The cell fate determinant Llgl1 influences HSC fitness and prognosis in AML

A unique characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSCs and potentially also in leukemia stem cells. Recently, studies have shed light on developmental molecules and evolutionarily conserved signals as regulators of stem cells in hematopoiesis and leukemia. In this study, we provide evidence that the cell fate determinant Llgl1 (lethal giant larvae homolog 1) plays an important role in regulation of HSCs. Loss of Llgl1 leads to an increase in HSC numbers that show increased repopulation capacity and competitive advantage after transplantation. This advantage increases upon serial transplantation or when stress is applied to HSCs. Llgl1−/− HSCs show increased cycling but neither exhaust nor induce leukemia in recipient mice. Llgl1 inactivation is associated with transcriptional repression of transcription factors such as KLF4 (Krüppel-like factor 4) and EGR1 (early-growth-response 1) that are known inhibitors of HSC self-renewal. Decreased Llgl1 expression in human acute myeloid leukemia (AML) cells is associated with inferior patient survival. Thus, inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.

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Hematopoietic Development from Human Embryonic Stem Cells

Hematology ◽

10.1182/asheducation-2007.1.11 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 11-16 ◽

Cited By ~ 13

Author(s):

Mickie Bhatia

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Self Renewal ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Cell Based Therapy

Abstract The most common human cell-based therapy applied today is hematopoietic stem cell (HSC) transplantation. HSCs can be defined by two essential properties: self-renewal and multilineage hematopoietic differentiation. These combined HSC properties allow them to differentiate into all blood cell types (multilineage) in a sustained manner for the lifetime of the animal, which requires their ability to make cellular copies of themselves (self-renewal). These features can be tested by transplantation from donor to recipient and provide a functional basis to define and identify HSCs. Currently, human bone marrow (BM), mobilized peripheral blood, and umbilical cord blood (CB) represent the major sources of transplantable HSCs, but their availability for use is limited by both quantity and compatibility. Although increasing evidence suggests that somatic HSCs can be expanded to meet current needs, their in vivo potential is concomitantly compromised after ex vivo culture. Pluripotent human embryonic stem cells (hESCs) may provide an alternative. hESCs possess indefinite proliferative capacity in vitro, and have been shown to differentiate into the hematopoietic cell fate, giving rise to erythroid, myeloid, and lymphoid lineages using a variety of differentiation procedures. In most cases, hESC-derived hematopoietic cells show similar clonogenic progenitor capacity and primitive phenotype to somatic sources of hematopoietic progenitors, but possess limited in vivo repopulating capacity when transplanted into immunodeficient mice. Although this suggests HSC function can be derived from hESCs, the efficiency and quality of these cells must be characterized using surrogate models for potential clinical applications.

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Cell Cycle Regulation and Cell Fate Decisions in Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.1349.1349 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1349-1349

Author(s):

Emmanuelle Passegue ◽

Amy J. Wagers ◽

Sylvie Giuriato ◽

Wade C. Anderson ◽

Irving L. Weissman

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Cell Cycle Regulation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Cycle Regulation

Abstract The blood is a perpetually renewing tissue seeded by a rare population of adult bone marrow hematopoietic stem cells (HSC). During steady-state hematopoiesis, the HSC population is relatively quiescent but constantly maintains a low numbers of cycling cells that differentiate to produce the various lineage of mature blood cells. However, in response to hematological stress, the entire HSC population can be recruited into cycle to self-renew and regenerate the blood-forming system. HSC proliferation is therefore highly adaptative and requires appropriate regulation of cell cycle progression to drive both differentiation-associated and self-renewal-associated proliferation, without depletion of the stem cell pool. Although the molecular events controlling HSC proliferation are still poorly understood, they are likely determined, at least in part, by regulated expression and/or function of components and regulators of the cell cycle machinery. Here, we demonstrate that the long-term self-renewing HSC (defined as Lin−/c-Kit+/Sca-1+/Thy1.1int/Flk2−) exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for HSC function in hematopoietic reconstitution; and a proliferative state, which represents almost a fourth of this population and is essential for HSC functions in differentiation and self-renewal. We show that when HSC exit quiescence and enter G1 as a prelude to cell division, at least two critical events occur: first, during the G1 and subsequent S-G2/M phases, they temporarily lose efficient in vivo engraftment activity, while retaining in vitro differentiation potential; and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). Together, these findings provide a direct link between HSC proliferation, cell cycle regulation and cell fate decisions that have critical implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

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Foxo3 Modulation of ATM and Oxidative Stress Mediates Distinct Functions in the Regulation of Hematopoietic Stem and Progenitor Cell Fate.

Blood ◽

10.1182/blood.v110.11.1272.1272 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1272-1272 ◽

Cited By ~ 1

Author(s):

Safak Yalcin ◽

Julia P. Luciano ◽

Xin Zhang ◽

Cecile Vercherat ◽

Reshma Taneja ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Hematopoietic Stem Cell ◽

Progenitor Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Atm Gene

Abstract FOXO transcription factors are required for hematopoietic stem cell self renewal. In this study, we demonstrate that Foxo3 plays a specific and essential function in the regulation of both hematopoietic stem and progenitor cell fate. Foxo3 null mice display a myeloproliferative syndrome characterized by splenomegaly, a major expansion of the myeloid compartment in the blood, bone marrow and spleen, cytokine hypersensitivity of progenitors in hematopoietic organs and associated with the repression of the B lymphoid compartment. In addition, loss of Foxo3 leads to significant defects in hematopoietic stem cell numbers and activity. In particular, the numbers of long-term culture initiating cells (LTC-IC) was significantly reduced and the ability to repopulate lethally irradiated mice was severely compromised in Foxo3-defcient mice. This effect was mediated at least partially by enhanced accumulation of reactive oxygen species (ROS) in Foxo3-deficient hematopoietic stem cells as demonstrated by reduced QRT-PCR expression of several anti-oxidant enzymes leading to accumulation of ROS, (as measured by chloromethyl,dichlorodihydrofluorescein diacetate assay) in Foxo3 null hematopoietic stem cells, and in vitro and in vivo rescue of the phenotype using ROS scavengers. Furthermore, we demonstrate that while ROS accumulation results in suppression of Foxo3 null hematopoietic stem cell compartment, it enhances the activity of multipotential cells. By measuring RNA versus DNA content, and BrdU uptake, we determined that Foxo3-deficient hematopoietic stem cells exit quiescence (G0) and are impaired in their cycling at the G2/M phase. In particular, we identified ROS activation of p19ARF/p53 pathway and ROS-independent modulation of ataxia telangiectasia mutated (ATM) gene and p16INK4a, as major contributors to the interference with Foxo3-deficient hematopoietic stem cell self renewal and cycling. Loss of ATM has been shown to lead to hematopoietic stem cell deficiency. Importantly, we show that ATM gene expression is significantly suppressed in Foxo3-deficient hematopoietic stem cells suggesting that ATM lies downstream of Foxo3. Retroviral expression of a constitutively active form of Foxo3 in Foxo3-deficient bone marrow mononuclear cells enhances significantly the ATM expression suggesting that Foxo3 regulate expression of ATM gene. These combined findings suggest that Foxo3 functions in a tumor suppressor network to protect hematopoietic stem cells against deleterious effects of oxidative damage, to maintain hematopoietic lineage fate determination and to restrict the activity of long term repopulating hematopoietic stem cells. These findings provide insights into the mechanisms underlying hematopoietic stem cell fate.

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An RNA Interference Screen Identifies the Cell Fate Determinants Msi2 and Prox1 as Novel Regulators of Hematopoietic Stem Cell Self-Renewal.

Blood ◽

10.1182/blood.v114.22.394.394 ◽

2009 ◽

Vol 114 (22) ◽

pp. 394-394

Author(s):

Kristin J Hope ◽

Sonia Cellot ◽

Stephen Ting ◽

Guy Sauvageau

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Self Renewal ◽

Hematopoietic Stem ◽

Control Shrna ◽

Fate Determinants ◽

Cell Fate Determinants

Abstract Abstract 394 Hematopoietic stem cells (HSC) can not yet be unambiguously prospectively identified, a fact which has made it difficult to determine whether a segregation of cell fate determinants underlies the asymmetric/symmetric self-renewal of these cells or whether deregulation of such determinants could contribute to the pathogenesis of hematopoietic malignancies by inducing constitutive symmetric self-renewal divisions. We have addressed these questions through a functional genetics approach taking advantage of systematic RNAi to evaluate the function of conserved polarity factors and cell fate determinants in HSCs. From a list of 72 of such factors identified in the literature, 30 murine homologues were chosen based on their differentially higher level of expression in HSC-enriched populations as measured by qRT-PCR. For each candidate we designed 3 unique short hairpin RNA (shRNA) encoding retroviral constructs also carrying EGFP for the purposes of following transduced cells. Primitive hematopoietic cells enriched for HSC were infected at high efficiency with the library in an arrayed 96-well format and their in vivo reconstituting potential was then evaluated through competitive repopulating unit assays. Genes for which shRNA vectors altered late transplant EGFP levels below or above thresholds as defined by a control shRNA to luciferase were considered as hits. Using this approach, we identified and comprehensively validated 4 genes, including the RNA binding protein Msi2, for which shRNA-mediated depletion dramatically impairs repopulation but does not induce cell death or a cell cycle block. Importantly, we show that the loss in the repopulating ability of these shRNA transduced cells is mediated at the stem cell level and is not due to progenitor or downstream cell toxicity or to any defect in the process of bone marrow homing. Subsequent expression profiling indicated that Msi2 is also upregulated in HOXB4-overexpressing symmetrically expanding HSC in line with our findings that it functions as a positive HSC regulator and further suggesting that it represents a potential novel HSC marker. As well as finding HSC agonists, the RNAi screen identified the homeodomain containing transcription factor Prox1 as a negative HSC regulator since its shRNA-mediated transcript loss consistently led to the dramatic in vivo accumulation of EGFP+ transduced cells. Grafts comprised of Prox1 shRNA-transduced cells did not exhibit any lineage skewing however, repeatedly contained an average of 10-fold more primitive Lin-Sca+CD150+48- cells as compared to non-transduced donor cells within the same recipient or to control shRNA-luciferase grafts indicating Prox1 knockdown leads to a significant in vivo expansion of phenotypic HSCs. Moreover, following a 7 day in vitro culture, cells infected with shRNAs to Prox1 were both morphologically and immunophenotypically more primitive than control cells and when transplanted at this time yielded a significantly enhanced engraftment level relative to control shRNAs (51+/-6% GFP vs 8+/-3% GFP). These results further suggest that Prox1 reduction by RNAi expands functional HSCs in vitro. Together these findings have identified conserved cell fate determinants as important and novel regulators of murine hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Epigenetic Erosion in Adult Stem Cells: Drivers and Passengers of Aging

Cells ◽

10.3390/cells7120237 ◽

2018 ◽

Vol 7 (12) ◽

pp. 237 ◽

Cited By ~ 7

Author(s):

Christian Kosan ◽

Florian Heidel ◽

Maren Godmann ◽

Holger Bierhoff

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Adult Stem Cells ◽

Healthy Aging ◽

Cell Function ◽

Functional Decline ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions

In complex organisms, stem cells are key for tissue maintenance and regeneration. Adult stem cells replenish continuously dividing tissues of the epithelial and connective types, whereas in non-growing muscle and nervous tissues, they are mainly activated upon injury or stress. In addition to replacing deteriorated cells, adult stem cells have to prevent their exhaustion by self-renewal. There is mounting evidence that both differentiation and self-renewal are impaired upon aging, leading to tissue degeneration and functional decline. Understanding the molecular pathways that become deregulate in old stem cells is crucial to counteract aging-associated tissue impairment. In this review, we focus on the epigenetic mechanisms governing the transition between quiescent and active states, as well as the decision between self-renewal and differentiation in three different stem cell types, i.e., spermatogonial stem cells, hematopoietic stem cells, and muscle stem cells. We discuss the epigenetic events that channel stem cell fate decisions, how this epigenetic regulation is altered with age, and how this can lead to tissue dysfunction and disease. Finally, we provide short prospects of strategies to preserve stem cell function and thus promote healthy aging.

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Relationship Between Self-Renewal and Differentiation Pathways in Stem Cells and Leukemia

Blood ◽

10.1182/blood.v124.21.4789.4789 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4789-4789

Author(s):

Robert S Welner ◽

Danielle E Tenen ◽

Henry Yang ◽

Deepak Bararia ◽

Giovanni Amabile ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Wnt Signaling ◽

Cell Fate ◽

Wnt Pathway ◽

Beta Catenin ◽

Self Renewal ◽

Hematopoietic Stem ◽

Over Expression

Abstract Hematopoietic stem cells are capable of perpetual self-renewal and multi-lineage differentiation, properties that are maintained throughout life by minimal cell cycle activity. Our work has focused on deciphering transcriptional driven differentiation versus self-renewal pathways in stem and progenitor cells. To this end, we have studied transcription factors that control the fate of hematopoietic stem cells by combining mouse models of activated self-renewal with models that can report transcription factor expression. We chose to study the Wnt pathway, activated in several types of leukemia, in combination with the ets family PU.1 transcription factor, vital to almost all myeloid and lymphoid lineages. PU.1 regulates a number of important myeloid specific genes that mediate differentiation to a specific cell fate. To understand the interaction of these pathways, we found that over-expression of Wnt signaling or beta-catenin, the downstream signaling component of the Wnt pathway, was able to inhibit PU.1-mediated differentiation in a PU.1-inducible cell line. There was little to no up-regulation of the myeloid markers Mac1 or Gr1 with activation of Wnt signaling upon induction with 4-hydroxy-tamoxifen (4-OHT). Additionally, many genes related to myeloid differentiation were not increased as compared to control-induced cultures. To understand how these interactions might function in vitro, we crossed a Cre-responsive activated beta-catenin (floxed allele Exon3) mouse to a PU.1-GFP knock-in mouse. From this model, we are able to see changes in PU.1 (GFP) expression in specific populations of hematopoietic progenitors upon activation of beta-catenin. Most importantly, in the LT-HSCs (defined by Lin- cKitHi Sca1+ CD150+ CD48-), we observed a significant increase in GFP (PU.1) intensity upon activation of active beta-catenin. Additionally, there was an increase in the total number of LT-HSCs, as defined by surface markers. LT-HSCs with active beta-catenin and GFP (PU.1) were found to be more in cycle and they express lower levels of transcription factors related to differentiation. These results demonstrate that when beta-catenin is activated, PU.1’s role is modified and the self-renewal program is enhanced at the expense of differentiation. Furthermore, activation of beta-catenin in the hematopoietic cells of mice has been shown to lead to impaired differentiation and eventual death. Even though active beta-catenin has been shown to be essential in several subtypes of myeloid leukemias using murine models, its over-expression is not sufficient to lead to leukemic development. However, heterozygous PU.1/GFP knock-in mice were crossed to the beta-catenin overexpression model, they rapidly developed leukemia post Cre induction. This is not observed in the PU.1/GFP knock-in mice in the absence of beta-catenin activation, suggesting that Wnt signaling adds to a block in differentiation needed for leukemic transformation. These mice show splenomegaly and increased myelocytic populations in the peripheral blood. The leukemia was transplantable to secondary mice and expressed high levels of GFP (PU.1) in the spleen, bone marrow and peripheral blood. These findings demonstrate that the interaction and crosstalk between these two pathways regulate hematopoietic stem cell fate. Future studies will focus on understanding how this interaction between transcription factor and self-renewal pathways becomes disrupted in leukemic stem cells. Disclosures No relevant conflicts of interest to declare.

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TGF-β signaling in the control of hematopoietic stem cells

Blood ◽

10.1182/blood-2014-12-618090 ◽

2015 ◽

Vol 125 (23) ◽

pp. 3542-3550 ◽

Cited By ~ 102

Author(s):

Ulrika Blank ◽

Stefan Karlsson

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Transforming Growth Factor ◽

Wide Spectrum ◽

Transforming Growth Factor Β ◽

The Body ◽

Self Renewal ◽

Hematopoietic Stem ◽

Downstream Signaling

Abstract Blood is a tissue with high cellular turnover, and its production is a tightly orchestrated process that requires constant replenishment. All mature blood cells are generated from hematopoietic stem cells (HSCs), which are the self-renewing units that sustain lifelong hematopoiesis. HSC behavior, such as self-renewal and quiescence, is regulated by a wide array of factors, including external signaling cues present in the bone marrow. The transforming growth factor-β (TGF-β) family of cytokines constitutes a multifunctional signaling circuitry, which regulates pivotal functions related to cell fate and behavior in virtually all tissues of the body. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from homeostasis of the immune system to quiescence and self-renewal of HSCs. Here, we review key features and emerging concepts pertaining to TGF-β and downstream signaling pathways in normal HSC biology, featuring aspects of aging, hematologic disease, and how this circuitry may be exploited for clinical purposes in the future.

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Dichotomous regulation of lysosomes by MYC and TFEB controls hematopoietic stem cell fate

10.1101/2021.02.24.432720 ◽

2021 ◽

Author(s):

Laura García-Prat ◽

Kerstin B. Kaufmann ◽

Florin Schneiter ◽

Veronique Voisin ◽

Alex Murison ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Nutrient Sensing ◽

Cell Fate Determination ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem Cell Fate ◽

Transcription Factor Eb

SummaryIt is critical to understand how quiescent long-term hematopoietic stem cells (LT-HSC) sense demand from daily and stress-mediated cues and transition into bioenergetically active progeny to differentiate and meet these cellular needs. Here, we show that lysosomes, which are sophisticated nutrient sensing and signaling centers, are dichotomously regulated by the Transcription Factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSC and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, which promotes quiescence, self-renewal and governs erythroid-myeloid commitment. By contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism to drive LT-HSC activation. Collectively, our study identifies lysosomes as a central regulatory hub for proper and coordinated stem cell fate determination.

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