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ABSTRACT: Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs), yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast, highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations, one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1), have little intravenous engraftment potential, and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However, varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect.
Experimental hematology 06/2012; 40(10):857-866.e5. · 3.11 Impact Factor
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ABSTRACT: Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34(+) and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34(+) cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.
Journal of Biological Chemistry 12/2011; 287(8):5600-14. · 4.77 Impact Factor
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Maura Gasparetto,
Sanja Sekulovic,
Chad Brocker,
Patrick Tang,
Anush Zakaryan,
Ping Xiang,
Florian Kuchenbauer,
Maggie Wen,
Katayoon Kasaian,
Marie France Witty,
Patty Rosten,
Ying Chen, Suzan Imren,
Gregg Duester,
David C Thompson,
Richard Keith Humphries,
Vasilis Vasiliou,
Clay Smith
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ABSTRACT: High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes.
Experimental hematology 12/2011; 40(4):318-29.e2. · 3.11 Impact Factor
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ABSTRACT: Leukemia-initiating cells (LICs) have been the subject of considerable investigation because of their ability to self-renew and maintain leukemia. Thus, selective targeting and killing of LICs would provide highly efficient and novel therapeutic strategies. Here we explored whether we could use a canine leukemia cell line (G374) derived from a dog that received HOXB4-transduced repopulating cells to study leukemia in the murine xenograft model and the dog.
G374 cells were infused in dogs intravenously and in nonobese diabetic/severe combined immunodeficient and nonobese diabetic/severe combined immunodeficient/IL2Rγ(null) mice either intravenously or directly into the bone cavity. Animals were bled to track engraftment and proliferation of G374 cells, and were sacrificed when they appeared ill.
We found that canine LICs are capable of sustained in vitro self-renewal while maintaining their ability to induce acute myeloid leukemia, which resembles human disease in both dogs and mice. Furthermore, we developed a novel strategy for the quantification of LIC frequency in large animals and showed that this frequency was highly comparable to that determined by limited dilution in mouse xenotransplants. We also demonstrated, using single-cell analysis, that LICs are heterogeneous in their self-renewal capacity and regenerate a leukemic cell population consistent with a hierarchical leukemia model.
The availability of this novel framework should accelerate the characterization of LICs and the translation of animal studies into clinical trials.
Experimental hematology 10/2010; 39(1):124-32. · 3.11 Impact Factor
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ABSTRACT: Myeloid ectropic viral integration site 1 (MEIS1) is a Hox cofactor known for its role in development and is strongly linked to normal and leukemic hematopoiesis. Although previous studies have focused on identifying protein partners of MEIS1 and its transcriptionally regulated targets, little is known about the upstream transcriptional regulators of this tightly regulated gene. Understanding the regulation of MEIS1 is important to understanding normal hematopoiesis and leukemogenesis.
Here we describe our studies focusing on the evolutionary conserved putative MEIS1 promoter region. Phylogenetic sequence analysis and reporter assays in MEIS1-expressing (K562) and nonexpressing (HL60) leukemic cell line models were used to identify key regulatory regions and potential transcription factor binding sites within the candidate promoter region followed by functional and expression studies of one identified regulator in both cell lines and primary human cord blood and leukemia samples.
Chromatin status of MEIS1 promoter region is associated with MEIS1 expression. Truncation and mutation studies coupled with reporter assays revealed that a conserved ETS family member binding site located 289 bp upstream of the annotated human MEIS1 transcription start site is required for promoter activity. Of the three ETS family members tested, only ELF1 was enriched on the MEIS1 promoter as assessed by both electrophoretic mobility shift assay and chromatin immunoprecipitation experiments in K562. This finding was confirmed in MEIS1-expressing primary human samples. Moreover, small interfering RNA-mediated knockdown of ELF1 in K562 cells was associated with a decreased MEIS1 expression.
We conclude that the ETS transcription factor ELF1 is an important positive regulator of MEIS1 expression.
Experimental hematology 09/2010; 38(9):798-8, 808.e1-2. · 3.11 Impact Factor
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ABSTRACT: Development of strategies to extensively expand hematopoietic stem cells (HSCs) in vitro will be a major factor in enhancing the success of a range of transplant-based therapies for malignant and genetic disorders. In addition to potential clinical applications, the ability to increase the number of HSCs in culture will facilitate investigations into the mechanisms underlying self-renewal. In this unit, we describe a robust strategy for consistently achieving over 1000-fold net expansion of HSCs in short-term in vitro culture by using novel engineered fusions of the N-terminal domain of nucleoporin 98 (NUP98) and the homeodomain of the hox transcription factor, HOXA10 (so called NUP98-HOXA10hd fusion). We also provide a detailed protocol for monitoring the magnitude of HSC expansion in culture by limiting dilution assay of competitive lympho-myeloid repopulating units (CRU Assay). These procedures provide new possibilities for achieving significant numbers of HSCs in culture, as well as for studying HSCs biochemically and genetically.
Current protocols in stem cell biology 02/2008; Chapter 2:Unit 2A.7.
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ABSTRACT: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo.
ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice.
Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38, CD56, CD66b, or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360.
Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that, in combination with other markers, allows easy isolation of the stem cell fraction at unprecedented purities.
Haematologica 10/2007; 92(9):1165-72. · 6.42 Impact Factor
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ABSTRACT: Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of beta-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.
Journal of Clinical Investigation 11/2004; 114(7):953-62. · 15.39 Impact Factor
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Luke R Shier,
Kirk R Schultz, Suzan Imren,
Jill Regan,
Andrew Issekutz,
Irene Sadek,
Andrew Gilman,
Zhijuan Luo,
Tony Panzarella,
Connie J Eaves,
Stephen Couban
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ABSTRACT: A recent phase III trial comparing granulocyte colony-stimulating factor (G-CSF)-stimulated bone marrow (G-BM) and G-CSF-mobilized peripheral blood (G-PB) in matched sibling allograft recipients showed that G-BM produced a similar hematologic recovery but a reduced incidence of extensive chronic graft-versus-host disease, indicating differences in the cell populations infused. As a first step toward identifying these differences, we treated a group of healthy adult humans with 4 daily doses of G-CSF 10 microg/kg and monitored the effects on various hematopoietic and immune cell types in the PB and BM over 12 days. G-CSF treatment caused rapid and large but transient increases in the number of circulating CD34+ cells, colony-forming cells, and long-term culture-initiating cells and in the short-term repopulating activity detectable in nonobese diabetic/severe combined immunodeficiency/beta2-microglobulin-null mice. Similar but generally less marked changes occurred in the same cell populations in the BM. G-CSF also caused transient perturbations in some immune cell types in both PB and BM: these included a greater increase in the frequency of naive B cells and CD123+ dendritic cells in the BM. The rapidity of the effects of G-CSF on the early progenitor activity of the BM provides a rationale for the apparent equivalence in rates of hematologic recovery obtained with G-BM and G-PB allotransplants. Accompanying effects on immune cell populations are consistent with a greater ability of G-BM to promote tolerance in allogeneic recipients, and this could contribute to a lower rate of chronic graft-versus-host disease.
Biology of Blood and Marrow Transplantation 10/2004; 10(9):624-34. · 3.87 Impact Factor
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Suzan Imren,
Emmanuel Payen,
Karen A. Westerman,
Robert Pawliuk,
Mary E. Fabry,
Connie J. Eaves,
Benjamin Cavilla,
Louis D. Wadsworth,
Yves Beuzard,
Eric E. Bouhassira,
Robert Russell,
Irving M. London,
Ronald L. Nagel,
Philippe Leboulch,
R. Keith Humphries
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ABSTRACT: Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains
an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by
means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid β globin gene expression
has not yet been achieved in β thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment
and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid
correction of severe β thalassemia in mice, resulting from a homozygous deletion of the β major globin gene, by transplantation
of syngeneic bone marrow transduced with an HIV-1-derived [β globin gene/LCR] lentiviral vector also containing the Rev responsive
element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually
all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted
mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time ≈95% of the red
blood cells in all mice contained human β globin contributing to 32 ± 4% of all β-like globin chains. Hematological parameters
approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All
circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free α globin
chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated
in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment
can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events
of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation
of human clinical trials in β thalassemia patients.
Proceedings of the National Academy of Sciences 10/2002; 99(22):14380-14385. · 9.68 Impact Factor
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ABSTRACT: Histoincompatibility between murine donors and recipients of bone marrow (BM) transplants reduces engraftment, and this compromises assessment of hematopoietic stem cells (HSCs) in certain transgenic mice. To study HSCs in the S+S-Antilles mouse model of human sickle cell disease (SCD), we developed an autotransplant protocol. Initial experiments showed no differences between S+S-Antilles mice and normal C57BL/6 (+/+) mice in their radiosensitivity or baseline hematopoietic progenitor numbers. The kinetics of red blood cell (RBC) replacement post-transplant in +/+ recipients of mixtures of transgenic and +/+ BM cells also showed no competitive advantage of the +/+ cells. BM cells were then aspirated from mice 4 days after 5-fluorouracil treatment, transduced with a green fluorescent protein (GFP)-encoding retrovirus, and transplanted into the same recipients that, just before transplant, were irradiated with 800 cGy. We subsequently detected high levels of GFP(+) RBCs (21-79%) and white blood cells (WBCs; 35-88%) in the blood for 11 months and showed that transduced HSCs regenerated in the primary mice also repopulated secondary mice. These findings provide a generally applicable protocol for performing autotransplants in mice and forecast the potential utility of this approach in assessing HSC-based gene therapy protocols in transgenic mouse models of many human diseases.
Molecular Therapy 10/2002; 6(3):422-8. · 6.87 Impact Factor
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Suzan Imren,
Emmanuel Payen,
Karen A Westerman,
Robert Pawliuk,
Mary E Fabry,
Connie J Eaves,
Benjamin Cavilla,
Louis D Wadsworth,
Yves Beuzard,
Eric E Bouhassira,
Robert Russell,
Irving M London,
Ronald L Nagel,
Philippe Leboulch,
R Keith Humphries
[show abstract]
[hide abstract]
ABSTRACT: Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid beta globin gene expression has not yet been achieved in beta thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free alpha globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.
Proceedings of the National Academy of Sciences 10/2002; 99(22):14380-5. · 9.68 Impact Factor
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ABSTRACT: Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.
Blood 09/2002; 100(4):1257-64. · 9.90 Impact Factor
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ABSTRACT: Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.
