a tetrodotoxin-sensitive, voltage-gated sodium current (INa ) similar to that found in neurons. Since the biophysical properties of neuronal INa change during development, we wondered whether the same also occurred in taste cells. Here, we used the patch-clamp recording technique to study INa in salt-sensing cells (sodium cells) of rat fungiform papillae. We identified these cells by exploiting the known blocking effect of amiloride on ENaC, the sodium (salt) receptor. Based on the amplitude of INa , which is known to increase during development, we subdivided sodium cells into two groups: cells with small sodium current (SSC cells; INa <1nA) and cells with large sodium current (LSC cells; INa >1nA). We found that: the voltage dependence of activation and inactivation significantly differed between these subsets; a slowly inactivating sodium current was more prominent in LSC cells; membrane capacitance in SSC cells was larger than in LSC cells. mRNA expression analysis of the α-subunits of voltage-gated sodium channels in fungiform taste buds supported the functional data. Lucifer Yellow labelling of recorded cells revealed that our electrophysiological criterion for distinguishing two broad groups of taste cells was in good agreement with morphological observations for cell maturity. Thus, all these findings are consistent with developmental changes in the voltage-dependent properties of sodium-taste cells. KEY POINTS: Taste cells are sensory receptors that undergo continuous turnover while they detect food chemicals and communicate with afferent nerve fibres. The voltage-gated sodium current (INa ) is a key ion current for generating action potentials in fully differentiated and chemo-sensitive taste cells, which use electrical signalling to release neurotransmitters. Here we show that, during the maturation of rat taste cells involved in salt detection (sodium cells), the biophysical properties of INa , such as voltage dependence of activation and inactivation, change significantly. Our results help reveal how taste cells gain electrical excitability during turnover, a property critical to their operation as chemical detectors that relay sensory information to nerve fibres.

Photon upconversion-assisted dual-band luminescence solar concentrators coupled with perovskite solar cells for highly efficient semi-transparent photovoltaic systems.

A luminescent solar concentrator-based photovoltaic system (LSC-PVs) is highly transparent because it harvests solar light via the LSC, a transparent panel containing only fluorophores, and is, therefore, promising as a PV window. However, for the practical use of LSC-PV, achieving high efficiency remains a challenge. Here, we demonstrate an LSC-PV, which is based on the combination of an upconversion (UC)-assisted dual band harvesting LSC and perovskite solar cells (PSCs). We prepare a dual LSC panel consisting of a downshift (DS) LSC that absorbs violet light and an LSC that upconverts the red light. We apply a highly efficient mixed halide PSC with an efficiency of 17.22%. We control the thickness of the LSC panel as well as the dye concentration to maximize the emission from dual LSCs. The dual LSCs coupled with a PSC exhibit a high average-visible-transmittance of 82% and achieve a maximum efficiency of 7.53% at 1 sun (AM 1.5G) illumination. The dual LSC-PSC exhibits a constant efficiency even under oblique solar light illumination and a stable operation with an efficiency retention of 80%.

Performance and degradation of an SOEC stack with different cell components

High temperature water electrolysis with solid oxide electrolysis cells (SOECs) is a promising method for hydrogen production. In order to study the performance and degradation behavior of cells with different cell components in electrolysis mode, a four-cell stack was assembled that used JÜLICH's F10 design with two types of air electrodes based on La0.6Sr0.4CoO3−δ (LSC) and La0.6Sr0.4Co0.2Fe0.8O3−δ (LSCF). A PVD-prepared GDC layer was applied to LSCF cells while a screen-printing GDC layer was used in the LSC cells. The performance of the stack was first characterized with IV-curve measurements in both SOFC and SOEC modes within the temperature range of 700–800 °C. The durability of the stack was investigated by conducting a long-term stationary electrolysis operation with a constant current density of −0.5 Acm−2 and steam conversion rate of 50% at 800 °C. Electrochemical Impedance Spectroscopy (EIS) was used to study the electrochemical performance of the stack, as well as the degradation behavior during long-term electrolysis operation. The method “Distribution of Relaxation Times” (DRT) was applied for the further analysis of the EIS data, and the DRT results have successfully supported the stack degradation analysis.

Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies

BackgroundResident stem and progenitor cells have been identified in the lung over the last decade, but isolation and culture of these cells remains a challenge. Thus, although these lung stem and progenitor cells provide an ideal source for stem-cell based therapy, mesenchymal stem cells (MSCs) remain the most popular cell therapy product for the treatment of lung diseases. Surgical lung biopsies can be the tissue source but such procedures carry a high risk of mortality.MethodsIn this study we demonstrate that therapeutic lung cells, termed “lung spheroid cells” (LSCs) can be generated from minimally invasive transbronchial lung biopsies using a three-dimensional culture technique. The cells were then characterized by flow cytometry and immunohistochemistry. Angiogenic potential was tested by in-vitro HUVEC tube formation assay. In-vivo bio- distribution of LSCs was examined in athymic nude mice after intravenous delivery.ResultsFrom one lung biopsy, we are able to derive >50 million LSC cells at Passage 2. These cells were characterized by flow cytometry and immunohistochemistry and were shown to represent a mixture of lung stem cells and supporting cells. When introduced systemically into nude mice, LSCs were retained primarily in the lungs for up to 21 days.ConclusionHere, for the first time, we demonstrated that direct culture and expansion of human lung progenitor cells from pulmonary tissues, acquired through a minimally invasive biopsy, is possible and straightforward with a three-dimensional culture technique. These cells could be utilized in long-term expansion of lung progenitor cells and as part of the development of cell-based therapies for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

Comparison of performance and degradation of large-scale solid oxide electrolysis cells in stack with different composite air electrodes

Three hydrogen electrode-supported, large-scale solid oxide electrolysis cells (SOECs) with different composite air electrodes, namely LSM–YSZ, LSC–GDC, and LSCF–GDC (LSM, LSC, and LSCF cells, respectively), were compared for performance and degradation in the same three cell stack with an H2O/H2 ratio of 90/10. The initial SOEC performance increased in the following order: LSM, LSCF, and LSC. The three cells were operated at a constant current density of 0.50 A cm−2 for 640 h during hydrogen production at 750 °C. After the operation, the 10 cm × 10 cm large-scale single cell was divided equally into four subparts from the two diagonal parts with the steam or air inlet area and steam or air outlet area for post-mortem analysis. Delamination mainly occurred in the steam and air inlet areas of the LSM and LSC cells, with more severe delamination in the steam inlet area than that in the air inlet area. Agglomeration of Ni in the Ni–YSZ hydrogen electrode was observed in the LSM and LSCF cells, whereas honeycomb Ni was observed in the steam and air inlet areas of the LSC cell.

Abstract B07: s-SHIP promoter expression identifies a subset of murine basal prostate cells as neonatal stem cells

Abstract The ability to isolate epithelial stem cells of the adult prostate (PrSCs) is crucial in terms of understanding their biology and purported role in tumor initiation. Previous isolations of PrSCs were based on subdividing total prostate epithelial cells by flow cytometry using antibodies to cell surface markers. In this study we have used a novel approach shown previously to successfully identify mammary stem cells (Bai &amp; Rohrschneider, 2010, Genes Dev. 24:1882). This method employs the s-SHIP (stem-SH2-containing 5'-Inositol Phosphatase) promoter to tag presumptive stem cells with GFP in a transgenic mouse model, called Tg11.5kb-GFP. Here we show that in Tg11.5kb-GFP mice, epithelial GFP+ (s-SHIP-expressing) cells are present transiently during early mouse prostate development: during the first week after birth, GFP expression was present in the growing buds in 1-3 % of total prostate cells, then decreased during the second week as the solid epithelial cords canalize. During this process, luminal and basal cells differenciate, and GFP+ cells localized in the basal cell layer of the epithelium. GFP expression was then totally turned off after 3 weeks. We therefore focused our study on epithelial GFP+ cells present between day 5 and day 7 after birth in order to determine if this population represents a population of putative stem cells. Cell surface marker analyses demonstrate an antigenic profile characteristic of basal epithelial prostate cells: lineage- CD24+ Sca-1+ CD49f+ (LSC). Interestingly, other research groups have shown that LSC population contains multipotent stem cells of the adult prostate; thus, the GFP+ cell population represent a subpopulation (~15%) of this basal/stem LSC population in the neonatal prostate. Moreover, in this LSC population, GFP+ cells showed ~20 fold more in vitro sphere-forming activity than the GFP- cells. Finally transplantation assays of 103 GFP+ cells in combination with urogenital sinus mesechymal cells demonstrated the potential of this GFP+ cell population to reconstitute in vivo prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for basal epithelial prostate cells exhibiting stem/progenitor cell properties. We are now performing transcriptional analysis of GFP+ and GFP- basal LSC cells in order to define gene sets that distinguish a basal cell profile from a tissue stem cell profile. Preliminary results showed interesting candidate genes including transcription factors. We anticipate that these results will lead to a better definition of adult PrSCs and will help to elucidate prostate cancer etiology. Citation Format: Guillaume Brocqueville, Hélène Bauderlique Le Roy, Larry R. Rohrschneider, Roland P. Bourette. s-SHIP promoter expression identifies a subset of murine basal prostate cells as neonatal stem cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B07.

Analysis of Wnt and Hedgehog Pathways Regulating Protein Kinases CK1 and CK2 in Acute Myeloid Leukemia Cells and Stem Cells: Correlation with the Expression of Wnt and Hedgehog Targets and Biological and Clinical Features.

AbstractAbstract 2501 Background.Leukemia stem cells (LSC) constitute the reservoir of acute and chronic leukemias, from which the relapse of the disease takes origin. The Wnt/βcatenin and the Hedgehog (Hh) signaling pathways regulate the balance between self-renewal and commitment of both normal hematopoietic stem cells (HSC) and LSC. Serine-Threonine protein kinases CK1 and CK2 phosphorylate members of Wnt and Hh and are therefore potential regulators of HSC and LSC biology; however, their function in HSC and LSC is unknown. Purpose.In the present work we have investigated the mRNA and protein levels of CK1 and CK2 in acute myeloid leukemia (AML) blasts and LSC isolated from patients. We have also integrated the expression data of CK1 and CK2 with the expression data of Wnt and Hh signaling pathways gene targets and with biological and clinical parameters of a group of AML patients and cell lines. We also tested the effects on the growth of AML blasts of CK1 and CK2 small chemical inhibitors. The aim of this study was to gain insights into the function of these pivotal protein kinases and on their expression levels relative to biological, clinical and prognostic variables that dictate the outcome of AML patients. Methods.mRNA was extracted from HSC and FACS-sorted LSC (as defined as Lineage- CD34+ CD38- CD123+ CD90+); proteins were obtained from the bulk of blasts from fifteen AML patients. Quantitative RT-PCR was employed to assess mRNA levels of: CK1α, CK1γ, CK2α (catalytic) and CK2β (regulatory subunit); Lef1, FoxO1, FoxO3, CyclinD1 (Wnt targets); Ptch, Gli1, Gli2, Gli3, Bmi1 (Hh targets); western blot analysis was performed to determine CK1α, CK2α, CK2β, p53, phospho-Ser529 NF-κB and phospho-Ser13 Cdc37 (two direct CK2 target sites) protein levels. Normal CD34+ bone marrow cells were used as controls. CK2 chemical inhibitors (CX4945 and tTBB) were assayed on LSC and AML cells. Results.CK1α, CK2α and CK2β mRNA and proteins were found overexpressed in LSC and in AML blasts. CK1γ mRNA levels were barely detectable. Notably, some AML cases displayed low levels of CK2β but high levels of CK2α, indicating a possible CK2β-independent function of CK2α. FoxO1, FoxO3 mRNAs were found upregulated while Lef1 and CyclinD1 ones were found slightly and Axin2 one strongly diminished, respectively. Ptch, Smo, Gli1, Gli2 and Gli3 mRNAs were unchanged or reduced, while we observed a strong upregulation of that of Bmi1. A correlation between the overexpression of CK1α and CK2 and high-risk cytogenetic groups was observed. Also, in most of CK2α high-expressing cases, TP53 was found downregulated, while phospho-Ser529 p65 upregulated. Importantly, inhibition of CK2 with selective compounds caused AML cell and LSC growth arrest, a restoration of TP53 and a downregulation of phospho-Ser529-NF-kB p65 and phospho-Ser13 Cdc37 protein levels. Conclusions.The present study is the first to report on the expression of CK1 and CK2 kinases in normal HSC and LSC. CK1 (α) and CK2 (α and β) mRNA and protein levels were higher in LSC (especially from high risk groups) than in HSC. CK1γ mRNA levels were low. Wnt and Hh pathways genes were differentially upregulated pointing to a gene specific LSC-associated function. Moreover, our data with CK2 inhibitors suggest that CK2 could be a suitable therapeutic target to eradicate residual AML LSCs. Future research will assess more in depth the in vivo functional role of CK1 and CK2 in HSC and LSC. Disclosures:No relevant conflicts of interest to declare.

La0.6Sr0.4CoO3-δ Thin Films Prepared by Pulsed Laser Deposition as Cathodes for Micro-Solid Oxide Fuel Cells

Lanthanum-strontium-cobalt oxide (LSC) thin films fabricated by pulsed laser deposition (PLD) can be used as electrodes for micro-solid oxide fuel cells (micro-SOFCs) in the intermediate temperature range of 450-550 °C. The LSC thin films deposited under a high oxygen chamber pressure are nanocrystalline after thermal treatment at 550 °C and have a nanoporous microstructure. The buckling pattern of the free-standing yttria-stabilised-zirconia (YSZ) membrane influences the survival rate of the free-standing LSC|YSZ|LSC cells. Electrochemical impedance spectroscopy is used to investigate the properties of the LSC/YSZ interface in free-standing quasi-symmetrical LSC|YSZ|LSC membranes. LSC electrodes with a thickness of ~150 nm had an area specific resistance (ASR) of 3.7 Ωcm2 at 550 °C.

ROCK Inhibitor Y-27632 Suppresses Dissociation-Induced Apoptosis of Murine Prostate Stem/Progenitor Cells and Increases Their Cloning Efficiency

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin−Sca-1+CD49fhigh (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells.

Specificity of Markers of Leukemia Initiating Cells with a New Multiparameter Flow Cytometry Based Appraoch; Impact for Prognostic and Therapeutic Applications

AbstractAbstract 1834Leukemia stem cells (LSC) are proposed to underly relapse of AML. In order to develop methods to specifically detect LSCs and to design LSC specific therapies, discrimination between LSC and normal hematopoietic stem cells (HSC) is a prerequisite. HSC and LSC may be either CD34-positive (CD34+CD38-) or CD34-negative. The latter may be contained in stem cell compartments defined by functional parameters: Hoechst 33342 efflux (side population, SP) and ALDH activity. To discriminate between LSCs and HSCs aberrant cell surface markers have been described. Additional flowcytometric parameters (scatter, and level of expression of CD34/CD45) allow further discrimination (Terwijn Blood 2009; 114:165). We applied this new approach to compare the specificity of expression of the LSC markers CD123, CLL-1, CD96, CD44, CD47, and CD33. We studied LSCs in diagnosis AML, HSCs in normal bone marrow (NBM) and regenerating BM (RBM) and HSCs present at diagnosis in the AML BM itself. The Table shows the results for the CD34+CD38-cells. Large differences were found for LSCs: CD44>CD33>CD47>CD123≥CLL-1 and CD96 (column 2; median and ranges shown in all columns). Expression on HSCs in NBM (column 4) shows the lack of LSC specificity of several of these markers: CD44>CD47>CD33>CD123>CD96 and CLL-1. A similar result was found for RBM for the three markers studied: CD33>CD123>CLL-1 (column 5). Expression in HSCs present in the AML BM itself was: CD33>CD123>CLL-1 and CD96 (column 3). CD44 and CD47 could not be properly analyzed due to extensive overlap of LSC and HSC (column 3). In order to determine whether expression on HSC would negatively impact diagnostic and targeted therapy, we defined an in vitro “therapeutic window” (TW=expr. in LSC/expr. in HSC) in columns 6–8: each individual value for LSC for a particular marker was divided by the median value of HSC in NBM (column 7), RBM (column 8) or divided by each corresponding HSC value in the AML BM itself (column 6). Overall, TW was slightly higher for CLL-1 than for CD96, CD123 and CD33, irrespective HSC source (CD33 had a high TW in diagnosis BM). The TW for CD44 and CD47 was low (column 7) and not analyzable in the AML BM itself. Experiments were repeated for SP cells: TW for CLL-1 (for the 3 HSC sources, resp) were 2.1, 3.8, and 3.8; for CD123: 2.0, 1.5, and 2.5 and for CD33: 1.9, 1.3, and 1.6. CLL-1 was thus slightly superior for SPs. The overall conclusions from this project are that, due to a high specificity, CD123, CLL-1, CD96 and CD33, but not CD44 and CD47 are suitable for diagnostic purposes. Whether or not a similar conclusion can be drawn for therapeutic applications remains to be investigated. Large differences exist in expression of the different antigens for most individual AML cases; for therapeutic applications, for each AML case, the optimal antigen or, most likely, combinations of antigens will be needed. The most important conclusion is that, by using our approach, it is now possible to discriminate between concomitantly present LSCs and HSCs. This approach may have high impact for both prognostic purposes (e.g. prediction of relapse) and prospective isolation of LSC and HSC from the same AML BM to design new therapeutic modalities. It may further serve as a surrogate marker to monitor effectiveness of a treatment and to design antibody/ligand combination therapies in a personalized setting. Disclosures:No relevant conflicts of interest to declare.

Dicer Ablation Impairs Prostate Stem Cell Activity and Causes Prostate Atrophy

Dicer is an RNase III enzyme essential for microRNA maturation. Dicer ablation in diverse tissues has been shown to block tissue differentiation, induce cell apoptosis, impair specialized cellular function, and perturb organ structures. To gain insight into the role of microRNAs in prostate tissue function and homeostasis, we conditionally disrupted Dicer activity in the mouse prostate using an ARR2PB-Cre. We demonstrated that Dicer activity is disrupted in both prostatic basal/stem cells and differentiated luminal cells. Dicer knockout murine prostates are smaller in size and mass and develop epithelial hypotrophy in ventral prostates by 4 months. Dicer ablation induces increased apoptosis in the prostate, predominantly in the differentiated luminal cells. Paradoxically, a concurrent increase in proliferation is observed in both basal/stem cells and luminal cells, presumably due to compensatory growth of the cells devoid of homologous recombination in response to the elevated cellular apoptosis. We have previously shown that Lin(CD31CD45Ter119)(-)Sca-1(+)CD49f(high) (LSC) cells enrich for prostate stem cell activity. Through proliferation and differentiation, some LSC cells are capable of forming prostate spheres composed of cells at various stages of differentiation. Although LSC cells were expanded by threefold in Dicer knockout mice, the sphere-forming units of Dicer knockout prostate cells decreased by more than half compared with wild-type cells. In addition, most prostate spheres in the Dicer knockout culture were derived from cells that did not undergo homologous recombination. Our results demonstrate a critical role of microRNAs for the proliferative capacity of prostate stem cells and the maintenance of prostate homeostasis.

Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc

The core 1 structure Galβ1-3GalNAcα1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (C1GalT) and its specific molecular chaperone, Cosmc. The chaperone function of Cosmc has been well investigated biochemically. In this study, we established monoclonal antibodies specifically recognizing either C1GalT or Cosmc, respectively, and investigated the sub-cellular localization of each protein to elucidate how they cooperate to synthesize the core 1 structure.A sequential immunocytochemical analysis of the human colon cancer cell line, LSB, demonstrated different localization of two proteins. C1GalT was localized in Golgi apparatus, while Cosmc was localized in endoplasmic reticulum. In contrast, the LSC cells, which do not have core 1 synthase activity due to a missense mutation in the Cosmc gene, did not express the C1GalT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of C1GalT protein, the distribution of C1GalT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not C1GalT lead to precise localization of C1GalT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of C1GalT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity.

Gene Expression Profiling Reveals Fundamental Differences between Leukemic Stem Cells (Lin-CD34+CD38−) and Mature Blasts (Lin−CD34+CD38+) of Acute Myeloid Leukaemia (AML) Patients.

The identification of LSC has important implications for future research as well as for the development of novel therapies. The phenotypic description of LSC now enables their purification and should facilitate the identification of genes that are preferentially expressed in these cells compared to normal HSC. However, gene-expression profiling is usually conducted on mononuclear cells of AML patients from either peripheral blood and/or bone marrow. These samples contain a mixture of blasts cells, normal hematopoietic cells and limited number of leukemic stem cells. Thus, this results in a composite profile that obscure differences between LSC and blasts cells with low proliferative potential. The aim of this study was to compare the gene expression profile of highly purified LSC versus leukemic blasts in order to identify genes that might have important roles in driving the leukemia. For this purpose, we analyzed the gene expression profiles of highly purified LSCs (Lin−CD34+CD38−) and more mature blast cells (Lin−CD34+CD38+) isolated from 7 adult AML patients. All samples were previously tested for the ability of the Lin−CD34+CD38− cells but not the Lin−CD34+CD38+ fraction to engraft using the non-obese diabetic/severe combined immuno-deficiency (NOD-SCID) repopulation assay. Affymetrix microarrays (U133A chip), containing 22,283 genes, were used for the analysis. Comparison of Lin-CD34+CD38- cell population to the Lin−CD34+CD38+ cell fraction showed 5421 genes to be expressed in both fractions. Comparative analysis of gene-expression profiles showed statistically significant differential expression of 133 genes between the 2 cell populations. Most of the genes were downregulated in the LSC-enriched fraction, compared to the more differentiated fraction. Gene ontology was used to determine the categories of the up-regulated transcripts. These transcripts, which are selectively expressed, include a number of known genes (e.g., receptors, signalling genes, proliferation and cell cycle genes and transcription factors). These genes play important roles in differentiation, self-renewal, migration and adhesion of HSCs. Among the genes showing the highest differences in expression levels were the following: ribonucleotide reductase M2 polypeptide, thymidylate synthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II, CDC20, nucleolar and spindle associated protein 1, Rac GTPase activating protein 1, leukocyte immunoglobulin-like receptor, proliferating cell nuclear antigen, myeloperoxidase, cyclin A1 (RRM2, TYMS, ZWINT, CTSG, AZU1, TOP2A, CDC20, NUSAP1, RACGAP1, LILRB2, PCNA, MPO, CCNA1). Some transcripts detected have not been implicated in HSC functions, and others have unknown function so far. This work identifies new genes that might play a role in leukemogenesis and cancer stem cells. It also leads to a better description and understanding of the molecular phenotypes of these 2 cell populations. Hence, in addition to being a more efficient way to further understand the biology of LSC, this should also provide a more efficient way of identifying new therapeutics and diagnostic targets.

Molecular Cloning and Characterization of a Novel UDP-Gal:GalNAcα Peptide β1,3-Galactosyltransferase (C1Gal-T2), an Enzyme Synthesizing a Core 1 Structure of O-Glycan

Recently, a UDP-Gal:GalNAc(alpha) peptide beta1,3-galactosyltransferase (core 1 synthase 1; C1Gal-T1) has been purified from rat liver and its complementary DNA cloned from several species. We isolated a second candidate for core 1 synthase from a Colo205 cDNA library and named it C1Gal-T2. The deduced amino acid sequence of C1Gal-T2, having 26% homology to C1Gal-T1, showed a topology typical of a type II membrane protein. Real time PCR analysis revealed that the expression of C1Gal-T2 transcripts was widespread in many tissues and of relatively high level in salivary gland, stomach, small intestine, kidney, testis, thymus, and spleen. LSC cells, having no core 1 synthase activity, were transfected stably with the C1Gal-T2 gene. Their microsome fraction showed beta1,3-galactosyltransferase activity toward GalNAc-alpha-para-nitrophenyl and GalNAc(alpha)1 peptides resulting in the synthesis of the core 1 structure. The core 1 synthesizing activity of C1Gal-T2 was also determined by flow cytometry and lectin blotting using the LSC cells stably expressing C1Gal-T2. Finally, LSC cells, and Jurkat cells that also lack the core 1 synthase activity, were found to have null alleles of C1Gal-T2. These results indicated that C1Gal-T2 is the second candidate for core 1 synthase that plays an important role in synthesizing O-glycans in digestive organs.

Enzymatic basis for sialyl-Tn expression in human colon cancer cells.

Sialyl-Tn antigen (SAalpha2-6 GalNAc alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc alpha-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal beta1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R beta3-Gal-transferase (core 1 beta3-Gal-transferase) was lacking. Core 1 beta3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc beta1-6 (Gal beta1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 beta3-Gal-transferase activity. The lack of core 1 beta3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 beta3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.

ヒト歯髄由来培養細胞のプラスミドｐＭＴ１‐ｎｅｏ遺伝子導入による不死化について

A human dental pulp tissue was explanted in culture and the outgrowing cells were transfected with the plasmid, pMT1-neo, which included the early region of SV 40 DNA and the neomycin-resistant gene. The transfected cells were cloned and cultured beyond the period of senescence for the non-transfected HOP cells, over 170 passages (360 population doubling levels and 730 days). The characteristics of the transfected LSC cells and the non-transfected HOP cells were investigated during in vitro aging. The results were as follows: 1. The alkaline phosphatase (ALPase) activity of the HOP cells decreased as the culture passages increased. In contrast, the ALPase activity of the LSC cells did not change during the entire serial culture period. 2. The ALPase inhibitory test indicated that the ALPase in the LSC cells was the bone/liver/kidney type. 3. The addition of 250 micrograms/ml of L-ascorbic acid resulted in an increased collagen synthesis compared with that of 50 micrograms/ml. 4. The growth rate and the ALPase activity of the LSC cells were affected by 1 alpha, 25-dihydroxyvitamin D3, L-ascorbic acid, beta-sodium glycerophosphate, epidermal growth factor or transforming growth factor-beta even after the serial culture. These results suggest that the LSC cells preserve some properties of the dental pulp and may be useful in the future research for exploring the mechanisms of the dental hard tissue formation and for testing the biocompatibility of the dental restorative materials.