Telomere Attrition and Chromosomal Instability During Long-Term Cultivation of Hematopoietic Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4001-4001
Author(s):  
Kathrin Lange ◽  
Beate Kuhlmann ◽  
Andrea Schienke ◽  
Axel Schambach ◽  
Ute Modlich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Chromosomal Instability ◽  
Insertional Mutagenesis ◽  
Erythroid Differentiation ◽  
Chromosomal Breakage ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Cd34 Cells

Abstract Abstract 4001 By means of a large RNA interference screen, Ebert et al. identified RPS14 as the target of del(5q), since haploinsufficiency of RPS14 in human hematopoietic stem cells (HSC) induces an erythroid differentiation defect, a characteristic feature of myelodysplastic syndromes (MDS) with isolated deletion in 5q (Nature451, 335–339 (2008)). However, to the best of our knowledge, these cells have not been observed for more than two weeks in culture. We have shown that telomere shortening and chromosomal instability play a crucial role, particularly during progression of MDS with del(5q) into acute myeloid leukemia (AML) (K Lange et al, Genes Chromosomes Cancer49, 260–269 (2010) and G Göhring et al, Leukemia, 2011 (in press)). Because of the limited proliferation of primary MDS cells in culture and because there is no appropriate MDS cell line, a good human cell culture model is urgently needed to investigate the relationship of telomere attrition and chromosomal instability during the progression of MDS with del(5q). Therefore, it was our aim was to create a long-term culture (LTC) model to observe the effects of RPS14 haploinsufficiency on differentiation capacity, DNA repair, telomere shortening and chromosomal instability in human HSCs. CD34+ cells were isolated from umbilical cord blood via magnetic cell separation. Knockdown of RPS14 to a level of 40 – 50% was performed via lentiviral transduction of shRNA vectors. As controls, in addition to untransduced mock cells, CD34+ cells were transduced with scrambled shRNA. LTC was performed on murine feeder layers (M2-10B4). In short-term culture, as expected, knockdown of RPS14 led to an erythroid differentiation defect, decreased proliferative activity and an increased level of apoptosis. Cultivation on murine feeder layers enabled expansion of CD34+ cells for more than 6 weeks. Generally, within 6 weeks RPS14 -deficient CD34+ cells showed a poorer proliferation than the control cells with a 214-fold versus a 6080-fold multiplication, respectively. Median numbers of γH2AX foci, indicators of DNA double-strand breaks, did not differ between RPS14-deficient and control cells with a median number of 9.4 and 8.2 foci/cell, respectively. Induction of γH2AC foci via Mitomycin C, a DNA cross-linking agent inducing double-strand breaks, did not reveal an altered DNA repair capacity with 14.3 and 11.6 foci/cell. Colony-forming assays showed that, even though RPS14 haploinsufficiency immediately led to an erythroid differentiation defect, this differentiation capacity decreased even further within the weeks of follow-up. This however, although with a delay of about 4 weeks, could also be observed in the control cells (Figure 1).Figure 1Figure 1. Telomere length decreased in RPS14- deficient cells as well as in control cells. Initially, telomeres showed a median length of about 12.5 kb, decreasing to a median length of 10.3 kb (range 8.4 – 11.4 kb) and 5.8 kb (range 5.7 – 8.9 kb) after 2 and 4 weeks, respectively, which afterwards elongated to a level of 8.7 kb (range 8.5 –8.9 kb) after 6 weeks. The mechanism underlying this primary shortening and subsequent re-elongation might be due to an up-regulation of telomerase or of the alternative lengthening of telomeres (ALT) mechanism. Cytogenetic investigations demonstrated an increase in chromosomal breakage in all cultures, pointing towards induction during LTC rather than due to RPS14 deficiency. Remarkably, chromosomal breakage and chromosome aberrations in single cells within the first 4 weeks of cultivation of hematopoietic stem cells transduced with scrambled shRNA was followed by clonal dominance of monosomy 7 after 6 weeks. This may be an effect caused by insertional mutagenesis. LM-PCR is currently under way to obtain insights into the affected insertion site(s). Monosomy 7 is one of the most frequent chromosome aberrations in MDS and frequently occurs as an additional aberration in MDS/AML with inversion 3q generating an MDS-EVI1 fusion transcript. Thus, it will be interesting to see whether monosomy 7 in our LTC model cooperates with EVI1 activation as in sporadic MDS and AML. In conclusion, a LTC model on feeder layer cells seems to be an appropriate system to analyze hematopoietic stem cells and MDS in culture. However, cell culture artefacts inducing telomere shortening and chromosomal instability or insertional mutagenesis have to be taken into account and regular cytogenetic analyses should always be performed. Disclosures: No relevant conflicts of interest to declare.

AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1778-1778
Author(s):  
Flavia O. Pinho ◽  
Dulcineia M. Albuquerque ◽  
Sara T.O Saad ◽  
Fernando F. Costa
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Erythroleukemia Cell ◽  
Human Red Cells

Abstract Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 5720-5726 ◽  
Author(s):  
John F. DiPersio ◽  
Edward A. Stadtmauer ◽  
Auayporn Nademanee ◽  
Ivana N. M. Micallef ◽  
Patrick J. Stiff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Hematopoietic Stem Cells ◽  
Primary Endpoint ◽  
Controlled Study ◽  
Double Blind ◽  
Hematopoietic Stem ◽  
Injection Site Reactions ◽  
Cd34 Cells ◽  
Stimulating Factor

Abstract This phase 3, multicenter, randomized (1:1), double-blind, placebo-controlled study evaluated the safety and efficacy of plerixafor with granulocyte colony-stimulating factor (G-CSF) in mobilizing hematopoietic stem cells in patients with multiple myeloma. Patients received G-CSF (10 μg/kg) subcutaneously daily for up to 8 days. Beginning on day 4 and continuing daily for up to 4 days, patients received either plerixafor (240 μg/kg) or placebo subcutaneously. Starting on day 5, patients began daily apheresis for up to 4 days or until more than or equal to 6 × 106 CD34+ cells/kg were collected. The primary endpoint was the percentage of patients who collected more than or equal to 6 × 106 CD34+ cells/kg in less than or equal to 2 aphereses. A total of 106 of 148 (71.6%) patients in the plerixafor group and 53 of 154 (34.4%) patients in the placebo group met the primary endpoint (P < .001). A total of 54% of plerixafor-treated patients reached target after one apheresis, whereas 56% of the placebo-treated patients required 4 aphereses to reach target. The most common adverse events related to plerixafor were gastrointestinal disorders and injection site reactions. Plerixafor and G-CSF were well tolerated, and significantly more patients collected the optimal CD34+ cell/kg target for transplantation earlier compared with G-CSF alone. This study is registered at www.clinicaltrials.gov as #NCT00103662.

scholarly journals AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
Author(s):  
André Larochelle ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Genetic Manipulation ◽  
Retroviral Vectors ◽  
Lymphoid Cells ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4622-4622
Author(s):  
Nadja Grund ◽  
Patrick Maier ◽  
Uwe Appelt ◽  
Heike Allgayer ◽  
Frederik Wenz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
In Silico ◽  
Integration Site ◽  
General Distribution ◽  
Cytostatic Drug ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.

Targeting Acute Myeloid Leukemia Stem Cells by MUC1-C Subunit Inhibition

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 848-848 ◽  
Author(s):  
Dina Stroopinsky ◽  
Jacalyn Rosenblatt ◽  
Keisuke Ito ◽  
Li Yin ◽  
Hasan Rajabi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Control Peptide ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 848 Introduction: Acute myeloid leukemia (AML) arises from a malignant stem cell population that is resistant to cytotoxic therapy and represents a critical reservoir of conferring disease recurrence. A major focus of investigation is the identification of unique markers on leukemia stem cells (LSCs) that differentiate them from normal hematopoietic stem cells and thereby serve as potential therapeutic targets. MUC1 is a high molecular weight transmembrane glycoprotein that is aberrantly expressed in many epithelial tumors and confers cell growth and survival. We have developed an inhibitor of the MUC1-C receptor subunit that blocks oligomer formation and nuclear localization. In the present study, we have examined expression of MUC1 on LSCs as compared to normal hematopoietic stem cells and studied the effect of MUC1-C inhibition on the functional properties of LSCs. Methods and Results: Using multichannel flow cytometric analysis, we isolated the LSC compartment as defined by CD34+/CD38-/lineage- cells from bone marrow specimens obtained from patients with active AML. The majority of LSCs strongly expressed MUC1 with a mean percentage of 77% (n=6). These findings were confirmed by immunocytochemical staining of LSCs isolated by flow cytometric sorting. MUC1 expression was not detectable on the CD34- fraction of AML cells, but was present on the granulocyte-macrophage progenitor (GMP) fraction (CD34+/CD38+ cells) (mean=83%; n=6). In contrast, MUC1 expression was not observed on CD34+ progenitors isolated from normal donors (18%, n=6). In concert with these findings, RT-PCR analysis for MUC1 RNA demonstrated expression in CD34+ cells isolated from AML patients, but not normal volunteers. Notably, we also found that MUC1 expression selectively identifies malignant hematopoietic progenitors in a patient with chimerism between normal and leukemia derived stem cells. The presence of MUC1+CD34+ cells was detected in a patient with AML who achieved a morphologic complete remission following sex mismatched allogeneic transplantation. Using Bioview technology, we found that MUC1 is expressed only in the recipient (XX) CD34+ cells, representing residual malignant cells, whereas the donor (XY) derived CD34+ cells, representing the majority of the progenitors, lacked MUC1 expression. We subsequently examined the effects of MUC1-C inhibition on the capacity of leukemic progenitors to proliferate and support colony formation. MUC1-C inhibition with the GO-203 cell-penetrating peptide resulted in downregulation of the β-catenin pathway, an important modulator of cell division and survival, which is known to support the LSC phenotype. No significant change was detected with a control peptide, or with MUC1-C inhibition of progenitors isolated from a normal control. Furthermore, MUC1-C inhibition resulted in apoptosis, as demonstrated by flow cytometric staining for AnnexinV in AML CD34+ cells, but not in CD34+ progenitors isolated from normal volunteers (mean Annexin positive cells 53% and 5%, respectively, n=4). Consistent with these findings, the MUC1-C inhibitor, but not the control, peptide resulted in cell death of CD34+ cells isolated from AML patients, but not normal controls. Most significantly, exposure of CD34+ AML cells to the MUC1-C inhibitor resulted in loss of their capacity for colony formation in vitro with mean colonies of 4 and 40 for those cells exposed to the MUC1 inhibitor and a control peptide (n=2). In contrast, colony formation by normal hematopoietic stem cells was unaffected. Conclusions: MUC1 is selectively expressed by leukemic progenitors and may be used to differentiate malignant from normal hematopoietic stem cell populations. MUC1-C receptor subunit inhibition results in (i) downregulation of b-catenin signaling, (ii) induction of apoptosis and cell death, and (iii) disruption of the capacity to induce leukemia colony formation. Disclosures: Stone: genzyme: Consultancy; celgene: Consultancy; novartis: Research Funding. Kufe:Genus Oncology: Consultancy, Equity Ownership.

Valproic Acid Results In Maintenance but Not Expansion of Transplantable Hematopoietic Stem Cells From Human Umbilical Cord Blood

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 827-827
Author(s):  
Hiroto Araki ◽  
Sudhakar Baluchamy ◽  
Benjamin Petro ◽  
Mirza Saqib Baig ◽  
Montha Suhangul ◽  
...  
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Molecular Mechanisms ◽  
Epigenetic Modifications ◽  
Human Umbilical Cord ◽  
Gene Transcript ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 827 Epigenetic modifications are considered to be important in determining the fate of hematopoietic stem cells (HSC). We previously demonstrated that the sequential addition of the chromatin-modifying agents (CMA) 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA) expands transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Others have shown that valproic acid (VPA), an HDAC inhibitor, also expands HSC (DeFelice et al. Cancer Res 2005). We thus compared the efficacy of 5azaD/TSA and VPA in promoting the ex vivo expansion of human cord blood (CB) HSC. Cells were incubated with cytokines alone (SCF, Flt3 ligand, TPO and IL-3) or with cytokines and either 5azaD/TSA or VPA, resulting in 2.2-fold, 10.7-fold or 65-fold expansion, respectively, of primitive CD34+CD90+ cells after 9 days (n=3, Cytokine alone vs. VPA p=0.004; Cytokine alone vs. 5azaD/TSA p=0.03; VPA vs. 5azaD/TSA p=0.003). Interestingly, the 10.7-fold expansion of CD34+CD90+ cells following 5azaD/TSA treatment correlated with a 10- and 10.5-fold expansion of short-term colony-forming cells (CFC) and long-term cobblestone area-forming cells (CAFC), respectively. However, the 65-fold expansion of CD34+CD90+ cells achieved with VPA treatment yielded only a 25.6- and 8.4-fold expansion of CFC and CAFC, respectively. These results suggest a marked discordance between the phenotype and function of CD34+CD90+ cells when they are expanded in VPA, but not in 5azaD/TSA. Thus, we examined the in vivo hematopoietic repopulation potential of CMA-expanded CB HSC by quantitating SCID mouse repopulating cells (SRC) using limiting dilution assays. The frequency of SRC was 1 in 22,000 in primary CB cells (n=29 mice), 1 in 123,315, in (cytokine) controls (n=16 mice), 1 in 21,720 with VPA-treatment (n=27 mice), and 1 in 3,147, in 5azaD/TSA-treated CD34+CD90+ cell cultures (n=22 mice). Unlike control, treatment with VPA prevents loss of SRC but only results in SRC maintenance, whereas 5azaD/TSA treatment leads to a 7-fold expansion of SRC. Furthermore, serial transplantation of bone marrow (BM) from primary recipients engrafted with unmanipulated CB cells resulted in engraftment in 2 of 5 secondary mice, while BM from mice engrafted with VPA-treated cells failed to display secondary engraftment (n=5 mice), whereas BM from mice engrafted with 5azaD/TSA-treated cells resulted in engraftment in 5 of 6 secondary mice. Hence, we conclude that treatment of CB CD34+ cells with 5azaD/TSA or VPA results in distinct SRC outcomes-expansion or maintenance, respectively. To dissect the molecular mechanisms that may mediate these distinct SRC fates, we examined genes implicated in HSC self-renewal including HoxB4, Bmi1, STAT3, Ezh2 and PU.1. These gene transcript levels were increased in CD34+ cells treated with either 5azaD/TSA or VPA when compared to control cultures as measured by real time quantitative PCR. In accordance with these studies, CHIP assays using antibody against acetylated histone H4 indicate increased acetylation of the promoters of HoxB4 and Bmi1 genes in both VPA- and 5azaD/TSA-treated cells. In addition, higher levels of HoxB4, Ezh2 and PU.1 proteins were observed in VPA- and 5azaD/TSA-expanded cells, compared to control cultures. Since VPA treatment does not result in SRC expansion, these observations raise questions as to the importance of the upregulation of these genes for HSC expansion. Since the pharmacologic activity of CMAs is short (hours) we hypothesize that temporal effects, including early epigenetic modifications, lead to changes in transcription factor expression, which directly or indirectly promote symmetric or asymmetric divisions ultimately resulting in expansion or maintenance of HSC. Importantly, our global microarray data (n=3) using a human genome affymetrix chip (U133 plus 2.0) revealed a set of differentially expressed genes present in 5azaD/TSA- but not in VPA-expanded CD34+ cells, thus uncovering a potential molecular signature for HSC expansion. Currently, we are examining the molecular interactions of these signature genes and the effects of silencing of these genes on HSC expansion or maintenance which should allow us to begin to unravel the molecular mechanisms involved. In summary our data indicate that treatment of HSCs with different CMAs results in distinct fates: expansion or maintenance of HSC, an observation of potential therapeutic importance. Disclosures: No relevant conflicts of interest to declare.

Decreased Expression of HOXB4 Gene in Aplastic Anemia; Regulatory Interaction with GATA-2,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3436-3436
Author(s):  
Tohru Fujiwara ◽  
Hisayuki Yokoyama ◽  
Yoko Okitsu ◽  
Mayumi Kamata ◽  
Shinichi Fujimaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Promoter Region ◽  
Expression Vector ◽  
Gene Promoter ◽  
K562 Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Gene Promoter Region ◽  
Chip Analysis

Abstract Abstract 3436 Background: Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells (HSCs). It has been proposed that immunological injury in HSCs leads to reduced numbers of stem cells in the bone marrow. In addition, expression of the critical regulator of hematopoiesis GATA-2 is decreased in CD34-positive cells in AA (Fujimaki et al. Br J Haematol 2001). Despite the compelling results described above, only limited information has emerged regarding intrinsic abnormalities of hematopoietic stem cells in AA. It has been demonstrated that HOXB4 induces HSC expansion ex vivo (Antonchuk et al. Cell 2002), and restoring HOXB4 protein in HSCs from bone marrow failure patients promotes HSC expansion (Tang et al. Br J Haematol 2009). In conjunction with the evidence that recent genome-wide analysis of GATA factor chromatin occupancy identified GATA-2 peak at HOXB4 promoter (Fujiwara et al. Mol Cell 2009), we hypothesized that GATA-2 directly regulates HOXB4 expression in HSCs, which might contribute to the pathogenesis of AA. Here, we investigated possible link between GATA-2 and HOXB4, and also tested if HOXB4 is deregulated in CD34-positive cells from patients with AA. Method: For GATA-2 overexpression, human GATA-2 coding sequence was cloned into pcDNA3.1 expression vector as well as MSCV retroviral expression vector (Clontech). For GATA-2 knockdown, siRNA specific for human GATA-2 was transfected into CD34-positive cells or K562 cells by Amaxa Nucleofector kit (Amaxa Inc.). For promoter assay, DNA fragment of the HOXB4 gene promoter region (up to −262 from 1st ATG) was cloned into pGL3-Basic (Promega), and the GATA deletion construct was subsequently created with QuickChange™Site-Directed Mutagenesis Kit (Stratagene). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using antibodies for GATA-2 (H-116, Santa Cruz). For analyzing clinical samples, informed consent was obtained in all cases and ethical considerations according to the declaration of Helsinki were followed. Results: To examine if GATA-2 and HOXB4 are functionally linked, we transfected a GATA-2 expression vector into K562 cells, and demonstrated that GATA-2 significantly upregulated endogeneous HOXB4 expression. Furthermore, siRNA-mediated GATA-2 knockdown in K562 cells significantly reduced HOXB4 expression, indicating that HOXB4 is a GATA-2 target gene. We overexpressed/reduced GATA-2 in cord blood-derived CD34+ cells, which also provided evidence for GATA-2 regulation of HOXB4 expression. Promoter analyses revealed that GATA sequence located at −160/-157 of the HOXB4 gene promoter region was required to confer luciferase activity in K562 cells. In vitro DNA binding studies and quantitative ChIP analysis revealed specific GATA-2 occupancy at a chromatin region containing this element. Finally, we demonstrated that HOXB4 gene expression was significantly decreased in CD34+ cells from patients with AA (n=10) compared to those with ITP (n=13). The expression levels of HOXB4 and GATA-2 also correlated in these populations (r=0.6573, p<0.01). Conclusion: Based on these findings, we propose that decreased expression of GATA-2 in hematopoietic stem cells of AA leads to reduced HOXB4 transcription, which may have an important role in the development and/or progression of the disease. Disclosures: No relevant conflicts of interest to declare.

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