LRF Maintains HSC Homeostasis by Preventing Lymphoid-Primed LT-HSCs From Excessive Differentiation

Abstract Abstract 44 Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in embryos and are critical regulators for lymphoid lineage fate determination. However, their role in adult HSC function is currently under debate. LRF (Leukemia/ Lymphoma Related Factor, also known as Zbtb7a/pokemon) is a transcriptional factor that plays a key role in lymphoid lineage fate determination, erythroid terminal differentiation and germinal center B cell proliferation. LRF loss at HSC/progenitor levels led to excessive differentiation of HSCs into T cells in the BM at the expense of B cell development. Concomitantly, the numbers of LT-HSCs (CD34−CD150+CD48−Flt3−IL7Rα−Lin−Sca-1+c-Kit+) were significantly reduced in LRFflox/floxMx1-Cre+ mice one month after LRF inactivation. Reduced LT-HSC numbers in LRFflox/floxMx1-Cre+ mice were almost completely rescued by the genetic loss of Notch1 (LRFflox/floxNotch1flox/floxMx1-Cre+) or anti-DLL4 antibody treatment, suggesting that the reduction in LT-HSCs numbers was caused by Notch1/DLL4-mediated mechanisms. Furthermore, immunohistochemical (IHC) analysis demonstrated a dramatic increase in DLL4 protein levels in BM hematopoietic cells in LRFflox/floxMx1-Cre+ mice, although the precise mechanisms for this remain unknown. To determine LRF function in HSCs, highly enriched 50 LT-HSCs were transplanted to lethally-irradiated CD45.1+ congenic recipient mice and contributions of donor-derived cells (CD45.2+) in recipients' peripheral blood (PB) had been examined over 4 months after transplant. Compared to wild-type (WT) cells, LRF-deficient LT-HSCs barely contributed to lymphoid development (both T and B) in the recipients, while myeloid reconstitutions were largely unaffected. Using antibodies raised against the ligand binding domains of Notch1 and Notch2, we found that Notch proteins are expressed in a gradient at the most primitive CD34−LT-HSCs in adult BM. The CD34−LT-HSCs expressing Notch1 at high levels (Notch1high LT-HSCs) were susceptible to LRF inactivation and disappeared upon LRF inactivation. Only Notch1low LT-HSCs remained in the BM of LRFflox/floxMx1-Cre+ mice after pIpC injections. To elucidate the qualitative difference between Notch1high and Notch1low LT-HSCs in normal hematopoiesis, we analyzed cell cycle status, gene expression profiles and in vitro colony forming capacities at the single cell level. We found that the Notch1high LT-HSCs were in more active cell cycle as compared to Notch1low fractions. Single cell q-PCR analysis demonstrated Notch1low LT-HSCs express stem cell-related genes (e.g. Gata2, Mpl and Runx1) at higher levels compared to Notch1high LT-HSCs. Furthermore, Notch1high LT-HSCs reconstituted hematopoietic system more quickly compared to the Notch1low cells when transplanted to lethally-irradiated recipient mice. There is no difference in colony forming capacities between two LT-HSC subtypes. Since Notch1lowLT-HSCs gave rise to Notch1highLT-HSCs (and vice versa) in recipients' BM, these two LT-HSC fractions are likely to be interchangeable. Taken together, our data suggest that the LT-HSCs expressing Notch1 at high levels (Notch1high LT-HSCs) are “lymphoid-primed”, which are susceptible to LRF loss. We propose a model in which LRF acts as a safeguard to prevent lymphoid-primed LT-HSCs from excessive T-cell differentiation in the BM micro-environment. Our study sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and lymphoid differentiation. Disclosures: Yan: Genentech Inc.: Employment.

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CXCR4 is required for the quiescence of primitive hematopoietic cells

Journal of Experimental Medicine ◽

10.1084/jem.20072513 ◽

2008 ◽

Vol 205 (4) ◽

pp. 777-783 ◽

Cited By ~ 243

Author(s):

Yuchun Nie ◽

Yoon-Chi Han ◽

Yong-Rui Zou

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Stromal Cells ◽

Hematopoietic Cells ◽

Regulatory Factor ◽

Cell Compartment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Stem Cell Compartment

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4−/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

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Pbx1 Regulates the Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence.

Blood ◽

10.1182/blood.v110.11.94.94 ◽

2007 ◽

Vol 110 (11) ◽

pp. 94-94 ◽

Cited By ~ 1

Author(s):

Francesca Ficara ◽

Mark J. Murphy ◽

Min Lin ◽

Michael L. Cleary

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Acute Leukemia ◽

B Cell ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Fate Decisions

Abstract Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemia. It codes for a homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. Lack of Pbx1 is associated with multiple patterning malformations, defects in organogenesis, and severe fetal anemia, however embryonic lethality has prevented an assessment of its roles in the adult hematopoietic stem cell (HSC) compartment and in lymphoid differentiation. The objective of this study was to characterize the physiological roles for Pbx1 in the hematopoietic system, specifically in the regulation of cell fate decisions involved in the timing and/or extent of postnatal HSC and progenitor proliferation, self-renewal or differentiation capacity. A genetic approach was employed to conditionally inactivate Pbx1 in the hematopoietic compartment in vivo using Cre recombinase expressed under the control of the Tie2 or Mx1 promoters. A crucial role for Pbx1 in the development of the lympho-hematopoietic system was evidenced by reduced size, cell number, and altered architectures of the thymus and spleen in mutant mice. A marked reduction was observed in the bone marrow (BM) pro- and pre-B cell compartment, as well as a striking reduction (up to 10-fold) in common lymphoid progenitors (CLP), suggesting a role for Pbx1 at a critical stage of lymphoid development where acute leukemia likely originates. Accordingly, abnormal T cell development was observed in the thymus. Common myeloid progenitors (CMP) and Lin-cKit+Sca1+ (LKS, enriched in HSCs) cells were also reduced, as well as long-term stem cells (LT-HSCs, reduced 7-fold on average). Assessment of the proliferation status of LT- and ST (short-term)-HSCs, as well as multi-potent progenitors (MPP), revealed that the reduction of the HSC compartment was associated with a higher number of stem cells exiting the G0 phase, thus losing their quiescent state. Strikingly, Pbx1-deficient BM cells failed to engraft in competitive transplants, but were able to reconstitute congenic recipients in the absence of competition, indicating a profound defect of functional HSCs, which nevertheless retained reconstitution potential. Importantly, Pbx1 deficient HSCs progressively disappeared from primary transplant recipients, and were unable to engraft secondary recipients, demonstrating that Pbx1 is crucial for the maintenance of LT-HSC self-renewal. Microarray studies performed on mutant and wt LT- and ST-HSCs, followed by bioinformatics analysis, showed that in the absence of Pbx1 LT-HSCs are characterized by premature expression of a large subset of ST-HSC genes. The up-regulated differentially expressed transcripts are enriched for cell cycle regulatory genes, consistent with the observed increased cycling activity. Notably, more than 8% of the down-regulated genes are related to the Tgf-beta pathway, which serves a major role in maintaining HSC quiescence. Moreover, B-cell specific genes, which are expressed in the wt LT-HSC compartment, are down-regulated in the absence of Pbx1, suggesting that the observed reduction in CLP and B-cell numbers ultimately arose from a stem cell defect in lymphoid priming. We conclude that Pbx1 is at the apex of a transcriptional cascade that controls LT-HSC quiescence and differentiation, thus allowing the maintenance of their self-renewal potential, crucial for the homeostasis of the lympho-hematopoietic system.

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The Side Population Contains Functionally Distinct Hematopoietic Stem Cell Subpopulations

Blood ◽

10.1182/blood.v112.11.2464.2464 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2464-2464

Author(s):

Grant Anthony Challen ◽

Margaret A Goodell

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Side Population ◽

Clonal Diversity ◽

Stem Cell Markers ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System

Abstract Over the decades since hematopoietic stem cells (HSCs) were first identified, the traditional view has been that the hematopoietic system is regenerated by a single pool of multipotent, quiescent HSCs that are sequentially recruited into cell cycle and which then progressively divide and differentiate until they are exhausted and ultimately replaced by the next cohort of stem cells. However, recent evidence has challenged this classical clonal succession model of HSC hierarchy by suggesting that the hematopoietic system is maintained by a pool of different HSC subtypes, with distinct self-renewal and differentiation potentials (the clonal diversity model, Figure 1). The side population (SP), characterized by Hoechst dye efflux, has been used as a method for isolating HSCs for over a decade and the SP has been shown to be highly enriched for HSC activity. While the entire SP is strikingly homogeneous with respect to expression of canonical stem cell markers such as Sca-1 and c-Kit, we recently observed heterogeneous expression for the SLAM family molecule CD150 within the SP, with CD150+ cells more prevalent in the lower SP and CD150− cell more prevalent in the upper SP. We decided to examine this observation further by investigating the properties of cells from different regions of the SP. Functional capacity was assessed by competitive bone marrow transplantation of upper SP cells, lower SP cells, and a combination of the two populations. Lower SP cells showed better engraftment than upper SP cells in recipient mice, a trend that continued when donor HSCs were isolated from primary recipients and re-transplanted into secondary hosts. Lower SP cells showed 3-fold better engraftment than upper SP cells in secondary transplants, suggesting better self-renewal capacity. However, analysis of the hematopoietic lineages formed by donor cells in recipient mice demonstrated that while both upper and lower SP cells were capable of forming all mature lineages, lower SP cells were biased towards myeloid differentiation while upper SP cells were biased towards lymphoid differentiation. The lineage biases observed from transplantation of one cell population alone were exacerbated when both upper and lower SP cells were co-transplanted into the same recipient mouse, suggesting that while both populations are capable of forming all hematopoietic lineages, in the presence of the other stem cell type (as would be the case in normal homeostasis) that the majority of the output from each HSC subtype is almost exclusively lymphoid or myeloid. The lineage contribution trends observed in the peripheral blood were also reproduced when bone marrow of transplanted mice was analyzed, including at the level of progenitors with lower SP cells showing greater ability to make myeloid progenitors (megakaryocyte-erythrocyte progenitors and granulocyte-macrophage progenitors) and upper SP cells producing proportionately more common lymphoid progenitors. Microarray analysis of upper and lower SP cells to determine the molecular signatures underlying these functional differences found many genes critical for long-term HSC self-renewal to be highly expressed in lower SP cells including Rb1, Meis1, Pbx1 and TGFbr2 while upper SP cells showed higher expression of cell cycle and activation genes. Cell cycle analysis showed upper SP cells to be approximately 2-fold more proliferative than lower SP cells (18.9% to 8.3% Ki-67+, 39.4% to 20.1% BrdU+ 3-days post-BrdU administration). The clonal diversity model which proposes the adult HSC compartment consists of a fixed number of different HSC subtypes each with pre-programmed behavior has important implications for using HSCs in experimental and clinical settings. While other studies have provided functional evidence for the clonal diversity model, this is the first study to prospectively isolate the functionally distinct HSC subtypes prior to transplantation. Figure Figure

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Single Cell-Resolution Analysis of HSC Dysfunction in Mir-146a knockout Mice

Blood ◽

10.1182/blood.v130.suppl_1.714.714 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 714-714

Author(s):

Jennifer Grants ◽

Joanna Wegrzyn ◽

David Knapp ◽

Tony Hui ◽

Kieran O'Neill ◽

...

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Transcriptome Profiling ◽

Self Renewal ◽

Hematopoietic Stem ◽

Proliferation And Differentiation ◽

Stem And Progenitor Cells ◽

Resolution Analysis ◽

Signaling Activation

Abstract MicroRNA miR-146a is frequently depleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Loss of miR-146a may be an initiating event in tumorigenesis, as miR-146a loss in mouse models is sufficient to cause features of MDS and eventual progression to AML. To define how miR-146a loss initiates tumorigenesis, we analyzed hematopoietic stem cell (HSC) function from miR-146a knockout (KO) mice prior to onset of an overt malignant phenotype. Tracking cell division kinetics, proliferation, and differentiation of single long-term HSC (LT-HSC; EPCR+CD45+CD48-CD150+) in culture, we found evidence that miR-146a KOreduces HSC quiescence and promotes differentiating cell divisions. Our data show that miR-146a KO HSC dysfunction may stem from loss of a CD150-bright EPCR-bright sub-population, which has previously been associated with robust HSC activity. In line with this, single cell DNA methylation profiling revealed a reduction in a primitive sub-population of LT-HSCs in miR-146a KO animals. In addition, single cell LT-HSC transplants revealed a myeloid repopulation bias. As reduced HSC cell cycle quiescence has been linked to impaired HSC self-renewal upon hematopoietic stress, such as serial transplantation, we assessed the frequency of serially transplantable HSCs by performing secondary transplants with limiting dilution. Serially transplantable HSC frequency was reduced in miR-146a KO compared to wild type, suggesting impaired HSC self-renewal. Transcriptome profiling of miR-146a KO hematopoietic stem and progenitor cells identified tumor necrosis factor (TNF) signaling activation as a potential driver of HSC dysfunction. LT-HSC cell cycle quiescence and the CD150-bright EPCR-bright LT-HSC sub-population were restored in miR-146a/TNF double KO mice, suggesting that aberrant TNF signaling activation drives HSC dysfunction upon loss of miR-146a. Gene expression levels in the TNF signaling network are inversely correlated with miR-146a levels in human AML, implying that TNF signaling may similarly disrupt HSC function in miR-146a- depleted myeloid malignancies. Overall, our findings suggest that miR-146a promotes HSC cell cycle quiescence and inhibits differentiation by antagonizing TNF signaling, in order to maintain a primitive sub-population of long-term self-renewing HSCs. Disclosures Eaves: Experimental Hematology: Other: Editor of journal; StemCell Technologies Inc: Other: Wife of owner.

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LRF Is Indispensable for Hematopoietic Stem Cell Function Via Blocking Notch1-Mediated T Cell-Instructive Signals in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v114.22.81.81 ◽

2009 ◽

Vol 114 (22) ◽

pp. 81-81 ◽

Cited By ~ 1

Author(s):

Sung-UK Lee ◽

Manami Maeda ◽

Nagisa Sakurai ◽

Freddy Radtke ◽

Takahiro Maeda

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Target Gene ◽

T Cell Development ◽

Cell Development ◽

B Cell Development ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System

Abstract Abstract 81 Hematopoietic stem cells (HSC) have the ability to self-renew and give rise to all hematopoietic lineage cells. Understanding signals that regulate the balance between self-renewal and differentiation of HSCs is an important issue in stem cell biology as well as regenerative medicine. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function is currently under debate. We explored the role of the LRF (Leukemia/Lymphoma Related Factor), a Notch repressor (also known as Zbtb7a, pokemon, OCZF and FBI-1) in HSC function, as it plays key roles in embryonic development, oncogenesis, and hematopoiesis. Conditional inactivation of the LRF gene in mouse HSCs (LRFF/FMx1-Cre mice) led to the development of CD4/CD8 DP (double positive) T-cells at the expense of B-cell development in the bone marrow (BM) in a Notch-dependent manner. Absolute numbers of the most primitive HSCs (LT-HSCs), defined as CD150+CD48−Flt3−Vcam-1+IL7Rα−LSK (Lin−Sca1+c-Kit+), were significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/−IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRFF/FMx1-Cre mice one month after pIpC injection. Enhanced T cell development and concomitant loss of B cell development was also seen in LRF−/− fetal liver (FL). Lin−IL7Rα+c-Kit+PIR+ (Paired Immunoglobulin-like receptors) T cell precursors were significantly increased in LRF−/− FL, indicating that Notch-mediated aberrant lymphoid fate determination also takes place during fetal hematopoiesis. To address which Notch gene(s) are targeted by LRF, we studied the HSC/progenitor population of conditional LRF knockout (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, normal B cell development was restored in LRFF/FMx1-Cre mice. Reduction of LT-HSCs in LRFF/FMx1-Cre resulted from high Notch1 activity, as loss of Notch1 rescued LT-HSC numbers, suggesting that LRF functions to maintain HSCs and normal lymphoid fate by blocking Notch1. HSCs in active cell cycle are sensitive to 5-fluoro-uracil (5-FU) treatment, which causes remaining dormant HSCs to be recruited into the cell cycle to rapidly produce new cells and to quickly re-establish the hematopoietic system. To examine the self-renewal capacity of LRF deficient LT-HSC, LRFF/FMx1-Cre mice were treated with 5-FU after pIpC injection and the recovery of LT-HSC numbers examined. While control LT-HSC numbers recovered to pretreatment levels 3 wk after 5-FU treatment, levels in LRFF/FMx1-Cre mice remained low, accompanied by DP T cell development in the BM. Furthermore, after 5-FU treatment, LT-HSC numbers of LRFF/FNotch1F/FMx1-Cre were compatible to those of control and LRFF/FMx1-Cre mice, indicating that lack of self-renewal capacity in LRF deficient LT-HSCs was due to excessive differentiation toward T cells caused by Notch1. In support of this idea, when mice were given 5-FU weekly as a challenge to assess their HSC function in vivo, the survival percentage in LRFF/FMx1-Cre mice was much lower than in controls (0% versus 50% in 1 month, P <0.0001) and that of LRFF/FNotch1F/FMx1-Cre mice was compatible to controls. Serial bone marrow transplant experiments further demonstrated functional defects of LRF deficient HSCs, as they failed to reconstitute the hematopoietic system in secondary recipients. Microarray analysis and subsequent Gene Set Enrichment Analysis demonstrated upregulation of genes that were enriched in progenitor compartments. Since LRF can act as a transcriptional repressor, mRNA levels of Notch receptors and Notch ligands were examined using the same data set. A Notch target gene Hes1, but not Notch1 itself, was upregulated, and increased levels of Hes1 was also confirmed by real-time q-PCR in FACS-sorted LT-HSCs, as well as in 10.5 d.p.c whole embryos. These data suggest that LRF does not transcriptionally regulate Notch1, as LRF loss led to Notch1 target gene activation at the LT-HSC level without affecting Notch1 mRNA. Our genetic studies clearly indicate that LRF is indispensable for the maintenance of the HSC pool by repressing T cell-instructive signals mediated by Notch1 in the BM niche. Our findings shed new light on the regulatory mechanisms underlying the balance between HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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Unravelling Cell Cycle and Ontogeny Transcriptional Heterogeneity in Hematopoietic Stem Cells through Integrated Single Cell RNA-Seq

Blood ◽

10.1182/blood.v130.suppl_1.769.769 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 769-769

Author(s):

Benjamin Povinelli ◽

Quin Wills ◽

Nikolaos Barkas ◽

Christopher Booth ◽

Kieran Campbell ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Single Cell ◽

Developmental Time ◽

Rna Seq ◽

Self Renewal ◽

Hematopoietic Stem ◽

Time Points ◽

Post Transplant ◽

Cell Cycle Genes

Abstract During fetal development, hematopoietic stem cells (HSCs) undergo a remarkable expansion through a combination of rapid proliferation and high rates of self-renewal. In contrast, adult HSCs are characterized by long-term quiescence. Understanding of the molecular mechanisms underlying these ontogeny-dependent differences in cell cycle and self-renewal is hampered by marked heterogeneity within the HSC compartment, making it difficult to distinguish overlapping signatures in bulk transcriptional data. Advances in single cell genomics provide a new opportunity to tease apart different sources of gene expression heterogeneity, including those relating to cell cycle and self-renewal capability. To address these questions, and improve resolution for cell-cycle annotation of individual HSCs, we developed an integrated single cell (sc)RNA-seq and live cell-cycle staining technique using Hoechst 33342 (DNA) and Pyronin-y (RNA) based FACS index sorting, followed by smart-seq2 based scRNA-seq. We validated our approach on 4 hematopoietic cell lines from mouse and human, using these data as a training set to apply a novel integrated pseudotime package that orders single cells by stage of cell cycle rather than developmental trajectory. By this approach we detected non-canonical cell cycle genes not apparent through bulk sorting of distinct cell cycle phases, and not previously annotated in published cell cycle gene sets (sc-pseudotime genes = 665, FDR < 0.05; non-annotated cell cycle = 487; non-bulk detected = 570). We then applied our technique to analyze primary mouse HSCs from 3 developmental time points (e15.5 fetal liver (FL), 2 week old bone marrow, and 6 week old adult bone marrow (ABM), n >1,500 single cells). Our cell cycle based integrated pseudotime analysis revealed distinct cell cycle signatures for FL, ABM, and common cell cycle related transcripts across distinct developmental time points that have not previously been described, including 555 unique cell cycle genes in FL; 401 unique cell cycle genes in ABM, and 93 novel cell cycle genes in common to both developmental time points e.g. Pclaf, Zfp367 and including long non-coding RNAs e.g. Lockd (FDR < 0.001). Our dataset uniquely allowed us to explore ontogeny related molecular signatures without the overriding effect of confounding cell cycle associated gene expression by directly comparing non-mitotic cells from FL and ABM groups. We identified 404 differentially expressed transcripts (FDR <0.001, >2FC), including genes of unknown HSC function (e.g., FL: Lgals1, Gmfg; ABM: Zfp36l1, Rgs1). Single cell qPCR confirmed aberrant expression of 26/29 (89.7%) selected ontogeny candidate genes. Furthermore, hallmark gene set enrichment analysis revealed upregulation of oxidative phosphorylation, MYC targets, and E2F targets in FL; and TNFA signaling, Hypoxia, and TGF-beta signaling, among others, in ABM (FDR <0.01, NES > 1.5). We then functionally reversed ABM quiescence through in vivo 5-FU treatment, and performed our single cell RNA-seq analysis on HSCs both 2 and 6 days post injection. This allowed us to identify genes and pathways associated with selective resistance of HSCs to chemotherapy, including upregulation of the hallmark gene sets for unfolded protein response, fatty acid metabolism, and MTOR signaling (FDR < 0.01, NES > 1.5 for each set). To functionally validate novel ontogeny related genes we utilized genetic mouse models for two unexplored ABM related genes, Zfp36L1, an RNA-binding zinc finger protein, and Rgs1 a regulator of G-protein coupled signaling. We transplanted Cre-ERT2 conditionally floxed Zfp36L1 bone marrow with CD45.1 competitor control and induced deletion by tamoxifen four weeks post transplant. Compared to the initial four-week post transplant time point we observed a significant reduction in chimerism from Zfp36L1 deleted bone marrow compared to Cre-ERT2 control (p < .05). Competitive transplantation of Rgs1 -/- and WT bone marrow at a 1:1 ratio with CD45.1 competitors resulted in significantly reduced myeloid chimerism at 16 weeks post transplant. Secondary transplant and single cell cycle molecular analysis of these mice are ongoing together with functional validation of a number of other candidate genes. Our results demonstrate the utility of single cell analysis to discover novel HSC regulators providing a unique dataset for further studies investigating regulators of HSC function. Disclosures Mead: BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

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Telomere Damage Maintains Hematopoietic Stem Cells (HSCs) in an Activated Metabolic State, Which Compromises Their Self-Renewal Capability

Blood ◽

10.1182/blood-2018-174 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 174-174

Author(s):

Andrea Santoni ◽

Elena Fiorini ◽

Fides D Lay ◽

Matteo Marchesini ◽

Yamini Ogoti ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Single Cell ◽

Molecular Mechanisms ◽

Poly I:C ◽

Rna Seq ◽

Metabolic State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Functional Studies

Abstract DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown. Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit+Sca+Lin-CD34-flk2-CD150+CD48-CD41-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability. To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERTER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERTER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p<10-3) that was associated with a decreased number of the lymphoid-biased MPP4 cells, and an increased number of both megakaryocyte-biased MPP2 and myeloid-biased MPP3 cells. HSC exhaustion and increased myeloid-to lymphoid output were reminiscent of stressed hematopoiesis and premature aging. G5/G6 HSCs exhibited a significant accumulation of telomere dysfunction-induced foci (p<10-5) but did not display increased levels of apoptosis in steady-state conditions. HSC exhaustion could result from apoptosis and/or senescence induced by telomere damage in HSCs entering the cell cycle or from an altered balance between self-renewal and differentiation. To distinguish between these two possibilities, we first investigated the effect of inducing young G5/G6 HSCs out of a homeostatic quiescent state. By tracking the real-time changes in the expression level of annexin V on HSCs induced to differentiate towards the myeloid lineage, we found that apoptosis was not the primary fate of G5/G6 HSCs upon entry into the cell cycle. Similarly, in vivo treatment with poly I:C induced the G5/G6 HSCs to enter into the cell cycle at the same rate as that of the G0 mice without inducing apoptosis. Transcriptomic analysis of poly I:C-treated G0 and G5/G6 HSCs, compared with vehicle-treated controls, revealed a significant enrichment of genes involved in the regulation of the cell cycle and platelet production, which is consistent with previous findings showing that megakaryocyte differentiation of HSCs occurs in response to poly I:C to replenish platelets that are lost during inflammatory insult. Importantly, we did not observe any significant change in gene expression between G0 and G5/G6 HSCs isolated from poly I:C-treated mice, which confirmed that telomeric damage did not limit HSCs' proliferation potential by activating programs of senescence or apoptosis. Next, we evaluated the capability of single HSCs isolated from G0 or G5/G6 mice to either self-renew or differentiate. An evaluation of Numb inheritance and expression in G0 and G5/G6 HSCs (n=133 and n=113, respectively) induced to proliferate in vitro showed that G5/G6 HSCs had a 2-fold lower frequency of symmetric self-renewal division (p<10-3) and a concomitant 2-fold higher frequency of symmetric commitment (p<10-4). Accordingly, PB analysis revealed that the CD45.2-derived reconstitution was severely compromised in mice competitively transplanted with G5/G6 HSCs (0.26% vs 77%; p<10-3). Single cell RNA-seq analysis of G0 and G5/G6 HSCs followed by the differential analysis of the clusters showed that 40% of G5/G6 HSCs were in an activated metabolic state associated with hyperactive OXPHOS and ROS signaling pathways, which are directly involved in HSC functional decline. Finally, we reactivated telomerase to investigate the possibility of restoring normal HSC function upon elimination of damage. Single cell RNA-seq and functional studies are ongoing to evaluate whether HSCs' activated metabolic state and compromised self-renewal capability are reversible processes. This study challenges the concept that telomeric damage limits HSC's proliferative potential and offers unparalleled opportunities for unraveling regenerative strategies to ameliorate their decline. Disclosures Colla: Abbvie: Research Funding.

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Evi1 Is a Stem Cell-Specific Regulator of Self-Renewal Capacity In the Definitive Hematopoietic System

Blood ◽

10.1182/blood.v116.21.838.838 ◽

2010 ◽

Vol 116 (21) ◽

pp. 838-838

Author(s):

Keisuke Kataoka ◽

Tomohiko Sato ◽

Akihide Yoshimi ◽

Susumu Goyama ◽

Takako Tsuruta ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Specific Loss ◽

Loss Of Self ◽

Colony Forming Capacity

Abstract Abstract 838 Self-renewal is a defining property of stem cells. Although a number of molecules have been implicated in the regulation of hematopoietic stem cell (HSC) self-renewal, loss of these genes is accompanied with other hematological abnormalities. Thus, it is unclear what will happen with a specific loss of self-renewal capacity of HSCs. Evi1 is an oncogenic transcription factor in myeloid malignancies. Evi1 expression is limited to hematopoietic stem/progenitor fraction, and Evi1 is essential for the maintenance of HSCs, but is dispensable for blood cell lineage commitment. Thus, we hypothesized that Evi1 expression could distinguish hematopoietic stem and progenitor cells, and reduction of Evi1 gene dosage might cause a specific loss of self-renewal activity. First, to elucidate Evi1 expression within the hematopoietic system, we have generated Evi1-IRES-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of Evi1 gene. We found that Evi1 was predominantly expressed in the hematopoietic stem/progenitor fraction (Lin- Sca-1+ c-kit+ (LSK)), but its expression was rapidly extinguished during early stages of lineage commitment. Among the LSK compartment, Evi1 was expressed at the highest level in long-term HSCs (LT-HSCs; Flk2- CD34-, CD48- CD150+, or SP-tip fractions in LSK cells). Next, we hypothesized that Evi1 would have the potential to mark LT-HSCs effectively. To test this, we compared GFP+ and GFP- cells in the LSK fraction, and revealed that GFP+ LSK cells were more immature and quiescent with a higher colony-forming capacity than GFP- LSK cells. In addition, in vivo long-term multilineage repopulating cells were exclusively enriched in the GFP+ LSK fraction. In the embryo, Evi1 was highly expressed in the hematopoietic stem/progenitor fraction; that is, CD34+ c-kit+ cells in embryonic day 10.5 (E10.5) aorta-gonad-mesonephros, CD34+ c-kit+ CD48- cells in E12.5 placenta, and Mac-1+ Sca-1+ Lin- (MSL) CD48- cells in E14.5 fetal liver (FL). In vivo competitive repopulation assay showed that, in the MSL fraction of FL, GFP+ MSL cells exclusively had a long-term multilineage repopulating capacity. These results implied that Evi1 plays a more specific role in HSCs than in other hematopoietic cells. To clarify this, we analyzed heterozygous Evi1 knockout mice (Evi1 +/− mice), as it seems difficult to elucidate the function of a small population of HSCs in Evi1 conditional knockout mice due to the leaky expression of Cre recombinase. We have previously showed that haploinsufficiency of Evi1 leads to decreased numbers of LSK and CD34- LSK cells, and impaired long-term repopulating activity. Here we demonstrated the number of each fraction in Evi1 +/− LSK cells was reduced in proportion to their expression level of Evi1. But, there were no significant differences in the numbers of lymphoid and myeloid progenitors between Evi1 +/+ and Evi1 +/− mice. Evi1 +/− CD34+ LSK cells had an equivalent in vitro colony-forming capacity and day 11 colony-forming unit-spleen activity to Evi1 +/+ CD34+ LSK cells. However, in vivo short-term repopulation assay using CD34+ LSK cells showed that the percentage of donor-derived cells from Evi1 +/− mice was significantly declined at 4 weeks after transplantation. Moreover, Evi1 +/− CD34- LSK cells had a pronouncedly impaired in vivo repopulating capacity. These data suggested that the differentiation capacity of Evi1 +/− HSCs was maintained, but their self-renewal capacity was specifically reduced. Although flow cytometric analysis of cell-cycle status and apoptosis showed no differences in CD34- LSK cells between Evi1+/+ and Evi1 +/− mice, the G0 fraction of Evi1 +/− CD34+ LSK cells was significantly reduced, indicating that these cells might proliferate more rapidly to compensate for the impaired self-renewal capacity of HSCs. In conclusion, we showed that Evi1 is predominantly expressed in HSCs and its expression can mark long-term repopulating HSCs in the fetal and adult hematopoietic system. Moreover, functional loss caused by haploinsufficiency of Evi1 is limited to a defect of self-renewal capacity of HSCs, and the increased cell-cycle progression of CD34+ LSK cells in Evi1 +/− mice seems to be the consequence of the impaired self-renewal capacity. Our data may help to understand the unrevealed effects of loss of self-renewal activity of HSCs and compensative mechanism of their defects. Disclosures: No relevant conflicts of interest to declare.

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Single-cell analyses show TGF-b1 reduces self-renewal and myeloid differentiation potentials in hematopoietic stem cells

Experimental Hematology ◽

10.1016/j.exphem.2017.06.269 ◽

2017 ◽

Vol 53 ◽

pp. S109-S110

Author(s):

Xiaofang Wang ◽

Fang Dong ◽

Sen Zhang ◽

Wanzhu Yang ◽

Zhao Wang ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Single Cell ◽

Myeloid Differentiation ◽

Self Renewal ◽

Hematopoietic Stem

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Deletion of PI3-Kinase Promotes Myelodysplasia Through Dysregulation of Autophagy in Hematopoietic Stem Cells

10.1101/2020.12.04.412593 ◽

2020 ◽

Author(s):

Kristina Ames ◽

Imit Kaur ◽

Yang Shi ◽

Meng Tong ◽

Taneisha Sinclair ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Myeloid Leukemia ◽

Chromosomal Abnormalities ◽

Akt Pathway ◽

Hematopoietic Growth Factors ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Phosphoinositide 3 Kinase

AbstractHematopoietic stem cells (HSCs) maintain the blood system through a delicate equilibrium between self-renewal and differentiation. Most hematopoietic growth factors and cytokines signal through phosphoinositide 3-kinase (PI3K) via three Class IA catalytic PI3K isoforms (P110α, β, and δ), encoded by Pik3ca, Pik3cb, and Pik3cd, respectively. The PI3K/AKT pathway is commonly activated in acute myeloid leukemia (AML), and PI3K is a common therapeutic target in cancer. However, it is not known whether PI3K is required for HSC differentiation or self-renewal. We previously demonstrated that individual PI3K isoforms are dispensable in HSCs1,2. To determine the redundant roles of PI3K isoforms in HSCs, we generated a triple knockout (TKO) mouse model with deletion of all three Class IA PI3K isoforms in the hematopoietic system. Surprisingly, we observed significant expansion of TKO HSCs after transplantation, with decreased differentiation capacity and impaired multilineage repopulation. Additionally, the bone marrow of TKO mice exhibited myelodysplastic features with chromosomal abnormalities. Interestingly, we found that macroautophagy (thereafter autophagy) is impaired in TKO HSCs, and that pharmacologic induction of autophagy improves their differentiation. Therefore, we have uncovered important roles for PI3K in autophagy regulation in HSCs to maintain the balance between self-renewal and differentiation.

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