The First Zinc Finger Domain of EVI1 Enhances Self-Renewal of Hematopoietic Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1237-1237
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Giuseppina Nucifora
Keyword(s):  
Bone Marrow ◽  
Zinc Finger ◽  
Cell Clone ◽  
Integration Site ◽  
The Self ◽  
Mouse Strains ◽  
Zinc Finger Domain ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract EVI1 was first identified as a preferential integration site of ecotropic retroviruses in the MDS1/EVI1 genomic locus leading to myeloid tumors in susceptible mice. Later studies showed that retroviral integration in the MDS1/EVI1 locus results in the emergence of a non-malignant dominant hematopoietic stem cell clone in non-susceptible mice strains, in non-human primates, and in patients, suggesting that a gene encoded by the locus could be deregulated by the retrovirus and affect the self-renewal potential of the cell. The locus encodes two genes. One of them, EVI1, has long been associated with myeloid leukemia and myelodysplastic syndrome. To understand whether EVI1 has a role in self-renewal control, we forcibly expressed EVI1 in the bone marrow progenitors of two mouse strains that differ in their proliferation and self-renewal potential. By comparing the response of the hematopoietic cells to EVI1, we show that EVI1 has a role in prolonging the self-renewal potential of the cells and that this ability of EVI1 is however limited and modulated by inherent strain-specific characteristics. To identify the region of EVI1 mediating this effect, we infected the bone marrow progenitors of the two murine strains with EVI1 wild type or with specific alternative EVI1 point mutants and compared the self-renewal potential of the cells. This approach allowed us to show that the first zinc finger domain of EVI1 is required to enhance the self-renewal of the hematopoietic progenitors. This function is mediated by two specific zinc finger motifs and their disruption by point mutations abolished this effect. These results suggest that the motifs interact with factors that regulate self-renewal. The cooperation between EVI1 and these unknown proteins in deregulation of self-renewal occurs inappropriately when EVI1 is deregulated, but based on EVI1 gene knock out studies it is likely that this function of EVI1 is required during embryonic development.

scholarly journals Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 386-386 ◽  
Author(s):  
Grant A. Challen ◽  
Deqiang Sun ◽  
Mira Jeong ◽  
Min Luo ◽  
Jaroslav Jelinek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Donor Cell ◽  
The Self ◽  
Blood Cell Count ◽  
Serial Transfer ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiation Capacity

Abstract Abstract 386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

EVI1 Is Activated During the Generation of Induced Pluripotent Stem (iPS) Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5048-5048
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Nuria Montserrat ◽  
Alessandra Giorgetti ◽  
Juan Carlos Izpisúa Belmonte
Keyword(s):  
Stem Cell ◽  
Conflicts Of Interest ◽  
Integration Site ◽  
Ips Cells ◽  
The Self ◽  
Genomic Locus ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Retroviral Integration ◽  
Induced Pluripotent

Abstract Abstract 5048 EVI1 gene was first identified as a common site of retroviral integration in murine leukemia models. This gene is part of a complex genomic locus, MDS1-EVI1, that has been described as a target for retroviral integration that may lead to the emergence of a non-malignant dominant hematopoietic stem cell (HSC) clone in mice, in primates, and in humans. These studies suggested that one of the genes encoded by this locus could affect the self-renewal potential of HSC. Recent studies in mice revealed that indeed EVI1 plays an essential role in cell proliferation and it also enhances the self-renewal ability of HSC. The intense attention focused on the MDS1-EVI1 locus as retrovirus integration site prompted us to investigate whether EVI1 might have a role in somatic cell re-programming generated with retroviruses. Recent developments in stem cell research have enabled the re-programming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cells types and that might be used in the future for clinical therapy. In order to elucidate the molecular events allowing the conversion of adult somatic into pluripotent stem cell, we evaluated EVI1 expression during this process. We found that EVI1 is activated in the early stages of re-programming and then it is silenced once the cells has been fully re-programmed. EVI1 seems to facilitate the initiation of cell re-programming by up-regulating a subset of genes previously described as potent stimulators of stem cells expansion. Disclosures No relevant conflicts of interest to declare.

Molecular Integration Of HoxB4 and STAT3 For Self-Renewal Of Hematopoietic Stem Cells: A Model Of Molecular Convergence For Stemness

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2444-2444
Author(s):  
Il-Hoan Oh ◽  
Kim Tae-Min ◽  
Jae-Seung Shim
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Functional Integration ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell State ◽  
Stem Cell State

Abstract Multiple transcription factors (TFs) that regulate the self-renewal/stem cell state of hematopoietic stem cells (HSCs) have been identified, but understanding the molecular interplay of these TFs for their functional coordination remains a challenging issue. In this study, we investigated the functional integration and transcriptional coordination of STAT3 and HoxB4, which are TFs known to have similar effects on the self-renewal of HSCs. We found that while STAT3 (STAT3-C) or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both STAT3-C and HoxB4 did not have any additive enhancing effects. In contrast, the overexpression of HoxB4 caused a ligand-independent Tyr-phosphorylation in STAT3, and the inhibition of the STAT3 activity in HoxB4-overexpressing bone marrow cells significantly abrogated the enhancing effects of HoxB4 on both the bone marrow repopulation and maintenance of the undifferentiated state, revealing a molecular integration of these two TFs for HSC self-renewal. Expression microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis (GSEA) for TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3 showed significant overlap in the candidate TFs. Interestingly, among these identified TFs were the puripotency-related genes, Oct-4 and Nanog. These results indicate the functional integration of tissue-specific TFs for HSC self-renewal and provide insights into the functional convergence of various TFs towards a conserved transcription program for the stem cell state. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice

2006 ◽  
Vol 203 (10) ◽  
pp. 2247-2253 ◽  
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Yohei Morita ◽  
Takehiko Kamijo ◽  
Maarten van Lohuizen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Polycomb Group ◽  
The Self ◽  
Differential Impact ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Loss Of Self ◽  
Deficient Mice

The polycomb group (PcG) protein Bmi1 plays an essential role in the self-renewal of hematopoietic and neural stem cells. Derepression of the Ink4a/Arf gene locus has been largely attributed to Bmi1-deficient phenotypes in the nervous system. However, its role in hematopoietic stem cell (HSC) self-renewal remained undetermined. In this study, we show that derepressed p16Ink4a and p19Arf in Bmi1-deficient mice were tightly associated with a loss of self-renewing HSCs. The deletion of both Ink4a and Arf genes substantially restored the self-renewal capacity of Bmi1−/− HSCs. Thus, Bmi1 regulates HSCs by acting as a critical failsafe against the p16Ink4a- and p19Arf-dependent premature loss of HSCs. We further identified a novel role for Bmi1 in the organization of a functional bone marrow (BM) microenvironment. The BM microenvironment in Bmi1−/− mice appeared severely defective in supporting hematopoiesis. The deletion of both Ink4a and Arf genes did not considerably restore the impaired BM microenvironment, leading to a sustained postnatal HSC depletion in Bmi1−/−Ink4a-Arf−/− mice. Our findings unveil a differential role of derepressed Ink4a and Arf on HSCs and their BM microenvironment in Bmi1-deficient mice. Collectively, Bmi1 regulates self-renewing HSCs in both cell-autonomous and nonautonomous manners.

The Role of Truncated c-kit Receptor (tr-kit) in Maintenance and Differentiation of Hematopoietic Stem Cells and Multipotent Hematopoietic Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4193-4193 ◽  
Author(s):  
Jie Yang ◽  
Candice I. Saltiel ◽  
Ronald G. Nachtman ◽  
Xin Jing ◽  
Roland Jurecic
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Signaling Pathways ◽  
Rna Binding ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Multipotent Progenitors ◽  
Kit Receptor

Abstract Intrinsic mechanisms that regulate self-renewal of mammalian stem cells are slowly being elucidated. Self-renewal of stem cells in Drosophila and C. elegans is regulated by members of the conserved Pumilio family of RNA-binding proteins. Previously, we have cloned and characterized two mouse and human Pumilio genes (Pum1 and Pum2), which are abundantly transcribed in hematopoietic stem cells (HSC). To study the function of mammalian Pum proteins in HSC and multipotent progenitors, the RNA-binding domain of Pum2 was over-expressed in a stem cell factor (SCF)-dependent HSC-like cell line EML. In the presence of SCF EML cells undergo SCF-dependent self-renewal and remain undifferentiated. In the presence of various cytokines (IL-3, GM-CSF, G-CSF, Epo, Tpo, IL-7, Flt3L) EML cells differentiate into erythroid, granulocytic, megakaryocytic and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and suppresses their mutilineage differentiation in the absence of SCF. This uncoupling of the maintenance and differentiation signals in EML cells is accompanied by (a) an increased expression of the full-length c-kit and a novel truncated c-kit receptor called tr-kit, (b) cell intrinsic, SCF-independent activation of c-kit, and (c) constitutive activation of MAPK, PI3K and PLCγ signaling pathways in the absence of SCF. These results indicate that Pum2 could be supporting maintenance of multipotent hematopoietic cells through regulation of SCF/c-kit signaling pathway. An in depth analysis of the pattern of tr-kit expression in murine fetal liver and bone marrow-derived HSC, multipotent progenitors, lineage-committed progenitors and immature blood cells has shown that tr-kit expression is restricted to cell populations highly enriched for HSC and multipotent progenitors. This observation and the finding that an increased expression of tr-kit protein correlates with SCF-independent maintenance of EML cells, suggest that tr-kit could play an important role in SCF-independent activation of full-length c-kit receptor, and participate in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. The fact that Pum2 and tr-kit are co-expressed in bone marrow cells enriched for HSC and early multipotent progenitors (e.g. Lin-Sca-1+c-kit+ cells), but not in later progenitors (e.g. Lin-Sca-1−c-kit− cells), suggests an exciting possibility that HSC and early multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways. In contrast, more differentiated progenitors that lack self-renewal ability and do not express tr-kit, utilize only the canonical SCF-induced c-kit signaling. In this hypothetical model, the survival and maintenance of HSC and multipotent hematopoietic progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c-kit signaling. We are currently studying the effects of Pum2 and tr-kit over-expression and attenuation on (a) HSC and progenitor cell maintenance and differentiation, (b) HoxB4 and Notch1 pathways, involved in HSC maintenance and expansion, and (c) maintenance and differentiation of HSC expressing SLAM receptor CD150. Further study of Pum2 and tr-kit function could provide important new insights into the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation.

Id2 Is Required for the Self-Renewal and Proliferation of Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1173-1173 ◽  
Author(s):  
Lei Sun
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditional Knockout ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The production of mammalian blood cells is sustained throughout life by the self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation in this system leads to different pathologies including anemia, bone marrow failure and hematopoietic malignancies. The Helix-Loop-Helix transcriptional regulator Id2 plays essential roles in regulating proliferation and cell fate of hematopoietic progenitors; however, its role in regulating HSC development remains largely unknown. To assess the function of Id2 in HSCs, we developed two mouse models, including an Id2 conditional knockout model and an Id2-EYFP model, in which EYFP expression is driven by endogenous Id2 promoter. When we examined HSC function by serial transplantation, we found that mice transplanted with Id2F/F Mx1-Cre+ conditionally deleted bone marrow cells became moribund more rapidly after primary and secondary transplantation, compared to those transplanted with Id2+/F Mx1-Cre+ bone marrow, suggesting that HSC self-renewal is impaired when Id2 is deleted. To further determine if self-renewal and maintenance of HSCs depends on the expression level of Id2, we purified HSCs with different levels of Id2 expression using Id2-EYFP mice to specifically address the role of Id2 in HSCs. First, we confirmed Id2 is highly expressed in HSCs in this model. Second, when HSCs with either low or high levels of Id2-EYFP were transplanted into irradiated mice, cells with high levels of Id2 reconstituted transplanted recipients faster than those with low levels of Id2 at 3 weeks and longer, suggesting that Id2 expression is associated with repopulation advantage. Furthermore, Ki-67 staining showed that HSCs with high levels of Id2 have 15-fold more cells in G2/M phase, and fewer cells in G0. BrdU staining also suggested that there are 5-fold more BrdU+ cells in HSCs with high levels of Id2, indicating that Id2 expression correlates with cell cycle progression in HSCs. In addition, p57 has been reported to be required for quiescence of HSCs. Our preliminary data showed that p57 is downregulated in HSCs with high levels of Id2, and p57 is correspondingly upregulated in Id2-null HSCs. Altogether, our data demonstrate that Id2 is required for the self-renewal and proliferation of HSCs, and suggest a link between Id2 and the transcriptional regulatory networks that regulate the functional hematopoietic system. Since Id2 is also expressed in other adult stem cells including muscle and neuronal stem cells, as well as cancer cells, we believe our results can improve our understanding of stem cell biology and cancer development, and contribute to the identification of novel molecules that may be targeted to eliminate cancer stem cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Deterministic regulation of hematopoietic stem cell self-renewal and differentiation

Blood ◽  
2002 ◽  
Vol 100 (4) ◽  
pp. 1302-1309 ◽  
Author(s):  
Christa E. Müller-Sieburg ◽  
Rebecca H. Cho ◽  
Marilyn Thoman ◽  
Becky Adkins ◽  
Hans B. Sieburg
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Lymphoid Cells ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Direct Corollary ◽  
Kinetics Of ◽  
Adult Bone

Most current theories assume that self-renewal and differentiation of hematolymphoid stem cells (HSCs) is randomly regulated by intrinsic and environmental influences. A direct corollary of these tenets is that self-renewal will continuously generate functionally heterogeneous daughter HSCs. Decisions about self-renewal versus commitment are made by individual, single HSCs and, thus, require examination on the clonal level. We followed the behavior of individual, clonally derived HSCs through long-term, serial repopulation experiments. These studies showed that daughter HSCs derived from individual clones were remarkably similar to each other in the extent and kinetics of repopulation. Moreover, daughter HSCs within a clone showed equivalent contributions to the myeloid or lymphoid lineages. Lineage contribution could be followed because of the discovery of a new subset of HSCs that gave rise stably to skewed ratios of myeloid and lymphoid cells. Overall, the data argue that self-renewal does not contribute to the heterogeneity of the adult HSC compartment. Rather, all HSCs in a clone follow a predetermined fate, consistent with the generation-age hypothesis. By extension, this suggests that the self-renewal and differentiation behavior of HSCs in adult bone marrow is more predetermined than previously thought.

scholarly journals Real-time Expression of Stemness and Self-Renewal Genes in Canine Hematopoietic Progenitor Cells

2019 ◽  
Vol 39 (03) ◽  
Author(s):  
S Nandhini ◽  
Gowri A Mangala ◽  
G R Baranidharan ◽  
T V Meenambigai
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Integration Site ◽  
The Body ◽  
The Self ◽  
Hematopoietic Progenitor Cells ◽  
Common Source ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem

Blood cells are responsible for constant maintenance and immune protection of every cell type of the body and this relentless and brutal work requires cells that have the greatest powers of self-renewal and are designated as Hematopoietic progenitor cells (HPCs).Peripheral blood stem cells in circulation have become the most common source of hematopoietic stem cells intended for transplantation after minimal manipulation. Homeo box (Hox), sonic hedgehog (SHH), and Wingless-type MMTV integration site family (Wnt) are known to modulate the self-renewal and expansion of hematopoietic progenitor/stem cells in humans and mice. Unlike cytokines, Hox, SHH, and Wnt are highly conserved among species from flies to humans but studies regarding the self-renewal and expansion of the HSC are extremely limited in dogs.

scholarly journals Identification of a lineage of multipotent hematopoietic progenitors

Development ◽  
1997 ◽  
Vol 124 (10) ◽  
pp. 1929-1939 ◽  
Author(s):  
S.J. Morrison ◽  
A.M. Wandycz ◽  
H.D. Hemmati ◽  
D.E. Wright ◽  
I.L. Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cd4 Cells ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Functional Activities ◽  
Multipotent Progenitors ◽  
Lethal Irradiation

All multipotent hematopoietic progenitors in C57BL-Thy-1.1 bone marrow are divided among three subpopulations of Thy-1.1(lo) Sca-1+ Lin(-/lo) c-kit+ cells: long-term reconstituting Mac-1- CD4- c-kit+ cells and transiently reconstituting Mac-1(lo) CD4- or Mac-1(lo) CD4(lo) cells. This study shows that the same populations, with similar functional activities, exist in mice whose hematopoietic systems were reconstituted by hematopoietic stem cells after lethal irradiation. We demonstrate that these populations form a lineage of multipotent progenitors from long-term self-renewing stem cells to the most mature multipotent progenitor population. In reconstituted mice, Mac-1- CD4- c-kit+ cells gave rise to Mac-1(lo) CD4- cells, which gave rise to Mac-1(lo) CD4(lo) cells. Mac-1- CD4- c-kit+ cells had long-term self-renewal potential, with each cell being capable of giving rise to more than 10(4) functionally similar Mac-1- CD4- c-kit+ cells. At least half of Mac-1(lo) CD4- cells had transient self-renewal potential, detected in the spleen 7 days after reconstitution. Mac-1(lo) CD4(lo) cells did not have detectable self-renewal potential. The identification of a lineage of multipotent progenitors provides an important tool for identifying genes that regulate self-renewal and lineage commitment.

CD41 and Mpl Positive Fraction Shows an Efficient Leukemia-Initiating Capacity in Evi1-Induced Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1295-1295
Author(s):  
Satoshi Nishikawa ◽  
Shunya Arai ◽  
Naoko Watanabe-Okochi ◽  
Mineo Kurokawa
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Leukemia Cells ◽  
Rank Test ◽  
Hematopoietic Progenitors ◽  
Aberrant Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Semisolid Medium

Abstract Abstract 1295 Ecotropic viral integration site 1 (Evi1) is a transcription factor which is highly expressed in hematopoietic stem cells and crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in several types of acute myeloid leukemia (AML), of which the prognosis is generally poor, mainly due to therapeutic resistance. Effective Evi1-targeted therapies, however, have not yet been developed. Therefore, cell surface molecules or their downstream signaling pathways specific to Evi1 high-expressing leukemia cells would be good candidates for novel targeted therapy. Here in this study, we first revealed by gene expression data of AML patients that the expression of several megakaryocytic differentiation markers including CD41, CD61 and thrombopoietin (Thpo) receptor, Mpl was positively correlated to that of Evi1. To validate the association between these surface markers and Evi1, murine c-kit(+) hematopoietic progenitors were transduced with Evi1 or other several leukemia oncogenes and serially replated in methylcellulose semisolid medium. FACS analysis showed that the CD41(+) fraction emerged after third or fourth replating in Evi1-transduced progenitors, but not in progenitors immortalized by MLL-ENL or AML1-ETO. Similarly, the CD41(+) fraction was clearly observed in the bone marrow and spleen mononuclear cells of Evi1-induced leukemia mice. These results indicated the association between Evi1 and CD41 in AML. We next investigated the functional significance of the CD41(+) population in the Evi1-induced leukemia mouse model. The CD41(+) leukemia cells were morphologically more immature than the CD41(-) cells. Interestingly, the CD41(+) cells were highly positive for c-kit, and showed an enhanced colony-forming capacity in semisolid medium than the CD41(-) cells. Moreover, the CD41(+) cells developed AML with shorter latency than the CD41(-) cells in a murine bone marrow transplantation model (p<0.05, log-rank test). These results demonstrated that leukemia-initiating cells are more enriched in the CD41(+) fraction compared with the CD41(-) counterpart. Although the CD41(+) fraction was also observed in hematopoietic progenitors transduced with another leukemia oncogene, PML-RARA, these CD41(+) cells had differentiated and lost self-renewal capacity, which was in stark contrast to the case of Evi1. Remarkably, we found that Mpl was predominantly expressed in the CD41(+) fraction of Evi1 leukemia cells. Consistent with this finding, when Evi1 leukemia cells were cocultured with OP9 stromal cells, the addition of Thpo stimulated the proliferation and suppressed the apoptosis of the CD41(+) cells whereas the CD41(-) cells were not affected. Furthermore, stimulation of Evi1 leukemia cells with Thpo resulted in a marked increase in phosphorylation of Stat3, Stat5 and Erk1/2, while the addition of stem cell factor instead of Thpo failed to exert those effects. Therefore, Thpo/Mpl pathway is critical for proliferation and survival of the CD41(+) cells of Evi1 leukemia mice. To test whether the CD41(+) cells are resistant to anticancer treatment, Evi1 leukemia cells maintained on OP9 layers were treated with Ara-C for 48 hours, and the CD41 positive rate was measured by FACS. Notably, the CD41(-) cells were decreased in number at a lower concentration of Ara-C than the CD41(+) cells. While both fractions had similar cell cycle status, the CD41(+) cells showed a higher expression of pro-survival protein, Bcl-xl than the CD41(-) cells. Given that Bcl-xl could be activated by Stat family or Erk1/2, the CD41(+) fraction of Evi1 leukemia cells possibly acquire antiapoptotic potential through Thpo/Mpl signaling. In conclusion, we revealed that the CD41(+) fraction of Evi1 leukemia mice has more efficient leukemia-initiating capacity than the CD41(-) fraction. Importantly, the CD41(+) cells express Mpl, and Thpo/Mpl signaling participates in growth and survival of the CD41(+) cells in vitro. In addition, upregulation of Bcl-xl could confer antiapoptotic properties and chemo-resistance on the CD41(+) Evi1 leukemia cells. Our data would provide a rationale for targeting CD41 or Mpl in the novel treatment of Evi1 high-expressing leukemia. Disclosures: No relevant conflicts of interest to declare.

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