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ABSTRACT: Key points Hematopoietic stem cell aging associate with stable transcriptional alterations that persist through transplantation.Somatic cell reprogramming of aged hematopoietic stem and progenitor cells reverses functional defects associated with hematopoietic aging.
Blood 03/2013; · 9.90 Impact Factor
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ABSTRACT: The E2A transcription factors promote the development of thymus-seeding cells but it remains unknown whether these proteins play a role in T lymphocyte lineage specification or commitment. Here we showed that E2A proteins were required to promote T lymphocyte commitment from DN2 thymocytes and to extinguish their potential for alternative fates. E2A proteins functioned in DN2 cells to limit expression of Gata3, which encodes an essential T lymphocyte transcription factor whose ectopic expression can arrest T cell differentiation. Genetic, or siRNA-mediated, reduction of Gata3 rescued T cell differentiation in the absence of E2A and restricted the development of alternative lineages by limiting the expanded self-renewal potential in E2A(-/-) DN2 cells. Our data support a novel paradigm in lymphocyte lineage commitment in which the E2A proteins are necessary to limit the expression of an essential lineage specification and commitment factor in order to restrain self-renewal and prevent an arrest in differentiation.
Blood 01/2013; · 9.90 Impact Factor
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ABSTRACT: Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D(+) cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D(+) progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D(+) CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D(+) CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.
Proceedings of the National Academy of Sciences 12/2012; · 9.68 Impact Factor
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ABSTRACT: Despite great progress in identification of mesenchymal stem cells (MSCs) from bone marrow (BM), our knowledge on their in vivo cellular identity remains limited. We here report that cells expressing the transcription factor Ebf2 in the adult BM display characteristics of MSCs. The Ebf2(+) cells are highly clonal and physiologically quiescent. In vivo lineage-tracing experiments, single cell clone transplantations and in vitro differentiation assay revealed their self-renewal and multilineage differentiation capacity. Gene expression analysis of the freshly sorted Ebf2(+) cells demonstrated expression of genes previously reported to be associated with MSCs and co-expression of multiple lineage-associated genes in a single cell level. Thus, Ebf2 expression is not restricted to committed osteoblast progenitor cells but rather marks a multipotent mesenchymal progenitor cell population in adult mouse BM. These cells do not appear to be completely overlapping with the previous reported MSC populations. The findings provide new insights into the in vivo cellular identity and molecular properties of BM mesenchymal stem and progenitor cells.
Molecular and cellular biology 11/2012; · 6.06 Impact Factor
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ABSTRACT: Human hematopoietic stem cells reside in the CD34+CD38-CD90+ population in cord blood and bone marrow. However, this cell fraction is heterogeneous, and the phenotype of the rare primitive stem cells remains poorly defined. We here report that primitive cord blood CD34+CD38-CD90+ stem cells, with the ability to reconstitute NOD/SCID-IL2Rγ(c) null mice long term, at 24 weeks after transplantation, can be prospectively isolated at an increased purity by using integrin α2 receptor as an additional stem cell marker. Using a limiting dilution transplantation assay, we found a highly significant enrichment of multilineage reconstituting stem cells in the CD34+CD38-CD90+ cell fraction expressing the integrin α2 receptor, with a frequency of 1/29 cells, as compared to a frequency of 1/157 in the corresponding integrin α2- cells. In line with this, long-term reconstituting stem cells within the cord blood CD34+CD38- cell population were significantly enriched in the integrin α2+ fraction, while stem cells and progenitors reconstituting short-term, at 8-12 weeks, were heterogeneous in integrin α2 expression. Global gene expression profiling revealed that the lineage-marker negative (Lin-) CD34+CD38-CD90+CD45RA- integrin α2+ cell population was molecularly distinct from the integrin α2- cell population and the more mature Lin-CD34+CD38-CD90-CD45RA- cell population. Our findings identify integrin α2 as a novel stem cell marker, which improves prospective isolation of the primitive human hematopoietic stem cells within the CD34+CD38-CD90+ cell population for experimental and therapeutic stem cell applications.
Stem Cells 11/2012; · 7.78 Impact Factor
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ABSTRACT: To better understand the process of B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early B-lineage cells from Pax-5-deficient animals crossed to a B-lineage-restricted reporter mouse, allowing us to identify B-lineage-specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of specified progenitor B-cells marked by expression of a λ5 (Igll1) promoter-controlled reporter gene. Gene expression analysis of ex vivo isolated progenitor cells revealed that Pax-5 deficiency has a minor impact on B-cell specification. However, single-cell in vitro differentiation analysis of ex vivo isolated cells revealed that specified B-lineage progenitors still displayed a high degree of plasticity for development into NK or T lineage cells. In contrast, we were unable to detect any major changes in myeloid lineage potential in specified Pax-5-deficient cells. By comparison of gene expression patterns in ex vivo isolated Pax-5- and Ebf-1-deficient progenitors, it was possible to identify a set of B-cell-restricted genes dependent on Ebf-1 but not Pax-5, supporting the idea that B-cell specification and commitment is controlled by distinct regulatory networks.
Proceedings of the National Academy of Sciences 09/2012; 109(39):15871-6. · 9.68 Impact Factor
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ABSTRACT: Aging causes profound effects on the hematopoietic stem cell (HSC) pool, including an altered output of mature progeny and enhanced self-propagation of repopulating-defective HSCs. An important outstanding question is whether HSCs can be protected from aging. The signal adaptor protein LNK negatively regulates hematopoiesis at several cellular stages. It has remained unclear how the enhanced sensitivity to cytokine signaling caused by LNK deficiency affects hematopoiesis upon aging. Our findings demonstrate that aged LNK(-/-) HSCs displayed a robust overall reconstitution potential and gave rise to a hematopoietic system with a balanced lineage distribution. Although aged LNK(-/-) HSCs displayed a distinct molecular profile in which reduced proliferation was central, little or no difference in the proliferation of aged LNK(-/-) HSCs was observed after transplantation when compared to aged WT HSCs. This coincided with equal telomere maintenance in WT and LNK(-/-) HSCs. Collectively, our studies suggest that enhanced cytokine signaling can counteract functional age-related HSC decline.
Aging cell 07/2012; · 7.55 Impact Factor
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ABSTRACT: Despite significant progress in our understanding of mesenchymal stem cell (MSC) biology during recent years, much of the information is based on experiments using in vitro culture-selected stromal progenitor cells. Therefore, the natural cellular identity of MSCs remains poorly defined. Numerous studies have reported that CD44 expression is one of the characteristics of MSCs in both humans and mice; however, we here have prospectively isolated bone marrow stromal cell subsets from both human and mouse bone marrow by flow cytometry and characterized them by gene expression analysis and function assays. Our data provide functional and molecular evidence suggesting that primary mesenchymal stem and progenitor cells of bone marrow reside in the CD44(-) cell fraction in both mice and humans. The finding that these CD44(-) cells acquire CD44 expression after in vitro culture provides an explanation for the previous misconceptions concerning CD44 expression on MSCs. In addition, the other previous reported MSC markers, including CD73, CD146, CD271, and CD106/VCAM1, are also differentially expressed on those two cell types. Our microarray data revealed a distinct gene expression profile of the freshly isolated CD44(-) cells and the cultured MSCs generated from these cells. Thus, we conclude that bone marrow MSCs physiologically lack expression of CD44, highlighting the natural phenotype of MSCs and opening new possibilities to prospectively isolate MSCs from the bone marrow.
Journal of Biological Chemistry 05/2012; 287(31):25795-807. · 4.77 Impact Factor
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ABSTRACT: Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.
Immunity 05/2012; 36(6):921-32. · 21.64 Impact Factor
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ABSTRACT: Recent studies have identified a number of transcriptional regulators, including E proteins, EBF1, FOXO1, and PAX5, that act together to orchestrate the B-cell fate. However, it still remains unclear as to how they are linked at the earliest stages of B-cell development. Here, we show that lymphocyte development in HEB-ablated mice exhibits a partial developmental arrest, whereas B-cell development in E2A(+/-)HEB(-/-) mice is completely blocked at the LY6D(-) common lymphoid progenitor stage. We show that the transcription signatures of E2A- and HEB-ablated common lymphoid progenitors significantly overlap. Notably, we found that Foxo1 expression was substantially reduced in the LY6D(-) HEB- and E2A-deficient cells. Finally, we show that E2A binds to enhancer elements across the FOXO1 locus to activate Foxo1 expression, linking E2A and FOXO1 directly in a common pathway. In summary, the data indicate that the earliest event in B-cell specification involves the induction of FOXO1 expression and requires the combined activities of E2A and HEB.
Proceedings of the National Academy of Sciences 10/2011; 108(42):17402-7. · 9.68 Impact Factor
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ABSTRACT: Even though B-lymphocyte development is one of the best understood models for cell differentiation in the hematopoetic system, recent advances in cell sorting and functional genomics has increased this understanding further. This has suggested that already early lymphoid primed multipotent progenitor cells (LMPPs) express low levels of lymphoid restricted transcripts. The expression of these genes becomes more pronounced when cells enter the FLT-3/IL-7 receptor positive common lymphoid progenitor (CLP) stage. However, the expression of B-lineage specific genes is limited to a B-cell restricted Ly6D surface positive subpopulation of the CLP compartment. The gene expression patterns also reflect differences in lineage potential and while Ly6D negative FLT-3/IL-7 receptor positive cells represents true CLPs with an ability to generate B/T and NK cells, the Ly6D positive cells lack NK cell potential and display a reduced T-cell potential in vivo. These recent findings suggest that the CLP compartment is highly heterogenous and that the point of no return in B-cell development may occur already in B220(-)CD19(-) cells. These findings have allowed for a better understanding of the interplay between transcription factors like EBF-1, PAX-5 and E47, all known as crucial for normal B-cell development. In this review, we aim to provide a comprehensive overview of B-cell fate specification and commitment based on the recent advances in the understanding of molecular networks as well as functional properties of early progenitor populations.
Seminars in Immunology 09/2011; 23(5):335-40. · 6.39 Impact Factor
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ABSTRACT: Recent studies have documented genome-wide binding patterns of transcriptional regulators and their associated epigenetic marks in hematopoietic cell lineages. In order to determine how epigenetic marks are established and maintained during developmental progression, we have generated long-term cultures of hematopoietic progenitors by enforcing the expression of the E-protein antagonist Id2. Hematopoietic progenitors that express Id2 are multipotent and readily differentiate upon withdrawal of Id2 expression into committed B lineage cells, thus indicating a causative role for E2A (Tcf3) in promoting the B cell fate. Genome-wide analyses revealed that a substantial fraction of lymphoid and myeloid enhancers are premarked by the poised or active enhancer mark H3K4me1 in multipotent progenitors. Thus, in hematopoietic progenitors, multilineage priming of enhancer elements precedes commitment to the lymphoid or myeloid cell lineages.
Immunity 09/2011; 35(3):413-25. · 21.64 Impact Factor
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ABSTRACT: Deficiencies in the IL-7 signaling pathway result in severe disruptions of lymphoid development in adult mice. To understand more about how IL-7 deficiency impacts early lymphoid development, we have investigated lineage restriction events within the common lymphoid progenitor (CLP) compartment in IL-7 knockout mice. This revealed that although IL-7 deficiency had a minor impact on the development of LY6D(-) multipotent CLPs, the formation of the lineage restricted LY6D(+) CLP population was dramatically reduced. This was reflected in a low-level transcription of B-lineage genes as well as in a loss of functional B-cell commitment. The few Ly6D(+) CLPs developed in the absence of IL-7 displayed increased lineage plasticity and low expression of Ebf-1. Absence of Ebf-1 could be linked to increased plasticity because even though Ly6D(+) cells develop in Ebf-1-deficient mice, these cells retain both natural killer and dendritic cell potential. This reveals that IL-7 is essential for normal development of Ly6D(+) CLPs and that Ebf-1 is crucial for lineage restriction in early lymphoid progenitors.
Blood 06/2011; 118(5):1283-90. · 9.90 Impact Factor
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ABSTRACT: Somatic stem cells mediate tissue maintenance for the lifetime of an organism. Despite the well-established longevity that is a prerequisite for such function, accumulating data argue for compromised stem cell function with age. Identifying the mechanisms underlying age-dependent stem cell dysfunction is therefore key to understanding the aging process. Here, using a model carrying a proofreading-defective mitochondrial DNA polymerase, we demonstrate hematopoietic defects reminiscent of premature HSC aging, including anemia, lymphopenia, and myeloid lineage skewing. However, in contrast to physiological stem cell aging, rapidly accumulating mitochondrial DNA mutations had little functional effect on the hematopoietic stem cell pool, and instead caused distinct differentiation blocks and/or disappearance of downstream progenitors. These results show that intact mitochondrial function is required for appropriate multilineage stem cell differentiation, but argue against mitochondrial DNA mutations per se being a primary driver of somatic stem cell aging.
Cell stem cell 05/2011; 8(5):499-510. · 23.56 Impact Factor
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ABSTRACT: In the absence of early B-cell factor 1 (EBF1), B-cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 and its DNA-binding partner Runx1 by evaluating B lymphopoiesis in single (EBF1(het) and Runx1(het)) and compound haploinsufficent (Ebf1(+/-) Runx1(+/-), ER(het)) mice. Here, we demonstrate that decreased Ebf1 gene dosage results in the inappropriate expression of NK-cell lineage-specific genes in B-cell progenitors. Moreover, prolonged expression of Ly6a/Sca-1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre-B and all immature B cells of ER(het) mice. Enforced EBF1 expression repressed promiscuous transcription in pro-B cells of ER(het) mice and in Ebf1(-/-) Pax5(-/-) fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B-cell identity by directing repression of non-B-cell-specific genes.
European Journal of Immunology 03/2011; 41(6):1787-93. · 5.10 Impact Factor
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ABSTRACT: The development of lymphoid cells from bone marrow progenitors is dictated by interplay between internal cues such as transcription factors and external signals like the cytokines Flt-3 ligand and Il-7. These proteins are both of large importance for normal lymphoid development; however, it is unclear if they act in direct synergy to expand a transient Il-7R(+)Flt-3(+) population or if the collaboration is created through sequential activities. We report here that Flt-3L and Il-7 synergistically stimulated the expansion of primary Il-7R(+)Flt-3(+) progenitor cells and a hematopoietic progenitor cell line ectopically expressing the receptors. The stimulation resulted in a reduced expression of pro-apoptotic genes and also mediated survival of primary progenitor cells in vitro. However, functional analysis of single cells suggested that the anti-apoptotic effect was additive indicating that the synergy observed mainly depends on stimulation of proliferation. Analysis of downstream signaling events suggested that although Il-7 induced Stat-5 phosphorylation, Flt-3L caused activation of the ERK and AKT signaling pathways. Flt-3L could also drive proliferation in synergy with ectopically expressed constitutively active Stat-5. This synergy could be inhibited with either receptor tyrosine kinase or MAPK inhibitors suggesting that Flt-3L and Il-7 act in synergy by activation of independent signaling pathways to expand early hematopoietic progenitors.
Journal of Biological Chemistry 11/2010; 285(47):36275-84. · 4.77 Impact Factor
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ABSTRACT: The maturation of B-lymphoid cells from hematopoietic stem cells in the bone marrow is a complex process under control of interplay between extrinsic and intrinsic factors. In addition to serving as a model for normal cell differentiation, disturbances in this process results in immunodeficiencies and malignancies, such as leukemia and lymphoma, making the subject of high relevance for modern medicine. Although the process of B lymphopoiesis has been under intense investigation, recent methodological developments within the area of cell sorting, genome-wide expression, and DNA-binding analysis has allowed for a rapid development of the understanding of B-lymphocyte differentiation. This has suggested that the path to B-lymphoid cell fate may be initiated by lineage priming reflected in the expression of lymphoid associated genes already in multipotent hematopoietic progenitors. Upon differentiation, the gene expression profile is changed to involve an increasing number of B-lineage-restricted genes linked to loss of alternative developmental potentials and B-lineage commitment. This review focuses on the molecular regulation of early B-lymphoid development and aims to provide an up to date summary of the current status of the research area.
Immunological Reviews 11/2010; 238(1):47-62. · 11.15 Impact Factor
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Yin C Lin,
Suchit Jhunjhunwala,
Christopher Benner,
Sven Heinz,
Eva Welinder,
Robert Mansson, Mikael Sigvardsson,
James Hagman,
Celso A Espinoza,
Janusz Dutkowski,
Trey Ideker,
Christopher K Glass,
Cornelis Murre
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ABSTRACT: It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.
Nature Immunology 07/2010; 11(7):635-43. · 26.01 Impact Factor
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ABSTRACT: Aging of the hematopoietic stem cell compartment is believed to contribute to the onset of a variety of age-dependent blood cell pathophysiologies. Mechanistic drivers of hematopoietic stem cell (HSC) aging include DNA damage accumulation and induction of tumor suppressor pathways that combine to reduce the regenerative capacity of aged HSCs. Such mechanisms do not however account for the change in lymphoid and myeloid lineage potential characteristic of HSC aging, which is believed to be central to the decline of immune competence and predisposition to myelogenous diseases in the elderly. Here we have prospectively isolated functionally distinct HSC clonal subtypes, based on cell surface phenotype, bearing intrinsically different capacities to differentiate toward lymphoid and myeloid effector cells mediated by quantitative differences in lineage priming. Finally, we present data supporting a model in which clonal expansion of a class of intrinsically myeloid-biased HSCs with robust self-renewal potential is a central component of hematopoietic aging.
Proceedings of the National Academy of Sciences 03/2010; 107(12):5465-70. · 9.68 Impact Factor
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ABSTRACT: Even though the development of B lymphoid cells from hematopoietic stem cells is one of the most carefully investigated models of cell differentiation in adult mammalians, a set of recent findings has to a large extent increased our understanding for how B lymphoid commitment is achieved. These include the identification of IKAROS, PU.1 and E2A as transcription factors responsible for lymphoid lineage priming in multipotent cells, as well as the identification of EBF1 dependent B lineage restricted progenitors among cells lacking expression of the classical B lineage markers CD19 or B220. The insight that the B cell identity may be defined at an earlier stage then previously thought, allows for an increased understanding of B lymphoid development likely to unravel molecular mechanisms of high relevance also for other differentiation processes within as well as outside of the hematopoietic system.
Current opinion in immunology 03/2010; 22(2):148-53. · 10.88 Impact Factor
