Maintenance Of Adult Hematopoietic Stem Cells Research Articles

HES1 is required for mouse fetal hematopoiesis

BackgroundHematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling.MethodsIn this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay.ResultsWe found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo.ConclusionTogether, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

Vascular Notch Signaling in Stress Hematopoiesis.

Canonical Notch signaling is one of the most conserved signaling cascades. It regulates cell proliferation, cell differentiation, and cell fate maintenance in a variety of biological systems during development and cancer (Fortini, 2009; Kopan and Ilagan, 2009; Andersson et al., 2011; Ntziachristos et al., 2014). For the hematopoietic system, during embryonic development, Notch1 is essential for the emergence of hematopoietic stem cells (HSCs) at the aorta-gornado-mesonephro regions of the dorsal aorta. At adult stage, Notch receptors and Notch targets are expressed at different levels in diverse hematopoietic cell types and influence lineage choices. For example, Notch specifies T cell lineage over B cells. However, there has been a long-lasting debate on whether Notch signaling is required for the maintenance of adult HSCs, utilizing transgenic animals inactivating different components of the Notch signaling pathway in HSCs or niche cells. The aims of the current mini-review are to summarize the evidence that disapproves or supports such hypothesis and point at imperative questions waiting to be addressed; hence, some of the seemingly contradictory findings could be reconciled. We need to better delineate the Notch signaling events using biochemical assays to identify direct Notch targets within HSCs or niche cells in specific biological context. More importantly, we call for more elaborate studies that pertain to whether niche cell type (vascular endothelial cells or other stromal cell)-specific Notch ligands regulate the differentiation of T cells in solid tumors during the progression of T-lymphoblastic lymphoma (T-ALL) or chronic myelomonocytic leukemia (CMML). We believe that the investigation of vascular endothelial cells' or other stromal cell types' interaction with hematopoietic cells during homeostasis and stress can offer insights toward specific and effective Notch-related therapeutics.

Regulation of the hematopoietic stem cell lifecycle by the endothelial niche.

Hematopoietic stem cells (HSCs) predominantly reside either in direct contact or in close proximity to the vascular endothelium throughout their lifespan. From the moment of HSC embryonic specification from hemogenic endothelium, endothelial cells (ECs) act as a critical cellular-hub that regulates a vast repertoire of biological processes crucial for HSC maintenance throughout its lifespan. In this review, we will discuss recent findings in endothelial niche-mediated regulation of HSC function during development, aging and regenerative conditions. Studies employing genetic vascular models have unequivocally confirmed that ECs provide the essential instructive cues for HSC emergence during embryonic development as well as adult HSC maintenance during homeostasis and regeneration. Aging of ECs may impair their ability to maintain HSC function contributing to the development of aging-associated hematopoietic deficiencies. These findings have opened up new avenues to explore the therapeutic application of ECs. ECs can be adapted to serve as an instructive platform to expand bona fide HSCs and also utilized as a cellular therapy to promote regeneration of the hematopoietic system following myelosuppressive and myeloablative injuries. ECs provide a fertile niche for maintenance of functional HSCs throughout their lifecycle. An improved understanding of the EC-HSC cross-talk will pave the way for development of EC-directed strategies for improving HSC function during aging.

Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sortm1(Cre/ERT)Nat/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation.

Role of SIRT1 in the growth and regulation of normal hematopoietic and leukemia stem cells.

Recent studies have enhanced our understanding of the role of the SIRT1 deacetylase in regulation of normal hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs), and its importance in regulating autophagy and epigenetic reprogramming in response to metabolic alterations. Studies employing conditional deletion mouse models indicate an important role of SIRT1 in maintenance of adult HSCs under conditions of stress. SIRT1 is significantly overexpressed in LSC populations from acute myeloid leukemia (AML) patients with the FLT3-ITD mutation, and maintains their survival, growth and drug resistance, as previously described for chronic myelogenous leukemia (CML). SIRT1 can also enhance leukemia evolution and drug resistance by promoting genetic instability. Recent studies indicate an important role of SIRT1 in regulating autophagy in response to oxidative stress and nutrient requirements, and have elucidated complex mechanisms by which SIRT1 regulates epigenetic reprogramming of stem cells. SIRT1 inhibition holds promise as a novel approach for ablation of LSCs in chronic phase CML or FLT3-ITD-associated AML. Additional studies to understand the role of SIRT1 in linking metabolic alterations to genomic stability, autophagy and epigenetic reprogramming of stem cells are warranted.

Concise Review: Genetic Dissection of Hypoxia Signaling Pathways in Normal and Leukemic Stem Cells

Adult hematopoiesis depends on rare multipotent hematopoietic stem cells (HSCs) that self-renew and give rise to progenitor cells, which differentiate to all blood lineages. The strict regulation of the fine balance between self-renewal and differentiation is essential for normal hematopoiesis and suppression of leukemia development. HSCs and progenitor cells are commonly assumed to reside within the hypoxic BM microenvironment, however, there is no direct evidence supporting this notion. Nevertheless, HSCs and progenitors do exhibit a hypoxic profile and strongly express Hif-1α. Although hypoxia signaling pathways are thought to play important roles in adult HSC maintenance and leukemogenesis, the precise function of Hif-dependent signaling in HSCs remains to be uncovered. Here we discuss recent gain-of-function and loss-of-function studies that shed light on the complex roles of hypoxia-signaling pathways in HSCs and their niches in normal and malignant hematopoiesis. Importantly, we comment on the current and often contrasting interpretations of the role of Hif-dependent signaling in stem cell functions.

P57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells

Quiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G(0) phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs.

Self-renewal related signaling in myeloid leukemia stem cells

A key characteristic of hematopoietic stem cells (HSC) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSC and potentially also in leukemic stem cells. Besides pathways such as Wnt signaling, Hedgehog signaling and Notch signaling, transcription factors (FoxOs) and cell fate determinants may also play a role in stem cells. While some of these pathways seem to be dispensable for maintenance of adult HSC, there may be a distinct requirement in leukemia stem cells for leukemic self-renewal. Here we will focus on self-renewal related signaling in myeloid leukemia stem cells and its therapeutic relevance.

The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance.

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.

Adult hematopoietic stem cells require NKAP for maintenance and survival

AbstractSteady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.

Cited2 Is an Essential Regulator of Adult Hematopoietic Stem Cells

SummaryThe regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches, we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast, conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16Ink4a and p19Arf) or Trp53 (encoding p53, a downstream target of p19Arf) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore, we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together, our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions, at least in part, via Ink4a/Arf and Trp53.

Inhibition of Notch Signaling in T Cells Prevents Immune-Mediated Bone Marrow Failure.

Abstract 180Aplastic anemia is a severe bone marrow disorder characterized by the loss of hematopoietic stem cells (HSC). HSC destruction is thought to be T cell-mediated in a majority of patients with aplastic anemia. Global immunosuppression and HSC transplantation can induce disease remission, but these treatments are not effective in all patients and can promote life-threatening complications. Thus, novel immunomodulatory approaches are needed in this disorder. Notch is a conserved cell-cell communication pathway that can regulate T cell differentiation and function with context-dependent effects. To study the role of Notch signaling in pathogenic T cells causing immune-mediated bone marrow failure, we inhibited canonical Notch signaling in mature T cells through conditional expression of the pan-Notch inhibitor DNMAML (ROSA-DNMAMLf × Cd4-Cre mice). We used two complementary mouse models of immune-mediated bone marrow failure that mimic features of aplastic anemia: administration of C57BL/6 (B6) T cells into sublethally irradiated (500 rads) minor histocompatibility antigen mismatched BALB/b recipients (Chen et al., J Immunol 2007; 178:4159), or infusion of B6 lymphocytes into unirradiated MHC-mismatched B6×DBA F1 recipients. In contrast to control B6 T cells which led to lethal bone marrow failure in virtually all recipients, DNMAML-expressing Notch-deprived T cells were profoundly deficient at inducing HSC loss in both disease models, leading to markedly improved long-term survival (>90%). Notch-deficient T cells showed a modest decrease in overall expansion within secondary lymphoid organs, but their accumulation in the target bone marrow was preserved. Upon restimulation with anti-CD3 and anti-CD28 antibodies, DNMAML T cells had decreased production of IL-2 and interferon gamma. Activated CD4+ and CD8+ DNMAML T cells had reduced interferon gamma, granzyme B, and perforin transcripts despite preserved induction of the master transcription factors Tb×21 (encoding T-bet) and Eomes. In vivo infusion of CFSE-labeled host-type target cells revealed a decreased cytotoxicity in DNMAML as compared to control B6 T cell recipients. These observations point to a novel spectrum and mechanism of Notch action in mature T cells. Since we have shown recently that canonical Notch signaling is dispensable for the maintenance of adult HSCs (Maillard et al., Cell Stem Cell 2008, 2:356), our findings suggest that Notch inhibition could represent a novel therapeutic modality to target the T cell response and reverse immune-mediated HSC destruction in aplastic anemia. Disclosures:Shan:American Society of Hematology: Research Funding. Zhang:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding. Maillard:Damon Runyon Cancer Research Foundation: Research Funding; American Society of Hematology: Research Funding; University of Michigan Comprehensive Cancer Center: Research Funding.

Zfx Controls the Self-Renewal of Embryonic and Hematopoietic Stem Cells

Stem cells (SC) exhibit a unique capacity for self-renewal in an undifferentiated state. It is unclear whether the self-renewal of pluripotent embryonic SC (ESC) and of tissue-specific adult SC such as hematopoietic SC (HSC) is controlled by common mechanisms. The deletion of transcription factor Zfx impaired the self-renewal but not the differentiation capacity of murine ESC; conversely, Zfx overexpression facilitated ESC self-renewal by opposing differentiation. Furthermore, Zfx deletion abolished the maintenance of adult HSC but did not affect erythromyeloid progenitors or fetal HSC. Zfx-deficient ESC and HSC showed increased apoptosis and SC-specific upregulation of stress-inducible genes. Zfx directly activated common target genes in ESC and HSC, as well as ESC-specific target genes including ESC self-renewal regulators Tbx3 and Tcl1. These studies identify Zfx as a shared transcriptional regulator of ESC and HSC, suggesting a common genetic basis of self-renewal in embryonic and adult SC.

Analysis of the Human Fetal Liver Hematopoietic Microenvironment

In the adult, hematopoietic stem cells (HSCs) are resident in the bone marrow (BM) compartment and are in direct association with the BM stromal microenvironment. However, human adult HSCs are largely quiescent and undergo limited self-renewal. This is in contrast to the higher frequency of cycling HSCs undergoing self-renewal during fetal development when hematopoiesis is transiently localized to the fetal liver (FL), suggesting that FL provides a more conducive microenvironment to support HSCs. Here, we provide phenotypic and molecular characterization of primary human FL stromal cells capable of supporting human repopulating progenitors. Qualitative and quantitative analysis revealed several properties unique to FL stromal cells compared to adult BM-derived stroma that included a greater than 10-fold enhanced proliferative capacity of FL stromal vs adult BM, and a 2-fold increase in the number of N-cadherin- and osteopontin-expressing cells. Supportive of extrinsic influences likely to modulate HSC expansion, global gene expression microarray analysis revealed that FL stroma has higher expression of regulators of the Wnt signaling pathway compared to adult BM stroma, which demonstrated an increased expression of the Notch signaling pathway. Our results suggest that human FL stromal cells provide a unique microenvironment to HSCs compared to adult BM stroma by controlling Wnt signaling of HSCs during human fetal hematopoietic development, while Notch signaling is tightly regulated by the HSC microenvironment in the adult. We propose that the human HSC niche is ontogenically controlled during human development to provide appropriate expansion of fetal HSCs and subsequent maintenance of adult HSCs.

Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival.

Hematopoietic stem cells (HSCs) sustain blood formation throughout life. Pathways regulating maintenance of adult HSCs are largely unknown. Here we report that the Ets-related transcription factor Tel/Etv6, the product of a locus frequently involved in translocations in leukemia, is a selective regulator of HSC survival. Following inactivation of Tel/Etv6, HSCs are lost in the adult bone marrow but their progeny are unaffected and transiently sustain blood formation. Accordingly, absence of Tel/Etv6 after lineage commitment is ostensibly without consequence except for unexpected impairment of maturation of megakaryocytes. Thus, we establish Tel/Etv6 as a selective and essential regulator of postembryonic HSCs.