Quiescent Fraction Research Articles

Filament profiles from WISExSCOS galaxies as probes of the impact of environmental effects

The role played by large-scale structures in galaxy evolution is not very well understood yet. In this study, we investigated properties of galaxies in the range 0.1 < z < 0.3 from a value-added version of the WISExSCOS catalogue around cosmic filaments detected with DisPerSE. We fitted a profile of galaxy over-density around cosmic filaments and found a typical radius of rm = 7.5 ± 0.2 Mpc. We measured an excess of passive galaxies near to the spine of the filament that was higher than the excess of transitioning and active galaxies. We also detected star formation rates (SFR) and stellar mass (M⋆) gradients pointing towards the spine of the filament. We investigated this result and found an M⋆ gradient for each type of galaxy, that is active, transitioning, and passive; we found a positive SFR gradient for passive galaxies. We also linked the galaxy properties and gas content in the cosmic web. To do so, we investigated the quiescent fraction fQ profile of galaxies around the cosmic filaments. Based on recent studies about the effect of the gas and the cosmic web on galaxy properties, we modelled fQ with a β model of gas pressure. The slope obtained in this work, β = 0.54 ± 0.18, is compatible with the scenario of projected isothermal gas in hydrostatic equilibrium (β = 2/3) and with the profiles of gas fitted in Sunyaev-Zel’dovich data from the Planck satellite.

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

Chronic myeloid leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the BM of patients having achieved remission under treatment. We demonstrate by single-cell RNASequencing analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38– leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their BM microenvironment.

Abstract C50: Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state

Abstract Tumors contain a heterogeneous mixture of live cells that are either undergoing proliferation or reside in a nonproliferative state. Cells that do not divide are often associated with increased chemoresistance, challenging therapies that target rapidly proliferating cells. In pancreatic cancer, paucity of blood vessels frequently generates hypoperfused, “arid” regions that constrain tumor cell metabolism and proliferation. Nonetheless, experimental conditions for studying tumor cells in vitro typically mimic well-nourished, “fertile” environments optimized for supporting proliferating cells rather than the more prevalent quiescent state. Characterization of the poorly vascularized KPC model confirmed that the quiescent phenotype is common and is strongly correlated to reduced tissue perfusion. To formulate culture conditions that will emulate the arid microenvironment associated with the nonproliferative state in vitro, we systematically deprived various nutrients from tumor cells and established the necessary components for attaining cell quiescence. Deprivation of amino acids on top of glucose, oxygen, and serum levels was essential for obtaining a complete and reversible cell cycle arrest without loss of viability. Cell cycle arrest was accompanied by a number of physiologic adaptations. Metabolomic analysis indicated reduction in the levels of TCA cycle and mevalonate pathway intermediates indicative of increased reliance on nonglycolytic metabolism. In addition, free amino acid pools were diminished and tumor cells exhibited a significant increase in autophagic flux and micropinocytosis, suggesting that amino acid availability is limited under arid conditions. Concomitant with reduced proliferation and metabolic rewiring, tumor cells under arid conditions exhibited remarkable resistance to gemcitabine. To explore alternative strategies to target these nonproliferating pancreatic tumor cells, we performed a comprehensive genome-wide CRISPR/Cas9-based genetic screen and compared vulnerabilities under ”fertile” and “arid” conditions. Functional annotation of sgRNAs depleted under arid conditions indicated a striking dependence on oxidative phosphorylation for survival. Remarkably, our analysis also revealed that a number of targetable genetic and epigenetic programs driving cell cycle progression were not essential and, in some instances, even hazardous for cells under arid conditions. These results suggest that pancreatic tumor cells that cease proliferation in a nutrient-depleted environment are physiologically rewired and may exhibit a distinct drug response profile from rapidly proliferating cells. The experimental model we developed allows investigation of this dominant yet understudied cell population and may serve as a platform for identification of agents targeting the quiescent fraction of tumors. Citation Format: Yogev Sela, Jinyang Li, Miriam Doepner, Shivahamy Maheswaran, Clementina Mesaros, Ian Blair, Ophir Shalem, Ben Stanger. Dissecting vulnerabilities of pancreatic tumors’ silent fraction unravels secrets of an alternative cell state [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C50.

Discrimination of Dormant and Active Hematopoietic Stem Cells by G0 Marker Reveals Dormancy Regulation by Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G0 marker (G0M) mouse line that visualizes quiescent cells and identify a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of thesepopulations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations.

More connected, more active: galaxy clusters and groups at z ∼ 1 and the connection between their quiescent galaxy fractions and large-scale environments

ABSTRACT High-redshift galaxy clusters, unlike local counterparts, show diverse star formation activities. However, it is still unclear what keeps some of the high-redshift clusters active in star formation. To address this issue, we performed a multiobject spectroscopic observation of 226 high-redshift (0.8 < z < 1.3) galaxies in galaxy cluster candidates and the areas surrounding them. Our spectroscopic observation reveals six to eight clusters/groups at z ∼ 0.9 and z ∼ 1.3. The redshift measurements demonstrate the reliability of our photometric redshift measurements, which in turn gives credibility for using photometric redshift members for the analysis of large-scale structures (LSSs). Our investigation of the large-scale environment (∼10 Mpc) surrounding each galaxy cluster reveals LSSs – structures up to ∼10 Mpc scale – around many of, but not all, the confirmed overdensities and the cluster candidates. We investigate the correlation between quiescent galaxy fraction of galaxy overdensities and their surrounding LSSs, with a larger sample of ∼20 overdensities including photometrically selected overdensities at 0.6 < z < 0.9. Interestingly, galaxy overdensities embedded within these extended LSSs show a lower fraction of quiescent galaxies ($\sim 20{{\ \rm per\ cent}}$) than isolated ones at similar redshifts (with a quiescent galaxy fraction of $\sim 50 {{\ \rm per\ cent}}$). Furthermore, we find a possible indication that clusters/groups with a high quiescent galaxy fraction are more centrally concentrated. Based on these results, we suggest that LSSs are the main reservoirs of gas and star-forming galaxies to keep galaxy clusters fresh and extended in size at z ∼ 1.

Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically Relevant Ex Vivo Gene Therapy Settings.

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit ex vivo LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow–derived CD34+ cells while better preserving their engraftment capacity in vivo. CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G0 fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels in vivo. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC ex vivo culture time.

PS1436 IFN‐Α TREATMENT NORMALIZES THE PLATELET COUNTS AND REDUCES QUIESCENT STEM CELLS IN A CALR‐DEL52 TRANSGENIC MOUSE MODEL, IN VIVO

Background:Treatment options for Myeloproliferative Neoplasms (MPN) have mainly been limited to disease managing risk of thromboembolic events and long‐term transformation to acute myelogenous leukemia and myelofibrosis. To date, type I interferons (IFNs) are the only class of drugs with curative potential in MPN as they can actually reduce the allelic burden of mutant clones in patients. The mechanism of action of IFN in MPNs remains elusive. This is mainly because modelling IFN treatment in mouse models has proven to be difficult due to the short half‐life of murine non‐pegylated IFNs. This shortcoming was solved by the murine pegylated IFN‐α (murine ropeginterferon‐a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) which can be used to model IFN treatment in mouse models of MPN.Aims:Generation of a conditional transgenic mouse model for CALR‐del52 induced MPN and modelling IFN treatment in these mice using murine pegylated IFN‐α (mRopeg).Methods:The CALR‐del52 transgenic mice (with human mutant sequence) were generated on a C57BL/6 background by homologous recombination of embryonic stem cells at Ozgene (Perth, Australia). The CALR‐del52 floxed mice were bred with vavCre transgenics, thereby allowing the expression of CALR‐del52 specifically in the hematopoietic cells. MPN phenotype in the mice was characterized by peripheral blood counts and flow cytometry. IFN treated mice received mRopeg weekly (600 ng) by subcutaneous injection for 4 weeks. Control mice received the same volume of vehicle (in this case, phosphate buffered saline – PBS).Results:Heterozygous CALR‐del52fl/+;vavCre mice displayed elevated platelet counts starting from 6 weeks of age, with normal white blood cell and red blood cell counts. However, homozygous CALR‐del52fl/fl;vavCre mice developed much higher platelet counts associated with increased white blood cell and decreased red blood cell counts. Homozygous CALR‐del52 mice also developed splenomegaly starting from 6 months of age. Also at 6 months, there was significant increase in the megakaryocyte population in the bone marrow, along with significant increase in LSK cells (Lin− Sca+ kit+) and the quiescent (Fraction A) stem cell population. IFN‐α treatment of CALR‐del52 transgenic mice for 4 weeks resulted in the normalization of the platelet counts to wild type levels. Interestingly, the total stem cell population (LSK cells) in the bone marrow increased upon IFN‐α treatment. However, the percentage of quiescent stem cells (Fraction A) reduced, while the percentage of cycling stem cells (Fraction B) and multipotent stem cells (Fraction C) was increased.Summary/Conclusion:Expression of CALR‐del52 in hematopoietic cells leads to the development of MPN phenotype in mice, and the severity positively correlated with the number of mutant alleles. Heterozygous CALR‐del52 mice developed an Essential Thrombocythemia (ET) like phenotype, with high platelet counts but normal white blood and red blood cell counts. However, the homozygous CALR‐del52 mice developed signs of myelofibrosis with age, including reduced red blood cell counts and splenomegaly. IFN‐α treatment resulted in normalization of platelet numbers to wild type levels within 4 weeks of treatment. Importantly, it also resulted in significant reduction of the quiescent hematopoietic stem cells, while cycling and multipotent stem cells were increased. This suggests that IFN‐α induces proliferation of quiescent stem cells into cycling and multipotent stem cells in CALR‐del52 mutant cells.image

A systematic search for galaxy proto-cluster cores at z ∼ 2

AbstractA proto-cluster core is the most massive dark matter halo (DMH) in a given proto-cluster. To reveal the galaxy formation in core regions, we search for proto-cluster cores at z ˜ 2 in ˜1.5deg2 of the COSMOS field. Using pairs of massive galaxies (log (M*/Mʘ) ≥ 11) as tracers of cores, we find 75 candidate cores. A clustering analysis and the extended Press-Schechter model show that their descendant mass at z = 0 is consistent with Fornax-like or Virgo-like clusters. Moreover, using the IllustrisTNG simulation, we confirm that pairs of massive galaxies are good tracers of DMHs massive enough to be regarded as proto-cluster cores. We then derive the stellar mass function and the quiescent fraction for member galaxies of the 75 candidate cores. We find that stellar mass assembly and quenching are accelerated as early as z ˜ 2 in proto-cluster cores.

Galaxy populations in the most distant SPT-SZ clusters

We present the first results from a galaxy population study in the highest redshift galaxy clusters identified in the 2500 deg2 South Pole Telescope Sunyaev Zel’dovich effect (SPT-SZ) survey, which is sensitive to M500 ≳ 3 × 1014 M⊙ clusters from z ∼ 0.2 out to the highest redshifts where such massive structures exist. The cluster selection is to first order independent of galaxy properties, making the SPT-SZ sample particularly well suited for cluster galaxy population studies. We carried out a four-band imaging campaign with the Hubble and Spitzer Space Telescopes of the five z ≳ 1.4, S/NSZE > 5 clusters, that are among the rarest most massive clusters known at this redshift. All five clusters show clear overdensities of red galaxies whose colors agree with the initial cluster redshift estimates, although one (SPT-CLJ0607–4448) shows a galaxy concentration much less prominent than the others. The highest redshift cluster in this sample, SPT-CLJ0459–4947 at z ∼ 1.72, is the most distant M500 > 1014 M⊙ cluster discovered thus far through its intracluster medium, and is one of only three known clusters in this mass range at z ≳ 1.7, regardless of selection. Based on UVJ-like photometric classification of quiescent and star-forming galaxies, we find that the quiescent fraction in the cluster central regions (r/r500 < 0.7) is higher than in the field at the same redshift, with corresponding environmental quenching efficiencies typically in the range ∼0.5 − 0.8 for stellar masses log(M/M⊙) > 10.85. We have explored the impact of emission from star formation on the selection of this sample, concluding that all five clusters studied here would still have been detected with S/NSZE> 5, even if they had the same quiescent fraction as measured in the field. Our results thus point towards an efficient suppression of star formation in the central regions of the most massive clusters, occurring already earlier than z ∼ 1.5.

De Novo PITX1 Expression Controls Bi-Stable Transcriptional Circuits to Govern Self-Renewal and Differentiation in Squamous Cell Carcinoma.

Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining genetargeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analysesreveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression toprevent TPC expansion. This bi-stable, multi-inputnetwork reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCCgrowth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.

Discrimination of Dormant and Active Hematopoietic Stem Cells by G <sub>0</sub> Markers Reveals Dormancy Regulation by Basal Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generated a G0 marker mouse line that visualizes quiescent cells. The G0 marker identifies a population of active HSCs (G0 markerlow) which are rare and distinct from dormant HSCs (G0 markerhigh) within the conventional quiescent HSC fraction. Single-cell RNA-sequencing analyses showed that the gene expression profiles of these populations were nearly identical, yet differed in their Cdk4/6 activity. Furthermore high-throughput small molecule screening revealed that high levels of basal cytoplasmic calcium (Ca2+) protected the dormancy of HSCs from cell cycle activation induced by calcium influx. These findings indicate that the G0 marker separates dormant and active adult HSCs, which are regulated by Cdk4/6 and Ca2+ levels. This G0 marker mouse line represents a useful new resource for investigating physiologically important stem cell subpopulations.

Inhibition of the Hedgehog Pathway Decreases the Quiescent CD34+CD38- Population in Acute Myeloid Leukemia

IntroductionAcute myeloid leukemia (AML) is a clonal disease with a reduced life expectancy due to a high relapse rate. One explanation is that leukemic stem cells (LSC) evade the action of conventional chemotherapy due to their quiescent state. Several mechanisms have been proposed that regulate their quiescence, however, by analogy with normal hematopoietic stem cells, a key role may be carried out by the signaling pathways Notch and Hedgehog (Hh). The objectives of this study are to analyze the role of Notch and Hh pathways in the quiescence of LSC and to verify if the pharmacological inhibition of the Notch and Hh pathways decreases the percentage of quiescent LSC. In this way, LSCs would be sensitized to chemotherapy treatments and the high relapse rate of AML could be reduced.MethodsExpression of GLI1, a transcription factor of the Hh signaling pathway and NOTCH Internal Cleaved Domain (NICD) were analyzed in the hematopoietic stem and progenitor cells of four patients diagnosed with AML. The selection of quiescent fraction was performed by flow cytometry using anti-CD34-FITC, anti-CD117-PerCP, anti-CD45PE-Cy7, anti-CD38-APC-Cy7 and anti-KI67-BV510 antibodies. KI67 negative cells were considered quiescent. The activation of NOTCH and Hh pathways was studied using rabbit anti-GLI, anti-NICD primary antibodies and anti-rabbit BV421 secondary antibodies. Results were expressed in median (range) or mean ± standard deviation.Dose-response curves of inhibitors of the Notch pathway (BMS-906024, inhibitor of γ-secretase), Hh pathway (BMS-833923, SMO inhibitor and GANT61, GLI inhibitor), and cytarabine (AraC) were made to study drug potency in the OCI-AML3 cell line. We also analyzed its synergistic behavior in combination with Arac by calculating the combination index (CI) of each of them. These experiments were conducted in triplicate and values were expressed as the mean ± standard deviation.Finally, the effect of BMS-833923 and BMS-906024 on the quiescence of the CD34+CD38- cells of two patients diagnosed with AML was studied by flow cytometry.The paired samples t-test was used in the statistical analysis of GLI and NICD expression between G0 and proliferating cells and in the statistical analysis of the decrease of quiescent cells due to Notch and Hh inhibitors.ResultsFirst of all, hematopoietic and progenitor cells were quantified in four AML patients: the median of the percentage of CD34+CD38- cells with respect to total cells in the bone marrow of the AML patient studied was 1.1% (range: 0.12%-9.05%), within which 71.50% (range: 64.30%-88.43%) are quiescent. Interestingly, we found a trend for a higher expression of NOTCH signaling pathway in the proliferating CD34+CD38- cells (relative median fluorescent intensity (MFI) = 1.91 (range: 1.51-3.34)) compared to the quiescent fraction (relative MFI=1.55 (range: 1.18-1.94); p=0.105). But no differences were found in expression of GLI1.Before studying the effect on cellular quiescence of Notch and Hh inhibitors in monotherapy and in combination with AraC, we evaluated their effect on cell viability. The most potent drug studied was AraC (IC50 = 4.055 μM), followed by inhibitors of the Hh pathway (IC50 BMS-833923 = 5.041 μM; IC50 GANT61 = 7.042 μM) on the OCI-AML3 cell line. In contrast, the γ-secretase inhibitor (BMS-906024) showed no effect. Moreover, it was found that the combination 0.8 μM AraC plus 8 μM BMS-833923 was the most synergistic (CI = 0.53, 15% viability with respect to DMSO control).Subsequently, the effect of the SMO inhibitor on the quiescent CD34+CD38- cells of two patients diagnosed with CD34+ AML was analyzed: BMS-833923 decreased the percentage of quiescent CD34+CD38- cells by 88.5±16.3% in monotherapy (p=0.083) and in presence of AraC by 85.8 ± 21.2% (p=0.113) (figure 1).ConclusionThe use of SMO inhibitors for the treatment of AML is promising because it increases the sensitivity of leukemic cells to chemotherapy and facilitates their action by reducing the percentage of quiescent LSC. This could mean a decrease in the probability of relapse in patients with AML treated with Hh inhibitors. These results were derived from an ongoing project and more patients are being studied in order to confirm the explained results.This work is partially funded by the Madrid Association of Hematology and Hemotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing

Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy.We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture.By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between.After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC.Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO).To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status.As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. DisclosuresNo relevant conflicts of interest to declare.

OPN Maintains Leukemia Stem Cells Survival By Suppressing PUMA

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. In the era of chemotherapy, the cure rate of AML is 35 to 40% of adult patients who are 60 years of age or younger while it is only 5 to 15% of patients who are older than 60 years of age could achieve relative long term survival. Leukemia stem cells (LSCs) population is a small proportion in leukemia cells but plays an important role in initiation of AML and is the main cause of the recurrence after chemotherapy due to the ability of maintaining quiescence and other mechanisms to sustain survival. How to eradicate LSCs was investigated intensively during past years, but how LSCs maintain survival is still elusive. Here we show that osteopontin(OPN), a member of extracellular matrix protein, is essential for AML stem cells survival and could be a novel and potent therapeutic target for eradicating LSCs.We used RNA interference system to knock down OPN in a mouse model of MLL-AF9 induced AML. We examined the survival of AML mice and found that OPN knockdown (KD) group showed a significant prolongation of survival. Colony forming assay showed that OPN KD AML cells formed colonies in a lower frequency than that of control AML cells. Flow cytometry showed the frequency of LSC in KD AML cells was lower than that in control AML cells(0.04±0.02% vs 3.61±0.32%) and the median fluorescence density of c-Kit is lower in KD AML cells than of control AML cells(759.2±80.8 vs 1353.5±27.5). Brdu assay indicated an increased quiescent fraction in G0/G1 phases (64.9±8.9% vs 53.6±3.4%) of KD AML cells. The apoptosis of KD AML cells is increased than that in the control AML cells(14.82±3.4% vs 4.9±1.2%). Most interestingly, the change of phenotype is much more prominent in the LSCs population, ( comparing KD group and control group, frequency of G0/G1 in LSCs: 50.2±7.5% vs 26.4±3.7%; frequency of apoptotic cells in LSCs: 58.42±5.38% vs 7.45±1.57%). Limiting dilution assay confirmed that lower frequency of LSCs in the KD AML cells than in control(1/1204 vs 1/214). Thus we concluded that OPN plays a very important role in maintain survival of LSCs.To determine whether OPN knockdown could induce the same phenotype in human AML cells. We constructed OPN knockdown cell-line model by RNA interference system. Human AML cell-lines (SKM-1, ML2, OCI-AML3, SKM-1 FL3-ITD knock in) presented the same phenotype such as the increase of apoptosis and the decrease of the fraction of cells entering cell cycle. Then we performed Realtime PCR and western blot in mouse model to reveal the underlying mechanisms. Realtime PCR showed a significantly up-regulation of P16, P18, P21, which is related to the cell cycle arrest; western blot showed a significantly increase of PUMA and cleaved-caspase3, which indicated that knockdown OPN could promote apoptosis through repress the function of BCL2 by up-regulate PUMA in protein level. Interestingly, incubation of recombinant OPN protein with OPN KD cells did not rescue the phenotype suggesting that intracellular function of OPN in LSCs is important for its survival. The detailed mechanism of how OPN regulate PUMA is still under investigation.In our current study, it showed that OPN knock down can significantly prolong the survival of leukemic mice and decrease the leukemia burden by decreasing the stemness of LSC and promoting LSC apoptosis by inducing PUMA protein function. Thus it could be a good therapeutic target for eradicating LSCs in AML. DisclosuresNo relevant conflicts of interest to declare.

Identification of Genes Involved in Bacteriostatic Antibiotic-Induced Persister Formation.

Persister cells are metabolically quiescent multi-drug tolerant fraction of a genetically sensitive bacterial population and are thought to be responsible for relapse of many persistent infections. Persisters can be formed naturally in the stationary phase culture, and also can be induced by bacteriostatic antibiotics. However, the molecular basis of bacteriostatic antibiotic induced persister formation is unknown. Here, we established a bacteriostatic antibiotic induced persister model and screened the Escherichia coli single gene deletion mutant library for mutants with defect in rifampin or tetracycline induced persistence to ofloxacin. Thirsty-seven and nine genes were found with defects in rifampin- and tetracycline-induced persister formation, respectively. Six mutants were found to overlap in both rifampin and tetracycline induced persister screens: recA, recC, ruvA, uvrD, fis, and acrB. Interestingly, four of these mutants (recA, recC, ruvA, and uvrD) mapped to DNA repair pathway, one mutant mapped to global transcriptional regulator (fis) and one to efflux (acrB). The stationary phase culture of the identified mutants and parent strain BW25113 were subjected to different antibiotics including ofloxacin, ampicillin, gentamicin, and stress conditions including starvation and acid pH 4.0. All the six mutants showed less tolerance to ofloxacin, but only some of them were more sensitive to other specific stress conditions. Complementation of five of the six common mutants restored the persister level to that of the parent strain in both stationary phase and static antibiotic-induced conditions. In addition to the DNA repair pathways shared by both rifampin and tetracycline induced persisters, genes involved in rifampin-induced persisters map also to transporters, LPS biosynthesis, flagella biosynthesis, metabolism (folate and energy), and translation, etc. These findings suggest that persisters generated by different ways may share common mechanisms of survival, and also shed new insight into the molecular basis of static antibiotic induced antagonism of cidal antibiotics.

Altered B-lymphopoiesis in mice with deregulated thrombopoietin signaling

Thrombopoietin (TPO) is the master cytokine regulator of megakaryopoiesis. In addition to regulation of megakaryocyte and platelet number, TPO is important for maintaining proper hematopoietic stem cell (HSC) function. It was previously shown that a number of lymphoid genes were upregulated in HSCs from Tpo−/− mice. We investigated if absent or enhanced TPO signaling would influence normal B-lymphopoiesis. Absent TPO signaling in Mpl−/− mice led to enrichment of a common lymphoid progenitor (CLP) signature in multipotential lineage-negative Sca-1+c-Kit+ (LSK) cells and an increase in CLP formation. Moreover, Mpl−/− mice exhibited increased numbers of PreB2 and immature B-cells in bone marrow and spleen, with an increased proportion of B-lymphoid cells in the G1 phase of the cell cycle. Conversely, elevated TPO signaling in TpoTg mice was associated with reduced B-lymphopoiesis. Although at steady state, peripheral blood lymphocyte counts were normal in both models, Mpl−/− Eµ-myc mice showed an enhanced preneoplastic phase with increased numbers of splenic PreB2 and immature B-cells, a reduced quiescent fraction, and augmented blood lymphocyte counts. Thus, although Mpl is not expressed on lymphoid cells, TPO signaling may indirectly influence B-lymphopoiesis and the preneoplastic state in Myc-driven B-cell lymphomagenesis by lineage priming in multipotential progenitor cells.

The Pan-STARRS1 Medium-deep Survey: Star Formation Quenching in Group and Cluster Environments

Abstract We make use of a catalog of 1600 Pan-STARRS1 groups produced by the probability friends-of-friends algorithm to explore how the galaxy properties, i.e., the specific star formation rate (SSFR) and quiescent fraction, depend on stellar mass and group-centric radius. The work is the extension of Lin et al. In this work, powered by a stacking technique plus a background subtraction for contamination removal, a finer correction and more precise results are obtained than in our previous work. We find that while the quiescent fraction increases with decreasing group-centric radius, the median SSFRs of star-forming galaxies in groups at fixed stellar mass drop slightly from the field toward the group center. This suggests that the main quenching process in groups is likely a fast mechanism. On the other hand, a reduction in SSFRs by ∼0.2 dex is seen inside clusters as opposed to the field galaxies. If the reduction is attributed to the slow quenching effect, the slow quenching process acts dominantly in clusters. In addition, we also examine the density–color relation, where the density is defined by using a sixth-nearest-neighbor approach. Comparing the quiescent fractions contributed from the density and radial effect, we find that the density effect dominates the massive group or cluster galaxies, and the radial effect becomes more effective in less massive galaxies. The results support mergers and/or starvation as the main quenching mechanisms in the group environment, while harassment and/or starvation dominate in clusters.

The Ages of Passive Galaxies in a z = 1.62 Protocluster

Abstract We present a study of the relation between galaxy stellar age and mass for 14 members of the z = 1.62 protocluster IRC 0218, using multiband imaging and HST G102 and G141 grism spectroscopy. Using UVJ colors to separate galaxies into star-forming and quiescent populations, we find that, at stellar masses , the quiescent fraction in the protocluster is , consistent with a ∼ 2× enhancement relative to the field value, . At masses , f Q in the cluster is , consistent with the field value of . Using galaxy values derived from the G102 spectroscopy, we find no relation between galaxy stellar age and mass. These results may reflect the impact of merger-driven mass redistribution—which is plausible, as this cluster is known to host many dry mergers. Alternately, they may imply that the trend in f Q in IRC 0218 was imprinted over a short timescale in the protocluster’s assembly history. Comparing our results with those of other high-redshift studies and studies of clusters at , we determine that our observed relation between f Q and stellar mass only mildly evolves between and , and only at stellar masses . Both the and results are in agreement that the red sequence in dense environments was already populated at high redshift, , placing constraints on the mechanism(s) responsible for quenching in dense environments at .

Physical drivers of galaxies’ cold-gas content: exploring environmental and evolutionary effects with Dark Sage

We combine the latest spectrally stacked data of 21-cm emission from the ALFALFA survey with an updated version of the Dark Sage semi-analytic model to investigate the relative contributions of secular and environmental astrophysical processes on shaping the HI fractions and quiescence of galaxies in the local Universe. We calibrate the model to match the observed mean HI fraction of all galaxies as a function of stellar mass. Without consideration of stellar feedback, disc instabilities, and active galactic nuclei, we show how the slope and normalisation of this relation would change significantly. We find Dark Sage can reproduce the relative impact that halo mass is observed to have on satellites' HI fractions and quiescent fraction. However, the model satellites are systematically gas-poor. We discuss how this could be affected by satellite--central cross-contamination from the group-finding algorithm applied to the observed galaxies, but that it is not the full story. From our results, we suggest the anti-correlation between satellites' HI fractions and host halo mass, seen at fixed stellar mass and fixed specific star formation rate, can be attributed almost entirely to ram-pressure stripping of cold gas. Meanwhile, stripping of hot gas from around the satellites drives the correlation of quiescent fraction with halo mass at fixed stellar mass. Further detail in the modelling of galaxy discs' centres is required to solidify this result, however. We contextualise our results with those from other semi-analytic models and hydrodynamic simulations.

Star Formation Quenching Timescale of Central Galaxies in a Hierarchical Universe

Abstract Central galaxies make up the majority of the galaxy population, including the majority of the quiescent population at . Thus, the mechanism(s) responsible for quenching central galaxies play a crucial role in galaxy evolution as whole. We combine a high-resolution cosmological N-body simulation with observed evolutionary trends of the “star formation main sequence,” quiescent fraction, and stellar mass function at to construct a model that statistically tracks the star formation histories and quenching of central galaxies. Comparing this model to the distribution of central galaxy star formation rates in a group catalog of the SDSS Data Release 7, we constrain the timescales over which physical processes cease star formation in central galaxies. Over the stellar mass range we infer quenching e-folding times that span 1.5–0.5 Gyr with more massive central galaxies quenching faster. For , this implies a total migration time of from the star formation main sequence to quiescence. Compared to satellites, central galaxies take longer to quench their star formation, suggesting that different mechanisms are responsible for quenching centrals versus satellites. Finally, the central galaxy quenching timescale we infer provides key constraints for proposed star formation quenching mechanisms. Our timescale is generally consistent with gas depletion timescales predicted by quenching through strangulation. However, the exact physical mechanism(s) responsible for this remain unclear.