Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34⁺ HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34⁺ cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2,603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5′ ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.     

The pattern of gene expression in human CD15+ myeloid progenitor cells

We performed a genome-wide analysis of gene expression in primary human CD15(+) myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.     

Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice

Over the past decade xenotransplantation systems have been used with increasing success to gain a better understanding of human cells that are able to initiate and maintain the hematopoietic system in vivo. The nonobese diabetic/severe combined immunodeficiency (SCID) mouse has been a particularly useful model. Human cells capable of hematopoietic repopulation in this mouse, termed SCID-repopulating cells, have been assumed to represent the most primitive elements of the hematopoietic system, responsible for long-term maintenance of hematopoiesis. However, we demonstrate that SCID-repopulating cells present in the CD34(+) cell fraction of cord blood can be segregated into subpopulations with distinct repopulation characteristics. CD34(+)/CD38(+) progenitors can repopulate recipients rapidly, but can only maintain the graft for 12 weeks or less and have no secondary repopulation potential. Conversely, the more primitive CD34(+)/CD38(-) subpopulation repopulates recipients more gradually, can maintain the graft for at least 20 weeks, and contains cells with serial repopulation potential throughout the engraftment period. Additionally, a much higher frequency of T cell precursors are found among SCID-repopulating cells in the CD34(+)/CD38(-) subpopulation. These findings demonstrate that cells with variable repopulation potential comprise the human CD34(+) population and that short- and long-term potential of human precursors can be evaluated in the mouse model.     

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na⁺- and K⁺-dependent pH homeostasis and in Na⁺ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H⁺, Na⁺/H⁺, and K⁺/H⁺ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H⁺ and Na⁺/H⁺ antiports is presented. K⁺ and Li⁺ inhibit Na⁺ uptake, but there is little cross-inhibition between Na⁺ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.     

Expression of the Na+/Ca2+ exchanger emerges in hepatic stellate cells after activation in association with liver fibrosis

Activation of hepatic stellate (Ito) cells is a final common pathway of liver fibrosis. The findings presented in this paper indicate that expression of Na⁺/Ca²⁺ exchanger (NCX) emerges in rat hepatic stellate cells after activation in vitro during primary culture or in vivo in response to intoxication with CCl₄. NCX mRNA became detectable by Northern blot analysis in cultured stellate cells on day 3, as was α-smooth muscle actin, an indicator not only of smooth muscle differentiation but also of stellate cell activation. Western blot analysis showed expression of the exchanger protein in the activated stellate cells. Functional expression of the exchanger, monitored by Ni²⁺-sensitive, verapamil-insensitive intracellular free Ca²⁺ increases in response to reduction of extracellular Na⁺ concentration, became sizable by using Fura-2 in stellate cells by 7 days in culture. Furthermore, increased expression of the exchanger mRNA was found predominantly in stellate cells freshly isolated from the CCl₄ model rat of hepatic fibrosis. Thus, it is concluded that NCX expression is closely associated with activation of hepatic stellate cells in vitro and in vivo. Because, even at the whole liver level, increased expression of NCX mRNA became observable after induction of liver fibrosis, it is suggested that NCX expression serves a useful diagnostic marker of liver fibrosis or cirrhosis.     

Prevalent CD8+ T cell response against one peptide/MHC complex in autoimmune diabetes

Spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice is the result of a CD4(+) and CD8(+) T cell-dependent autoimmune process directed against the pancreatic beta cells. CD8(+) T cells play a critical role in the initiation and progression of diabetes, but the specificity and diversity of their antigenic repertoire remain unknown. Here, we define the structure of a peptide mimotope that elicits the proliferation, cytokine secretion, differentiation, and cytotoxicity of a diabetogenic H-2K(d)-restricted CD8(+) T cell specificity (NY8.3) that uses a T cell receptor alpha (TCRalpha) rearrangement frequently expressed by CD8(+) T cells propagated from the earliest insulitic lesions of NOD mice (Valpha17-Jalpha42 elements, often joined by the N-region sequence M-R-D/E). Stimulation of splenic CD8(+) T cells from single-chain 8. 3-TCRbeta-transgenic NOD mice with this mimotope leads to preferential expansion of T cells bearing an endogenously derived TCRalpha chain identical to the one used by their islet-associated CD8(+) T cells, which is also identical to the 8.3-TCRalpha sequence. Cytotoxicity assays using islet-derived CD8(+) T cell clones from nontransgenic NOD mice as effectors and peptide-pulsed H-2K(d)-transfected RMA-S cells as targets indicate that nearly half of the CD8(+) T cells recruited to islets in NOD mice specifically recognize the same peptide/H-2K(d) complex. This work demonstrates that beta cell-reactive CD8(+) T cells mount a prevalent response against a single peptide/MHC complex and provides one peptide ligand for CD8(+) T cells in autoimmune diabetes.     

Increased rates of CD4+ and CD8+ T lymphocyte turnover in simian immunodeficiency virus-infected macaques

Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4⁺ and CD8⁺ BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4⁺ T cell BrdUrd-labeling and decay were closely correlated with those of CD8⁺ T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4⁺ and CD8⁺ T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4⁺ T cell depletion in AIDS.     

CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4⁺ T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4⁺ T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.     

T cells recognizing leukemic CD34+ progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [³H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34⁺ CML progenitor cells, and not of the more mature CD34⁻ CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34⁺ CML progenitor cells mediate the antileukemic effect of DLI.     

Efficient and persistent expression of β-glucuronidase gene in CD34+ cells from human umbilical cord blood by retroviral vector

Abstract: We succeeded in efficiently transferring the β-glucuronidase gene in a retroviral vector to human hematopoietic progenitor cells using a centrifugation enhancement protocol. The transduction efficiency in CFU–GM was highly variable (23–100%) with an average of 66.8%. In the case of BFU–E, efficiency was 83% and 76% in 2 separate experiments. In LTCIC (long-term culture-initiating cell), transduction efficiency were 20% and 50% in 2 experiments. The enzymatic activity of β-glucuronidase in transduced cells were increased above the control level up to 5 wk. Considering that correction of the enzyme deficiency in a small number of hematopoietic cells can be therapeutic for the Sly disease mouse, our data provide encouragement that human trials of gene therapy based on transferring β-glucuronidase gene to hematopoietic cells may be efficacious.     

Development of human lymphohematopoietic stem and progenitor cells defined by expression of CD34 and CD81

In this study, cord blood CD34(+) cells expressed CD81, a member of the transmembrane 4 superfamily, and were classified into 3 subpopulations on the basis of their expression levels: CD34(+)CD81(+), CD34(low)CD81(+), and CD34(+)CD81(high). The lymphohematopoietic activity of each subpopulation was then examined by using suspension and clonogenic cultures for hematopoietic potential, coculture with MS-5 cells for B-cell potential, organ cultures of thymus lobes from nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) fetal mice, coculture with stromal cells derived from NOD/SCID fetal-mouse liver tissue for natural killer (NK) cell and mast cell potentials, and xenotransplantation into NOD/SCID mice for long-term repopulating (LTR) ability. CD34(+)CD81(+) cells represented a heterogeneous population that had all the lymphohematopoietic activities, including NOD/SCID mouse-repopulating ability. CD34(low)CD81(+) cells were enriched in erythroid, megakaryocytic, and NK lineage potentials but had lost T-cell and B-cell potentials and LTR ability. The CD34(+)CD81(high) fraction was depleted of most lymphohematopoietic potentials except NK cell and mast cell potentials. Thus, along the differentiation cascade from CD34(+)CD81(+) lymphohematopoietic stem cells, an up-regulation of CD81 or a down-regulation of CD34 results in a change in lymphohematopoietic properties. CD81 may serve as a marker for defining developmental stages of lymphohematopoietic stem cells. (Blood. 2001;97:3755-3762)     

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (E_δ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by E_δ and the TCR α enhancer, and that it prevents E_δ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.     

CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern

Gene expression patterns of CD34(+)CD38(-) cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic epsilon-globin expression were observed-but no adult beta-globin message-in CD34(+)CD38(-) cells from ESCs, whereas high levels of beta-globin expression-but no embryonic epsilon-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34(+)CD38(-) cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.     

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca²⁺ concentration and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. Herein we show that inhibitors of the Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca²⁺ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.     

Differential gene expression analysis in early and late erythroid progenitor cells in β‐thalassaemia

β‐ thalassaemia is a disorder of globin gene synthesis resulting in reduced or absent production of the β‐globin chain in red blood cells. In this study, haematopoietic stem cells were isolated from the peripheral blood of six transfusion dependent β‐thalassaemia patients and six healthy controls. Following 7 and 14 d in culture, early‐ and late‐ erythroblasts were isolated and purified. No morphological difference in maturation was observed following 7 d in culture, while a delayed maturation was observed in the patient group after 14 d. Following RNA isolation and linear amplification, gene expression analyses were performed using microarray technology. The generated data were analysed by two methods: the BRB‐ArrayTools platform and the Bioconductor platform using bead level data. Following 7 d culture, there was no difference in gene expression between the control and patient groups. Following 14 d culture, 384 differentially expressed genes were identified by either analysis. A subset of 90 genes was selected and the results were confirmed by Quantitative‐Real‐Time‐polymerase chain reaction. Pathways shown to be significantly altered in the patient group include apoptosis, MAPKinase and the nuclear factor‐κB pathway.     

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na⁺/glucose cotransporter. The Na⁺/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na⁺/Cl⁻/γ-aminobutyric acid, Na⁺/iodide and H⁺/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.     

CD84 expression on human hematopoietic progenitor cells

OBJECTIVE: CD84 is a member of the CD2 subgroup of the immunoglobulin receptor superfamily. Members of this family have been implicated in the activation of T cells and NK cells. Expression of CD84 was originally described on most mononuclear blood cells as well as platelets. To elucidate its presence on other blood cell types, we analyzed the expression pattern of CD84 on human immature CD34+ and mature hematopoietic cells. METHODS: Expression analysis was carried out by flow cytometry. The differentiation potential of CD84+ progenitor cells was assessed by colony-forming assays and long-term cultures. RT-PCR was used to analyze CD84 mRNA isoforms. RESULTS: In addition to monocytes, macrophages, B cells, and some T cells, CD84 is expressed on the cell surface of the majority of granulocytes. In addition, 64%+/-5% of CD34+ progenitor cells isolated from peripheral blood and 30.5%+/-5% from bone marrow of healthy volunteers also express CD84. The majority of CD34+ cells coexpressing lineage antigens were CD84+. In methylcellulose CD34+CD84+ cells formed primarily erythroid colonies, whereas myeloid or mixed colonies were scarce. The frequency of long-term culture-initiating cells in peripheral blood was approximately fivefold higher in CD34+CD84- vs CD34+CD84+ cells. In short-term cultures, 95% of the initially CD34+CD84- cells became CD84+ after 72 hours. CONCLUSIONS: CD84 is expressed on cells from almost all hematopoietic lineages and on CD34+ hematopoietic progenitor cells. The proliferative potential of CD34+ cells decreases with increasing CD84 expression, suggesting that CD84 serves as a marker for committed hematopoietic progenitor cells.     

Polyclonal structure of intestinal adenomas in ApcMin/+ mice with concomitant loss of Apc+ from all tumor lineages

When tumors form in intestinal epithelia, it is important to know whether they involve single initiated somatic clones. Advanced carcinomas in humans and mice are known to be monoclonal. However, earlier stages of tumorigenesis may instead involve an interaction between cells that belong to separate somatic clones within the epithelium. The clonality of early tumors has been investigated in mice with an inherited predisposition to intestinal tumors. Analysis of Min (multiple intestinal neoplasia) mice chimeric for a ubiquitously expressed cell lineage marker revealed that normal intestinal crypts are monoclonal, but intestinal adenomas frequently have a polyclonal structure, presenting even when very small as single, focal adenomas composed of at least two somatic lineages. Furthermore, within these polyclonal adenomas, all tumor lineages frequently lose the wild-type Apc allele. These observations can be interpreted by several models for clonal interaction within the epithelium, ranging from passive fusion within regions of high neoplastic potential to a requirement for active clonal cooperation.