Decreased Expression of Stem Cell-Specific Genes in Aplastic Anemia; Possible Regulation of HOXB4 Gene by GATA-2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2821-2821
Author(s):  
Hisayuki Yokoyama ◽  
Hideo Harigae ◽  
Shinichiro Takahashi ◽  
Yoko Okitsu ◽  
Johji Yamamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Aplastic Anemia ◽  
Promoter Analysis ◽  
Consensus Sequence ◽  
Expression Level ◽  
Dependent Manner ◽  
Aberrant Expression ◽  
Hematopoietic Stem

Abstract Aplastic anemia (AA) is characterized by reduced hematopoiesis resulting in pancytopenia. It is suggested that a certain immunological attack to hematopoietic stem cells play an important role in developing AA. However, limited information is available for the intrinsic characteristics of stem cells in AA. Previous work in our laboratory showed decreased expression of GATA-2 gene in CD34 positive cells in AA, suggesting that there is an aberrant expression of stem cell-specific genes in stem cells in AA. Recently it is emerged that some genes such as HOXB4 and BMI-1, function for the proliferation and maintenance of stem cells. In this study, we examined expression levels of HOXB4 and BMI-1 in CD34 positive cells by quantitative PCR in 10 patients with AA and 13 with idiopathic thrombocytopenic purpura (ITP). Between these two factors, the expression level of HOXB4 was markedly decreased in AA compared with in ITP, whereas that of BMI-1 was not. Moreover, the expression level of GATA-2 in these populations was significantly correlated to HOXB4 gene expression (Spearman’s rank correlation, r=0.6573 p<0.01) compared to that of BMI-1(r=0.4107, p>0.05). As they functions as a transcription factor, these results raise the possibility that GATA-2 and HOXB4 regulate each other. To explore this possibility, first, we cloned HOXB4 5′flanking region by PCR and performed promoter analysis. Since the previous report showed that the region from −164 to −116 was important for promoter activity of HOXB4 gene, we focused on a GATA element located at −160. When this element was deleted, the reporter activity was decreased to 60% of wild-type in K562 cells. Furthermore, co-transfection of GATA-2 expression vector significantly activates the reporter gene in a dose dependent manner. EMSA revealed that GATA-2 binds specifically to this element. On the other hand, the active region both of exon 1S and 1G promoter of GATA-2 gene, which was identified by the promoter analysis, did not contain the consensus sequence recognized by HOXB4. These findings suggest that in stem cells in AA, the decreased expression of GATA-2 gene lead to the reduced HOXB4 gene expression, which may responsible for the development of the disease.

Evi-1 is a transcriptional target of mixed-lineage leukemia oncoproteins in hematopoietic stem cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6304-6314 ◽  
Author(s):  
Shunya Arai ◽  
Akihide Yoshimi ◽  
Munetake Shimabe ◽  
Motoshi Ichikawa ◽  
Masahiro Nakagawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Mixed Lineage Leukemia ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation, overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities, which indicates the presence of other mechanisms for Evi-1 activation. In this study, we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL–mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population, in which Evi-1 particularly contributes to the propagation of MLL-ENL–immortalized cells. Furthermore, gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell–like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell–derived MLL leukemic cells.

Leukemic CD34+CD38- Cells Display Conserved Differential Expression of Many ATP Binding-Cassette Transporter Genes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1380-1380
Author(s):  
Marc H.G.P. Raaijmakers ◽  
Elke P.L.M. de Grouw ◽  
Louis T.F. van de Locht ◽  
Bert A. van der Reijden ◽  
Theo J.M. de Witte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Abc Transporters ◽  
Cell Biology ◽  
Cell Fraction ◽  
Atp Binding ◽  
Stem Cell Biology ◽  
Hematopoietic Stem ◽  
Atp Binding Cassette

Abstract In most cases of acute myeloid leukemia (AML) CD34+CD38− cells are considered to be stem cells, responsible for the maintenance and relapse of AML. ATP binding cassette transporters function in the extrusion of xenobiotics and chemotherapeutical compounds, and may be involved in therapy resistance. Elucidation of mechanisms conferring drug resistance to CD34+CD38− cells is essential to provide novel targets for stem cell eradication in AML. We studied gene expression of all 45 transmembrane ABC transporters (the complete ABCA, B, C, D and G family) in human hematopoietic CD34+CD38− cells and more committed CD34+CD38+ progenitor cells, from healthy donors and patients with non-hematological diseases (N=11) and AML patients (N=11). Gene expression was assessed using a novel real-time RT-PCR approach with micro fluidic cards. In normal CD34+CD38− cells 36 ABC transporters were expressed, 22 of these displayed significant higher expression in the CD34+CD38− cell fraction compared to the CD34+CD38+ cell fraction. In addition to the known stem cell transporters (ABCB1, ABCC1 and ABCG2) these differential expressed genes included many members not previously associated with stem cell biology. In AML the ABC transporter expression profile was largely conserved, including expression of all 13 known drug transporters. These data suggest an important role for many ABC transporters in hematopoietic stem cell biology. In addition, the preferential expression of a high number of drug transport related transporters predicts that broad spectrum inhibition of ABC transporters is likely to be required for CD34+38− stem cell eradication in AML. This approach will, apart from affecting the leukemic stem cells, equally affect the normal stem cells.

Clonal Expansion in Paroxysmal Nocturnal Hemoglobinuria (PNH): Expression of Mutant HMGA2 Suggests That PNH Is a Benign Tumor of the Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3533-3533 ◽  
Author(s):  
Norimitsu Inoue ◽  
Tomohisa Izui ◽  
Yoshiko Murakmai ◽  
Yuichi Endo ◽  
Jun-ichi Nishimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Aplastic Anemia ◽  
Benign Tumor ◽  
Bone Marrow Cells ◽  
Clonal Expansion ◽  
Mesenchymal Tumors ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Mutant Cells

Abstract In PNH, a somatic mutation of PIGA in hematopoietic stem cells causes the deficiency of glycosylphosphatidylinositol-anchored proteins (GPI-AP), but the basis of clonal expansion of the PIGA-mutant cells is speculative. Because some patients with aplastic anemia develop PNH, GPI-AP deficient stem cells may have a survival advantage in the setting of immune-mediated bone marrow injury. However, in many patients with aplastic anemia, the GPI-AP deficient cell populations remain small or disappear. Therefore, we hypothesized that additional abnormalities in the PIGA-mutant stem cells account for clonal expansion. We previously reported a patient with PNH/aplastic anemia (J20) whose PIGA-mutant hematopoietic cells had a coexistent cytogenetic abnormality [46,XX,ins(12;12)(q14;q12q14)]. In this patient, the insertion disrupted the 3′ untranslated region of HMGA2, an architectural transcription factor whose aberrant expression causes benign mesenchymal tumors. Truncated HMGA2 transcripts lacking the acidic tail (the pathophysiologically relevant form) were identified in the double mutant cells. In the present study, we characterized a similar genetic abnormality in a patient with classical PNH. In this case, PIGA-mutant cells again had a concurrent der(12) [46,XX,ins(12)(p13q14q13)]. A 20 Mbp fragment from 12q13 to q14 and a 300bp fragment from 12q14 together containing exons 1–4 and part of exon 5 of HMGA2 were inserted inversely and directly, respectively, into intron 1 of TEL at 12p13. One of the breakpoints in the HMGA2 locus was at almost the same position as the HMGA2 breakpoint in patient J20. Truncated HMGA2 transcripts (lacking the acidic tail) were highly expressed in bone marrow cells. Full-length transcripts of TEL without any fusion partners were normally expressed and no other transcription units were disrupted by the breakpoints. That similar cytogenetic abnormalities were observed in these two patients suggests that aberrant expression of HMGA2, in concert with mutant PIGA, accounted for the clonal expansion and explains the benign tumor characteristics of PNH.

Predicting Response to Immunosuppressive Therapy in Patients with Acquired Aplastic Anemiausing EGFR and TWIST1 Expression Levels

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5076-5076 ◽  
Author(s):  
Sharat Damodar ◽  
Varun Bafna ◽  
Arati Khanna-Gupta ◽  
Shilpa Prabhu ◽  
Nataraj KS ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Partial Response ◽  
Aplastic Anemia ◽  
Clinical Response ◽  
Immunosuppressive Therapy ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Hsc Niche

Abstract INTRODUCTION EGFR (epidermal growth factor receptor) is important for the proliferation of stem cells across the body including the hematopoietic niche. However, the role of EGFR in aplastic anemia and subsequent responses to standard-of-care therapy is unknown.TWIST is a basic helix-loop-helix transcription factor recently found to regulate the hematopoietic stem cell (HSC) niche. The HSC niche is important for treatment of aplastic anemia. Telomerase and associated gene mutations have been reported in aplastic anemia, but these mutations are not present in all subjects and hence the cellular mechanisms of therapeutic responses observed is not explained by deregulated telomerase or associated genes. OBJECTIVE: To investigate the utility of measuring gene expression levels of EGFR and TWIST on the clinical response to immunosuppressive therapy in acquired Aplastic anemia patients without mutations in telomerase gene. METHODS: This was a single institution analysis of patients with acquired Aplastic anemia, in the age group of 16 to 60 years, who were treated with immunosuppressive therapy between June 2014 to December 2015. 15 patients who did not have homozygous TERT (telomerase catalytic subunit) or DKC (Dyskeratosiscongenita) mutations as determined by sequencing were included in this study. Diagnosis of Aplastic anemia was established with bone marrow aspiration and biopsy with normal cytogenetics. PNH was ruled out in all patients. 7 patients had very severe aplasticanemia (VSAA) and 8 had severe aplastic anemia. There were 11 males and 4 females.Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TWIST and EGFR expression compared to that of normal samples (n=6).15 patients underwent immunosuppressive therapy with horse ATG at 40mg/kg/day for 4 days followed by oral cyclosporine for at least 3 months. Clinical response was assessed at 3 months and 6 months post ATG administration. Total RNA from healthy donors (6) were used to establish normal baseline gene expression values. RESULTS: Out of the 15 patients that received ATG infusion, 10 patients (66%) had an 8-fold reduced expression of EGFR and TWIST compared to normal control. We grouped the study subjectsintoEGFR/TWIST-low (C1) and EGFR/TWIST-normal(C2). C1 subjects were younger in age (average age 29) compared with C2 (average age 40) andwere more likely to be diagnosed with a more severe form of the disease (VSAA). In the C1 group, all patients responded well to treatment with 4 (80%) patients achieving CR and 1 patient achieving a partial response. In C2, 8 (80%) patients showed no response, with 2 patients showing a partial response at the end of 6 months. CONCLUSION: Our data suggests that EGFR and TWIST may have significant impact on the ability of the hematopoietic stem cell niche to respond to immunosuppressive therapy in aplastic anemia, particularly in the absence of telomerase mutations. Therefore, low expression levels of EGFR/TWIST at diagnosis in may be useful in predicting response to immunosupressive therapy and thereby influence treatment decisions in aplastic anemia patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells

2021 ◽  
Author(s):  
Cai Ping Koh ◽  
Avinash Govind Bahirvani ◽  
Chelsia Qiuxia Wang ◽  
Tomomasa Yokomizo ◽  
Cherry Ee Lin Ng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Genetic Engineering ◽  
Developmental Stages ◽  
Genetic Element ◽  
Malignant Diseases ◽  
Hematopoietic Stem ◽  
Gene Ablation

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs which are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

scholarly journals Emilin-2 is a component of bone marrow extracellular matrix regulating mesenchymal stem cell differentiation and hematopoietic progenitors

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Francesco Da Ros ◽  
Luca Persano ◽  
Dario Bizzotto ◽  
Mariagrazia Michieli ◽  
Paola Braghetta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Adipogenic Differentiation ◽  
Stem Cell Differentiation ◽  
Dependent Manner ◽  
Hematopoietic Stem

Abstract Background Dissection of mechanisms involved in the regulation of bone marrow microenvironment through cell–cell and cell–matrix contacts is essential for the detailed understanding of processes underlying bone marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma. Methods Immunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry. Results Emilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging. Conclusion Our data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.

Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 894-901 ◽  
Author(s):  
Christopher A. Klug ◽  
Samuel Cheshier ◽  
Irving L. Weissman
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter. Limiting dilution reconstitution of lethally irradiated recipient mice with 100% transduced, GFP+ LT-HSC showed that silencing of gene expression occurred rapidly in most integration events at the LT-HSC level, irrespective of the initial levels of GFP expression. When inactivation occurred at the LT-HSC level, there was no GFP expression in any hematopoietic lineage clonally derived from silenced LT-HSC. Inactivation downstream of LT-HSC that stably expressed GFPin long-term reconstituted animals was restricted primarily to lymphoid cells. These observations suggest at least 2 distinct mechanisms of silencing retrovirally expressed genes in hematopoietic cells.

MicroRNA Expression Profiling in Sorted AML Subpopulations: A Possible Role for miR-155/BIC in Stem Cell Maintenance and Leukemogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 466-466 ◽  
Author(s):  
Eric R. Lechman ◽  
Kristin J. Hope ◽  
Fernando J. Suarez Saiz ◽  
Katsuto Takenaka ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
B Cell ◽  
Lentiviral Vectors ◽  
Array Analysis ◽  
Hematopoietic Stem ◽  
Cell State ◽  
Mirna Array ◽  
Stem Cell State

Abstract MicroRNAs (miRNAs) are a new class of non-coding small RNAs that negatively regulate the expression of protein-encoding genes. Mature miRNAs are excised sequentially from primary miRNA (pri-miRNA) foldback precursor transcripts, and regulate gene expression at the post-transcriptional level. miRNAs functionally suppress gene expression by either inhibition of protein synthesis or by direct cleavage of the target mRNA. miRNA expression is tissue and developmental stage restricted, suggesting important roles in tissue specification and/or cell lineage determination. miRNAs are implicated in the regulation of diverse processes including cell growth control, apoptosis, fat metabolism and insulin secretion, and may be involved in the maintenance of the embryonic stem cell state. Several recent lines of evidence suggest a role for miRNAs in hematological malignancies. Many characterized miRNAs are located at fragile sites, minimal loss of heterozygosity regions, minimal regions of amplification or common breakpoint regions in human cancers. For example, chromosomal translocation t(8;17) in an aggressive B-cell leukemia results in fusion of miR-142 precursor and a truncated MYC gene. Furthermore, both miR-15 and miR-16 are located within a 30 kb deletion in CLL, and in most cases of this cancer both genes are deleted or underexpressed. In addition, mice transplanted with hematopoietic stem cells (HSC) overexpressing both c-Myc and the miR-17–92 polycistron developed cancers earlier with a more aggressive nature when compared to lymphomas generated by c-myc alone. To address the role of miRNAs in the regulation and maintenance of the hematopoietic stem cell state and leukemogenesis, we sorted 6 primary AML patient samples into 4 populations based on the expression of CD34/CD38 and performed miRNA array analysis. We identified a subset of miRNAs whose expression profile could discriminate the CD34+/CD38- fractions from more mature populations. In particular, BIC/miR-155 was found to be over-expressed in leukemic stem cells (LSC). Validation by qRT-PCR revealed this expression pattern in 5 of the 6 sorted AML samples. Furthermore, within umbilical cord blood (CB) cells, BIC/miR-155 is more highly expressed in the primitive CD34+38- fraction as compared to mature sub-fractions as assessed by Affymetrix microarray. miRNA array analysis also revealed elevated levels of miR-155 in bulk primary AMLs as compared to normal BM. Intriguingly, BIC/miR-155 was first identified as a common retroviral insertion site in avian leucosis virus induced B cell lymphomas, and BIC/miR-155 overexpression has been observed in all subtypes of Hodgkin’s lymphoma. To test the hypothesis that miR-155 is important in LSC/HSC function, we designed lentiviral vectors for RNAi mediated knockdown of BIC/miR-155. Knockdown of BIC/miR-155 within a novel CD34+ leukemic cell line resulted in a loss of CD34 expression and reduced proliferative potential. Additionally, knockdown within CB led to alterations in colony forming capacity. Additionally, we have recently generated lentiviral vectors for the enforced overexpression of BIC/miR-155. In vivo studies to investigate the effects of BIC/miR-155 over-expression and knockdown are ongoing and will be discussed.

A New Flowcytometry-Based Method to Discriminate Malignant From Normal Stem Cells in CML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3245-3245
Author(s):  
Jeroen J.W.M. Janssen ◽  
Wendy Deenik ◽  
Karlijn G.M. Smolders ◽  
Monique Terwijn ◽  
Angele Kelder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Risk Scores ◽  
Fish Analysis ◽  
Leukemic Stem Cells ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Culture Techniques

Abstract Abstract 3245 Poster Board III-182 Tyrosine kinase inhibitor (TKI) insensitivity of CML hematopoietic stem cells prevents eradication of the disease by these drugs and is presumably implicated in development of TKI resistance. Probably, improvement of treatment results will involve leukemic stem cell directed therapy. Therefore, more knowledge of stem cell specific targets would be instrumental. Previously, leukemic stem cells could only be identified indirectly by using culture techniques. We developed a new flowcytometric approach that enables to directly distinguish CML stem cells from their normal counterparts within single patient samples. In 24 newly diagnosed CML patients CML CD34+CD38- stem cells could be discriminated from normal stem cells by higher CD34 and CD45 expression and different forward/sideward light scatter properties, reflecting differences in size and granularity. In addition, aberrant expression of CD7, CD11b and CD56 was demonstrated on malignant stem cells, allowing clear discrimination from benign stem cells, that were always negative for these markers. Above all, in all tested CML patients we were able to demonstrate that high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. FISH analysis on FACS sorted cells proved that populations were BCR-ABL positive (in case of high CD34 and CD45 expression and high CD90 expression) or negative (in case of low CD34 and CD45 expression and low CD90 expression), while long term liquid culture assays with subsequent CFU assays and FISH analysis proved their malignant/normal stem cell character. Patients with a large proportion of non-leukemic stem cells had significantly lower clinical risk scores (Sokal, Euro) than patients with few remaining normal stem cells. This new technique will expand our possibilities to identify new CML stem cell specific targets and may improve efficacy assessment of CML treatment as well. Disclosures No relevant conflicts of interest to declare.

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