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1
Puzzarini, Cristina. "The HCS∕HSC and HCS+∕HSC+ systems: molecular properties, isomerization, and energetics." Journal of Chemical Physics 123, no. 2 (July 8, 2005): 024313. http://dx.doi.org/10.1063/1.1953367.
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Ropa, James, Scott Cooper, Lindsay Beasley, David M. Markovitz, Hal E. Broxmeyer, and Maegan L. Capitano. "Extracellular DEK Treatment Mimics Hypoxic Blockade of Extra Physiologic Oxygen Stress in Human and Mouse Hematopoietic Cells." Blood 138, Supplement 1 (November 5, 2021): 2152. http://dx.doi.org/10.1182/blood-2021-151171.
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Abstract Hematopoietic stem cell (HSC) and progenitor cell (HPC) self-renewal, proliferation, survival, and function are regulated by extracellular signals from cytokines and chemokines. DEK, a nuclear regulator of chromatin availability, was recently shown to also function as an extracellular protein that regulates HSC and HPC by inducing signaling through the CXC chemokine receptor 2 (CXCR2), enhancing pools of long-term stem cells while decreasing pools of functional HPC [Capitano et al., JCI, 2019, 129(6):2555-70]. Due to differing effects of extracellular (ec)DEK treatment on HSC growth and function compared to the effects on HPC, we hypothesized that DEK differentially regulates HSC compared to HPC. To explore this, we pulse-treated purified human cord blood (CB) CD34+ cells for 18 hours with recombinant human ecDEK protein. We then isolated by flow cytometry stringently immunophenotypically-defined human HSC, multipotent progenitors (MPP), common myeloid progenitors (CMP), and granulocyte-macrophage progenitors (GMP) and performed mRNA-sequencing to profile ecDEK-dependent transcriptomes of these separate populations of HSC/HPC. Interestingly, there are widely different ranges of effects on the transcriptomes of the different isolated HSC/HPC populations. CMP had the most differentially expressed (DE) genes induced by DEK treatment, followed by HSC and MPP, and GMP had relatively few changes, suggesting DEK treatment may primarily affect HSC and earlier HPC. HSC exhibited the strongest magnitude of changes in DE genes. Fast gene set enrichment analysis (FGSEA) revealed that GMP showed upregulation of gene programs associated with leukocyte migration and downregulation of gene programs regulated by MYC, CMP showed upregulation of gene programs associated with myeloid cell development and downregulation of lysine methyltransferase activity, and MPP and HSC both upregulated cytokine signaling responses in response to DEK treatment, though HSC also exhibited a downregulation of genes associated with cell cycle. Despite the identified unique changes upon DEK treatment in these different HSC/HPC populations, we were most interested to discover that all examined cell types demonstrated upregulation of genes associated with hypoxic responses in cells, and a concordant downregulation of genes that are frequently downregulated by hypoxic exposure following DEK treatment. This suggested to us that DEK may stimulate overlapping pathways related to hypoxia responses and/or response to exposure to extra physiologic oxygen. In fact, the effects on HSC and HPC induced by ecDEK is similar with previous work demonstrating that isolating HSC/HPC at 3% O 2 compared to ambient air conditions (~21% O 2) leads to a preserved pool of functional HSC but a lower number of functional HPC, due to a phenomenon termed extracellular physiologic shock/stress (EPHOSS) [Mantel et al., Cell, 2015, 161(7):1553-65]. To explore whether DEK regulates similar stress response pathways that are associated with EPHOSS, we assessed the effects of ecDEK treatment on HSC/HPC isolated at 3% O 2. Mice were treated with DEK in vivo, then bone marrow cells were isolated at 3% O 2 in a hypoxia chamber. Cells were kept at 3% O 2 or split to ambient air to allow equilibration to a higher oxygen tension and then assessed for HSC and functional HPC numbers. In contrast to untreated HSC/HPC isolated at 3% O 2, ecDEK treated cells isolated at 3% O 2 did not exhibit further increases in LT-HSC numbers or decreases in pools of functional HPC over ecDEK treatment alone identified by colony assays. Isolation at 3% O 2 followed by in vitro treatment with DEK showed similar results. This suggests ecDEK may neutralize the effects of EPHOSS by acting on similar gene programs that are induced by ambient air exposure. We also demonstrate that ecDEK prevents the ex vivo apoptosis of CB HSC/HPC populations, again suggesting that ecDEK is preventing the cellular stress associated with ambient air exposure and ex vivo growth, resulting in a preservation of HSC. Taken together, these data show that ecDEK acts as an extracellular signaling protein that prevents cellular stress by activating gene programs that overlap with hypoxia associated gene programs. This has important implications for the role of ecDEK in normal and diseased hematopoiesis, as well as clinical implications wherein ecDEK may be used to mimic the HSC preserving effects of isolation at 3% O 2. Disclosures Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct.
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Agúndez, M., N. Marcelino, J. Cernicharo, and M. Tafalla. "Detection of interstellar HCS and its metastable isomer HSC: new pieces in the puzzle of sulfur chemistry." Astronomy & Astrophysics 611 (March 2018): L1. http://dx.doi.org/10.1051/0004-6361/201832743.
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We present the first identification in interstellar space of the thioformyl radical (HCS) and its metastable isomer HSC. These species were detected toward the molecular cloud L483 through observations carried out with the IRAM 30 m telescope in the λ3 mm band. We derive beam-averaged column densities of 7 × 1012 cm−2 for HCS and 1.8 × 1011 cm−2 for HSC, which translate into fractional abundances relative to H2 of 2 × 10−10 and 6 × 10−12, respectively. Although the amount of sulfur locked by these radicals is low, their detection allows placing interesting constraints on the chemistry of sulfur in dark clouds. Interestingly, the H2CS/HCS abundance ratio is found to be quite low, ~1, in contrast with the oxygen analog case, in which the H2CO/HCO abundance ratio is around 10 in dark clouds. Moreover, the radical HCS is found to be more abundant than its oxygen analog, HCO. The metastable species HOC, the oxygen analog of HSC, has not yet been observed in space. These observational constraints are compared with the outcome of a recent model of the chemistry of sulfur in dark clouds. The model underestimates the fractional abundance of HCS by at least one order of magnitude, overestimates the H2CS/HCS abundance ratio, and does not provide an abundance prediction for the metastable isomer HSC. These observations should prompt a revision of the chemistry of sulfur in interstellar clouds.
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He, Fang, and Guanping Yao. "Ginsenoside Rg1 as a Potential Regulator of Hematopoietic Stem/Progenitor Cells." Stem Cells International 2021 (December 31, 2021): 1–11. http://dx.doi.org/10.1155/2021/4633270.
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Ginsenoside Rg1 (Rg1), a purified, active component of the root or stem of ginseng, exerts positive effects on mesenchymal stem cells (MSCs). Many recent studies have found that hematopoietic stem cells (HSCs), which can develop into hematopoietic progenitor cells (HPCs) and mature blood cells, are another class of heterogeneous adult stem cells that can be regulated by Rg1. Rg1 can affect HSC proliferation and migration, regulate HSC/HPC differentiation, and alleviate HSC aging, and these findings potentially provide new strategies to improve the HSC homing rate in HSC transplantation and for the treatment of graft-versus-host disease (GVHD) or other HSC/HPC dysplasia-induced diseases. In this review, we used bioinformatics methods, molecular docking verification, and a literature review to systematically explore the possible molecular pharmacological activities of Rg1 through which it regulates HSCs/HPCs.
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Chen, Xiao Bo, Jian Yin, and Wei Min Song. "Autogenous Volume Deformation and Creep Properties Analysis of C60 High Performance Concrete and C60 High Strength Concrete." Advanced Materials Research 639-640 (January 2013): 364–67. http://dx.doi.org/10.4028/www.scientific.net/amr.639-640.364.
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Based on engineering practice, autogenous volume deformation and creep properties of C60 high performance concrete(C60 HPC) and C60 high strength concrete(C60 HSC) were evaluated in the study. The results showed that the cement partly-replaced with fly ash could significantly decrease the creep deformation, creep coefficient and creep degree. In comparison with C60 HSC, the creep coefficient and creep degree of C60 HPC were decreased 17.9％and15.8％ in 28 days, 22.9% and 21.0% in 270 days. For C60 HPC and C60 HSC at the same age, autogenous volume deformation of C60 HPC is greater than that of C60 HSC, but they were both less than 80×10-6 , and the autogenous volume deformation was basically completed in 7 days.
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Mailloux, Adam, and Pearlie Epling-Burnette. "Collagen matrix deposition by hepatic stellate cells protects hepatocellular carcinoma from NK-mediated cytotoxicity (P2013)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 53.7. http://dx.doi.org/10.4049/jimmunol.190.supp.53.7.
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Abstract Liver fibrosis (LF), a leading risk factor for hepatocellular carcinoma (HCC), is caused by collagen-producing hepatic stellate cells (HSC) activated during chronic inflammation secondary to hepatitis-C infection, or alcohol liver disease (ALD). NK cells promote disease resolution via RAE1-dependant HSC clearance. A defect in NK function by an ill-defined mechanism contributes to a pro-fibrotic response. Using an engineered bioartifical collagen matrix, the expression of inhibitory leukocyte associated IgG-like receptor-1 (LAIR-1) was increased on dividing NK cells and IL-2-induced NK proliferation was inhibited. Thus, we hypothesized that the accumulation of HSC-derived collagen directly inhibits NK activity and protects both HSC and HCC from innate clearance. We found that deposition of native collagen matrix for 72h protected HSCs from NK lysis in Cr51 cytotoxicity assays. A HCC cell line (HEPG2), which produced no collagen when cultured in the absence of HSCs, was susceptible to activated NK killing and was unaffected by collagenase. To test if HSC-derived collagen protects HEPG2 cells, the populations were differentially labeled with ipophilic dyes and co-cultured for 72h for use in flow-based assays. Collagen matrix protected both targets, while collagenase pretreatment restored NK sensitivity. These results suggest that HSC-derived collagen may contribute to NK dysfunction in LF and HCC, and identifies LAIR-1 as a potential mediator of tumor-induced immune suppression.
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Das, Amitava, Uday Shergill, Lokendra Thakur, Sutapa Sinha, Raul Urrutia, Debabrata Mukhopadhyay, and Vijay H. Shah. "Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment." American Journal of Physiology-Gastrointestinal and Liver Physiology 298, no. 6 (June 2010): G908—G915. http://dx.doi.org/10.1152/ajpgi.00510.2009.
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Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 μg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase ( P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 μM), an inhibitor of MEK, which is a kinase upstream from Erk ( P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity ( P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation ( P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.
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Calvanese, Vincenzo, Sacha L. Prashad, Mattias Magnusson, and Hanna K. A. Mikkola. "Analysis of Highly Self-Renewing GPI-80+ Human Fetal Hematopoietic Stem Cells Identifies Novel Regulators of Stemness." Blood 124, no. 21 (December 6, 2014): 4314. http://dx.doi.org/10.1182/blood.v124.21.4314.4314.
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Abstract Achievements in pluripotent stem cell and reprogramming strategies provide hope for generating hematopoietic stem cells (HSC) in culture and for obtaining unlimited sources of transplantable cells. To reach this goal, deeper understanding of the regulatory mechanisms that distinguish the self-renewing HSC from non-self-renewing progenitors during human development is required. We analyzed the molecular signature of GPI-80 (VNN-2) expressing human second trimester fetal liver HSPC, the only population that harbors the truly self-renewing fetal HSC. Microarray and RNAseq analysis comparing CD34+CD38-CD90+GPI-80+ HSC to CD34+CD38-CD90+GPI80- HPC demonstrated remarkable molecular similarity of these two functionally distinct populations, including comparable expression of many key transcription factors involved in HSC development and maintenance (e.g. SCL, RUNX1, MLL1, HOXA9, BMI1, GFI1, ETV6 etc.). Nevertheless, this analysis identified a subset of transcriptional regulators uniquely up-regulated in GPI80+ HSC, such as MYCT1, HLF, MLLT3 and HIF3A. These factors were down-regulated in human fetal liver HSPC upon expansion on MSC stroma culture, during which they become compromised in in vivo engraftment ability despite maintaining HSC surface immunophenotype. multipotency and expression of most known HSC regulators. Moreover, these factors were absent or expressed at low levels in human ES cell derived HPC, which can acquire HSC surface phenotype but are unable to self-renew. The expression of MYCT1, MLLT3 and HLF was also enriched in undifferentiated HSPC subset as compared to progenitors in the adult bone marrow, while HIF3a expression was low post-natally. Altogether, these analyses implied strong correlation of the expression of these factors with HSC self-renewal properties. Strikingly, knockdown of MYCT1, HLF, MLLT3 and HIF3A in human fetal liver HSPC using lentiviral shRNA impaired maintenance of undifferentiated HSPC in MSC stroma co-culture, while their inducible overexpression augmented HSPC expansion and prevented their premature exhaustion. Uncovering how these novel HSC regulators protect the highly self-renewing fetal HSC will help define the pre-requisites for establishing and maintaining stemness in developing human HSC, and for generating HSC for therapeutic use. Disclosures No relevant conflicts of interest to declare.
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Butler, John T., Sherif Abdelhamed, Lina Gao, Jeong Lim, Terzah M. Horton, and Peter Kurre. "Leukemic Stress Targets the mTOR Pathway to Suppress Residual HSC in the BM Microenvironment." Blood 134, Supplement_1 (November 13, 2019): 3730. http://dx.doi.org/10.1182/blood-2019-125682.
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The remodeling of the bone marrow (BM) microenvironment that occurs along with the progressive spread of acute myeloid leukemia (AML) cells can be considered a constitutive aspect of leukemogenesis. To date most studies have focused on the functional and in part inflammatory adaptation of stroma, and its potential role in extrinsic chemotherapy resistance. Much less is known about the impact of leukemic stress on residual hematopoietic cells. We previously identified the trafficking of select microRNAs (miRs-) in extracellular vesicles (EVs) between AML cells and hematopoietic progenitor cells (HPC). These studies revealed the mechanism underlying the suppression of HPC function in the AML niche (Hornick, Doron et. al., Science Signaling 2016). Several groups, including ours also noted the relative resistance of residual hematopoietic stem cells (HSC) to elimination from the BM of xenografted animals. In the current study we set out to understand how leukemic stress in the AML xenograft niche shapes HSC fate and function. Using AML cell lines (Molm14, U937, HL60) we established NSG xenografts, systematically tracking peripheral blood AML chimerism to recover murine hematopoietic cells at low or negative tumor burden, and replicating key assays using purified EV for intrafemoral injections. In immunofluorescent studies we initially confirmed the uptake of GFP labeled xenograft-derived EVs across the spectrum of HPC and HSC (KSL/CD150+/CD48-), as well as the successive loss and peripheral displacement of HPCs, and gains in HSC frequency in the leukemic niche. These HSC were found to be enriched for G0 cell cycle status with an increase in phospho- p53, but showed no evidence of apoptosis or senescence. To understand the mechanism underlying their apparent quiescence, we performed in vitro proteomics studies of AML EV exposed HPSC identified downregulation of ribosomal biogenesis pathways. We then confirmed in vivo that residual HSC from AML xenografts experienced a loss of protein synthesis (OPP assay). We next reasoned that deficits in ribosome dysfunction and protein synthesis may reflect deregulation by specific miRNAs highly abundant in AML EV. Here, we had an opportunity to profile EV miRNA from the plasma of 12 unselected AML patients at diagnosis versus 12 control samples, and we confirmed a significant enrichment for specific miRNAs, including miR-1246. Raptor is a component of the mTOR pathway and an annotated target of miR-1246. We demonstrated in a series of experiments that miR-1246 translationally suppresses Raptor and downregulates protein synthesis in residual HSC from AML xenografts. The transfection of synthetic anti-miR1246 sequences on the other hand reversed the effects of AML EV in murine HSC. In aggregate we show that direct crosstalk between AML and hematopoietic cells adds to the adaptive changes that occur in the AML niche. Our experiments suggest a functional significance for EV miRNA that can be detected in AML patient plasma in the regulation of residual BM HSC. More broadly, the mechanisms by which leukemic stress alters hematopoietic function remain underexplored, but our observations suggest that leukemia derived EV contribute to changes in competitive fitness of residual HSC. Disclosures No relevant conflicts of interest to declare.
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Capitano, Maegan L., Scott Cooper, Bin Guo, Xinxin Huang, Carol Sampson, Safa Mohamad, Edward F. Srour, Christie M. Orschell, and Hal E. Broxmeyer. "Collection and Processing of Bone Marrow at 3% Oxygen Significantly Alters the Manifestation of Aged Mouse Hematopoietic Stem Cell Phenotype." Blood 134, Supplement_1 (November 13, 2019): 1202. http://dx.doi.org/10.1182/blood-2019-128642.
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Aging is an inevitable process associated with eventual deterioration of normal physiological functions. Aged hematopoiesis is associated with increased numbers of hematopoietic stem cells (HSC), but with decreased HSC functional activity (e.g. decreased engrafting capability in lethally irradiated mice and a shift in the myeloid:lymphoid bias of the engrafting HSC of the old mice, such that there are more myeloid but fewer lymphoid cells generated from HSC of the old mice). Production of HSC and progenitor (HPC) cells ex vivo is more efficient when cells are cultured in a hypoxic environment of ~ 5% oxygen tension than when cells are grown at ambient air (~21% oxygen). The bone marrow (BM) microenvironment niche that nurtures the survival and production of HSC and HPC and hematopoiesis during adult life is a hypoxic environment (~1-5% oxygen tension) compared to that of ambient air. However, almost all results of studies of young and aged mouse hematopoiesis have been based on numbers and activity of HSC and HPC that have been collected and processed in ambient air. Our recent work evaluating hematopoiesis in BM cells of young adult mice and with human cord blood cells found, through a phenomenon we designated Extra Physiological Oxygen Shock/Stress (EPHOSS), that there is a large loss of HSC with an increase of HPC within minutes of the collection of these cells in ambient air (Mantel et al., Cell, 2015). This led us to reason that perhaps what we know about aging hematopoiesis might not be entirely accurate and that a re-evaluation of aged HSC, HPC, and hematopoiesis was in order. We hypothesized that hematopoiesis in aged (~20-27 months of age) mice may not be as dysregulated as reported but that collection and processing of BM from the aged mice is more sensitive than similar cells from young (~6-16 weeks) mice to EPHOSS-induced events generated by the collection of the cells in ambient air. We evaluated BM from three different mouse strains (CB6, BALB/c, and C57Bl/6) at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or hypoxia (3% oxygen). BM from old mice collected/processed under hypoxic conditions exhibited phenotypically increased long-term HSC and common lymphoid progenitor (CLP) numbers and decreased common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) numbers when compared to old BM collected/processed under ambient air conditions. BM collected from old C57Bl/6 mice under hypoxia had increased engrafting capability more closely matching that of young BM. This was associated with a 3.14-fold increase in the number of competitive repopulating units (representative of functional HSC) in old BM collected under hypoxic conditions compared to old BM collected in ambient air as determined through limiting dilution analysis. The myeloid:lymphoid ratio of old BM collected under hypoxia matched that of young BM collected under air. This was associated with decreased cycling of CFU-GM, BFU-E, and CFU-GEMM in old BM collected/processed in hypoxia. Enhanced numbers/function of old BM HSCs collected in hypoxia is associated with changes in expression of CXCR4 (and HSC homing capability), CCR5, stress protein levels (e.g. HSP40 etc) and ROS (both total and mitochondrial). All of these noted changes demonstrated that the old BM collected/processed under hypoxic conditions more closely resemble functionally young BM. Thus, age-related differences between the HSC/HPC populations are not as drastic as previously reported and reflect the increased sensitivity of hematopoiesis from aged mice to an artificial ambient air collection procedure. Disclosures No relevant conflicts of interest to declare.
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Capitano, Maegan L., Nirit Mor-Vaknin, Maureen Legendre, Scott Cooper, David Markovitz, and Hal E. Broxmeyer. "Recombinant DEK Enhances Ex Vivo expansion of Human Cord Blood and Mouse Bone Marrow Hematopoietic Stem Cells in a CXCR2- and Hspg-Dependent Manner." Blood 126, no. 23 (December 3, 2015): 779. http://dx.doi.org/10.1182/blood.v126.23.779.779.
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Abstract DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin remodeling, and mRNA processing. Endogenous DEK regulates hematopoiesis, as BM from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Moreover, recombinant mouse (rm) DEK inhibits HPC colony formation in vitro. We now show that rmDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers (~2 fold) and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being more sensitive than control mice to this suppression. In contrast, in vivo administration of rmDEK to wild type and DEK-/- mice enhanced numbers of phenotypic LT-HSC. This suggests that DEK may enhance HSC numbers by blocking production of HPCs. We thus assessed effects of DEK on ex vivo expansion of human CD34+ cord blood (CB) and mouse Lin- BM cells stimulated with SCF, Flt3 ligand, and TPO. DEK significantly enhanced ex vivo expansion of rigorously-defined HSC by ~3 fold both on day 4 (~15 fold increase from day 0) and 7 (~29 fold increase from day 0) when compared to cells expanded without DEK. Expanding HSC with DEK also resulted in a decrease in the percentage of apoptotic HSC. Further studies were done to better define how DEK works on HSC and HPC. As extracellular DEK can bind to heparan sulfate proteoglycans (HSPG), become internalized, and then remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 673; Saha et al., 2013, PNAS, 110: 6847), we assessed effects of DEK on the heterochromatin marker H3K9He3 in the nucleus of purified mouse lineage negative, Sca-1 positive, c-Kit positive (LSK) BM cells by imaging flow cytometry. DEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK cells, indicating that rmDEK can be internalized by LSK cells and mediate heterochromatin formation. We also investigated whether inhibiting DEK's ability to bind to HSPG would block the inhibitory function of DEK in HPC. Blocking the synthesis of, the surface expression of, and the binding capability of HSPG blocked the inhibitory effect of DEK on colony formation. Blocking the ability of DEK to bind to HSPG also blocks the expansion of HSC in ex vivo expansion assays, suggesting that DEK mediates its function in both HSC and HPC by binding to HSPG but with opposing effects. To further evaluate the biological role of rmDEK, we utilized single-stranded anti-DEK aptamers that inactivate its function. These aptamers, but not their control, neutralized the inhibitory effect of rmDEK on HPC colony formation. Moreover, treating BM cells in vitro with truncated rmDEK created by incubating DEK with the enzyme DPP4 (DEK has targeted truncation sites for DPP4) eliminated the inhibitory effects of DEK, suggesting that DEK must be in its full- length form in order to perform its function. Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, and as DEK is a chemoattractant for mature white blood cells, we hypothesized that DEK may manifest at least some of its actions through CXCR2, the receptor known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case, we first confirmed expression of CXCR2 on the surface of HSC and HPC and then determined if neutralizing CXCR2 could block DEK's inhibitory function in HPC. BM treated in vitro with rmDEK, rhIL-8, or rmMIP-2 inhibited colony formation; pretreating BM with neutralizing CXCR2 antibodies blocked the inhibitory effect of these proteins. DEK inhibition of CFU-GM colony formation is dependent on Gai-protein-coupled receptor signaling as determined through the use of pertussis toxin, which is a mechanism unique to DEK, as we have previously reported that IL-8 and MIP-1a are insensitive to the inhibitory effects of pertussis toxin. Blocking the ability of DEK to bind to CXCR2 also inhibited the expansion of HSC in an ex vivo expansion assay. This suggests that DEK binds to CXCR2, HSPG or both to mediate its function on HPC and HSC, enhancing HSC but decreasing HPC numbers. Therefore, DEK may be a crucial regulatory determinant of HSC/HPC function and fate decision that is utilized to enhance ex vivo expansion of HSC. Disclosures No relevant conflicts of interest to declare.
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Hoggatt, Jonathan, Khalid S. Mohammad, Brahmananda Reddy Chitteti, Pratibha Singh, Jennifer M. Speth, Peirong Hu, Bradley Poteat, Edward Srour, Theresa Guise, and Louis M. Pelus. "Bone Marrow Niche Attenuation by Non-Steroidal Anti-Inflammatory Drugs Mobilizes Hematopoietic Stem and Progenitor Cells by Differing Mechanisms." Blood 118, no. 21 (November 18, 2011): 724. http://dx.doi.org/10.1182/blood.v118.21.724.724.
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Abstract Abstract 724 Hematopoeitic stem (HSC) and progenitor cells (HPC) are localized in niches contained within the bone marrow (BM) microenvironment. To facilitate acquisition of HSC/HPC for hematopoietic transplantation, donors can be treated with granulocyte-colony stimulating factor (G-CSF) to “mobilize” HSC/HPC from the BM niche to the peripheral system. Our laboratory has recently discovered that short term administration of non-steroidal anti-inflammatory drugs (NSAIDs) also mobilizes HSC/HPC and works in synergy with G-CSF. To understand the mechanisms underlying this novel therapeutic utility of NSAIDs we analyzed the BM niche post-NSAID and G-CSF treatment. Femurs of mice were analyzed after a 4 day regimen of the NSAID Meloxicam (3mg/kg, bid) and compared to control or G-CSF treated mice. Gross histological analysis showed a remarkable osteoblast (OB) “flattening” similar to that seen with G-CSF. Goldners trichrome staining revealed significantly reduced osteoid bone surfaces, with ∼3-fold increase in quiescent surfaces. Similarly, dynamic bone formation analysis using calcein/tetracycline labeling demonstrated reductions in mineral apposition rate and bone formation rate. There were no significant alterations in trabecular bone as determined by MicroCT. TRAP+ osteoclasts (OCs) were slightly elevated in both Meloxicam and G-CSF treated groups. To further assess the role of OCs, mice were mobilized with or without zoledronic acid (ZA) treatment, which inhibits OC activity. Similar to a recent report (Winkler, Blood, 2010), ZA resulted in an increase in HSC/HPC mobilization by both Meloxicam and G-CSF, suggesting that increased OC activity is not a mitigating mechanism for NSAID-mediated mobilization. Immunohistochemical (IHC) staining showed marked reductions in osteopontin (OPN), stromal-derived factor-1 (SDF-1) and N-cadherin expression by Meloxicam and G-CSF treated mice. In a separate set of experiments, OBs and mesenchymal stem cells (MSCs) were sorted by flow cytometry and gene expression assessed by Taq-Man assay. Similar to IHC analysis, Meloxicam treatment resulted in reduced SDF-1, OPN, Jagged-1, Runx2 and VCAM-1 gene expression. While the interaction of SDF-1 with its hematopoietically expressed receptor CXCR4 is a well known mediator of niche retention, both HSC and HPC were significantly mobilized by Meloxicam in CXCR4 knockout (KO) mice, suggesting that while reduced SDF-1 expression may concurrently play a role in NSAID mediated mobilization, it is not the definitive mechanism. In contrast, when OPN KO mice were mobilized with Meloxicam or G-CSF, Meloxicam unexpectedly increased mobilization of HPC only, and not HSC, while both HPC and HSC were mobilized by G-CSF. This surprising result indicates that NSAID-mediated OPN reduction is specifically responsible for the observed HSC mobilization, while HPC mobilization appears to be mediated by another mechanism(s). Recently, it has been reported that G-CSF reduces resident F4/80+ monocytes/macrophages (MOs), which normally support niche OBs and MSCs, and this reduction is at least partially responsible for niche attenuation and hematopoietic mobilization. However, in contrast to G-CSF, IHC analysis showed no reduction in F4/80+ cells after Meloxicam treatment, nor was there a reduction in CD169+ BM macrophages as assessed by flow cytometry. Therefore, while NSAIDs and G-CSF attenuate the niche microenvironment similarly, these agents function through independent mechanisms, perhaps explaining the synergistic mobilization seen by the two. In conclusion, NSAID treatment results in significant attenuation of the BM niche, including the niche components SDF-1, OPN, and N-cadherin. NSAID-mediated mobilization is independent of reduced SDF-1/CXCR4 signaling, as CXCR4 KO mice can be mobilized by NSAID. OPN KO specifically blocks the NSAID mobilization of HSC, but not HPC, suggesting differing mechanisms, or possibly multiple niche locations for these two populations; an intriguing finding suggesting that other mobilization strategies may be able to specifically target HSC or HPC. While G-CSF treatment results in reduced MOs, NSAIDs do not alter BM levels of these supportive cells. These results define not only a novel strategy for mobilization of HSC/HPC, but also suggest a possible strategy for targeted niche attenuation for other therapeutic applications, such as reduced conditioning regimens. Disclosures: Hoggatt: Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.
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Harun, Nadia, Marilyn Thien, Julius G. Juarez, Kenneth Francis Bradstock, and Linda J. Bendall. "S1P1 Agonists for Use as Adjunct Mobilizing Agents." Blood 116, no. 21 (November 19, 2010): 826. http://dx.doi.org/10.1182/blood.v116.21.826.826.
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Abstract Abstract 826 Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P1 receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB. Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P1 expression and an S1P1 conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models. Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P1 conditional knock-out mouse. When mobilised with AMD3100, these S1P1 knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P1 agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF. S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P1 conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes. Disclosures: No relevant conflicts of interest to declare.
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Huang, Xinxin, Scott Cooper, and Hal E. Broxmeyer. "Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression." Blood 124, no. 21 (December 6, 2014): 4332. http://dx.doi.org/10.1182/blood.v124.21.4332.4332.
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Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.
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Christopherson, Kent W., Nehal K. Porecha, Giao Hangoc, and Hal E. Broxmeyer. "Enhanced Functional Response to CXCL12/SDF-1 through Retroviral Overexpression of CXCR4: Implications for Hematopoietic Stem & Progenitor Cell Homing and Engraftment." Blood 104, no. 11 (November 16, 2004): 1198. http://dx.doi.org/10.1182/blood.v104.11.1198.1198.
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Abstract The chemokine CXCL12 (stromal cell-derived factor 1/SDF-1) induces the migration of hematopoietic stem and progenitor cells (HSC/HPC) through the corresponding chemokine receptor, CXCR4. CXCL12 is thought to be important for proper homing and engraftment of HSC/HPC to the bone marrow (BM) and mobilization of HSC/HPC out of the BM. Previous studies have suggested that breaking the CXCL12-CXCR4 interaction mobilizes both human and mouse HPC (Liles WC, et al. Blood. 2003 102(8): 2728-30.) (Broxmeyer HE, et al. ASH 44th Annual Meeting. 2002 #2397). Other studies suggest that blocking CXCR4 inhibits homing (Peled A, et al. Science. 1999 283(5403): 845-8) and CXCR4 overexpression by lentiviral vector increases repopulation (Kahn J, et al. Blood. 2004 103(8): 2942-9) of CD34+ cells to the BM of NOD/SCID recipient mice. Expression of CXCR4 on the surface of HSC/HPC appears to be variable, depending highly on the cytokine and growth factor composition of the environment. This may partially explain the reduction in homing efficiency observed during the expansion of cord blood (CB). The efficiency of engraftment therefore appears to be dependent on the response of HSC/HPC to CXCL12 which is in turn dependent upon levels of CXCR4 expressed on HSC/HPC. In order to study the functional cellular response of HSC/HPC independent of variable levels of CXCR4 expression on the surface of cells we utilized the MSCV-based bicistronic (EGFP) retroviral vector, MIEG3, to overexpress human CXCR4 in M07e cells. The human megakaryocytic leukemia cell line, M07e, (a CD34+, c-Kit+, growth-factor-dependent human cell line) has been established as a model for human HPC. Cells infected with the MIEG3 empty vector or the MIEG3-CXCR4 construct were sorted based on their expression of GFP and CXCR4 expression was measured by flow cytometric analysis. MIEG3-CXCR4 M07e cells (GFP+) and MIEG3-CXCR4 bright M07e cells (GFP+++) express significantly higher levels of CXCR4 than M07e cells, MIEG3 M07e cells (GFP+), or MIEG3 bright M07e cells (GFP+++). Migratory response of cells to CXCL12 was assessed by chemotaxis assay. Increased CXCL12 induced chemotaxis was observed in MIEG3-CXCR4 M07e cells (p<0.05) and MIEG3-CXCR4 bright M07e cells (p<0.01). Overexpression of CXCR4 also resulted in a significant increase in CXCL12 induced cell survival during growth factor withdrawal, most markedly at low doses of CXCL12 (10 and 1.0ng/ml) (p<0.05). Thus, retroviral overexpression of CXCR4 enhances both the migratory response and survival of the human HPC line, M07e, to CXCL12. Most importantly cells respond to levels of CXCL12 that are normally too low. This data validates the ability of the MIEG3-CXCR4 construct to efficiently overexpress CXCR4 and validates the future use of MIEG3-CXCR4 M07e cells for further study of CXCR4 in HPC. This is important given the previously established ability of MIEG3 to infect CD34+ CB cells (Tao W et al. Gene Ther. 2004 11(1): 61-9). This information also suggests that previously reported increases in engraftment resulting from CXCR4 overexpression is a function of both increased HSC/HPC settlement and increased HSC/HPC survival in the transplant recipient’s BM. This information may have potential therapeutic application for improvements in overall transplant efficiency.
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Wang, Pei-Wen, Tung-Yi Lin, Pei-Ming Yang, Chau-Ting Yeh, and Tai-Long Pan. "Hepatic Stellate Cell Modulates the Immune Microenvironment in the Progression of Hepatocellular Carcinoma." International Journal of Molecular Sciences 23, no. 18 (September 15, 2022): 10777. http://dx.doi.org/10.3390/ijms231810777.
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Hepatocellular carcinoma (HCC) is a major cause of increases in the mortality rate due to cancer that usually develops in patients with liver fibrosis and impaired hepatic immunity. Hepatic stellate cells (HSCs) may directly or indirectly crosstalk with various hepatic cells and subsequently modulate extracellular remodeling, cell invasion, macrophage conversion, and cancer deterioration. In this regard, the tumor microenvironment created by activated HSC plays a critical role in mediating pathogenesis and immune escape during HCC progression. Herein, intermediately differentiated human liver cancer cell line (J5) cells were co-cultured with HSC-conditioned medium (HSC-CM); changes in cell phenotype and cytokine profiles were analyzed to assess the impact of HSCs on the development of hepatoma. The stage of liver fibrosis correlated significantly with tumor grade, and the administration of conditioned medium secreted by activated HSC (aHSC-CM) could induce the expression of N-cadherin, cell migration, and invasive potential, as well as the activity of matrix metalloproteinases in J5 cells, implying that aHSC-CM could trigger the epithelial-mesenchymal transition (EMT). Next, the HSC-CM was further investigated and network analysis indicated that specific cytokines and soluble proteins, such as activin A, released from activated HSCs could remarkably affect the tumor-associated immune microenvironment involved in macrophage polarization, which would, in turn, diminish a host’s immune surveillance and drive hepatoma cells into a more malignant phenotype. Together, our findings provide a novel insight into the integral roles of HSCs to enhance hepatocarcinogenesis through their immune-modulatory properties and suggest that HSC may serve as a potent target for the treatment of advanced HCC.
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Suh, Hyung Chan, Ming Ji, John Gooya, Michael Lee, Kimberly Klarmann, and Jonathan R. Keller. "Id1 Provides a Proper Hematopoietic Progenitor Niche Function." Blood 112, no. 11 (November 16, 2008): 2427. http://dx.doi.org/10.1182/blood.v112.11.2427.2427.
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Abstract Development of hematopoietic stem cells (HSC) and their progeny is maintained by the interaction with cells in the microenvironment. In addition to hematopoietic cells, Id1 is expressed in stromal cells known to support hematopoiesis, and is involved in cell proliferation, differentiation and senescence. Therefore, to investigate the role of Id1 in hematopoiesis, we examined hematologic phenotypes of Id1−/− mice. In this study, we found increased neutrophils and macrophages, and decreased B cells and platelets in peripheral blood, and decreased BM cellularity. While the percentages of hematopoietic stem cells (HSC) in Id1−/− mice were increased relative to the Id1+/+ mice, their total numbers and function appeared normal. For example, Id1 was not required for self-renewal or repopulation of HSC. In contrast, we found that there were increased numbers of hematopoietic progenitor cells (HPC) in S phase of cell cycle in Id1−/− mice BM, suggesting that the loss of Id1 within HPC promotes proliferation. However, purified Id1−/− HPC had the same proliferation potential as Id1+/+ HPC when cultured in vitro. In transplantation experiments, we proved that BM microenvironment in Id1−/− mice is defective by showing that the Id1+/+ HSC showed impaired hematopoietic development in Id1−/− mice, while the Id1−/− HSC had normal repopulation potential in an Id1+/+ microenvironment. In agreement with these findings, Id1−/− BM stromal cell cultures supported enhanced proliferation of hematopoietic progenitors. Furthermore, quantitative PCR showed that SCF, M-CSF, OPN, SDF-1 and TGF-α mRNA expression was decreased in Id1−/− stromal cells relative to Id1+/+ stromal cells, while G-CSF, GM-CSF, and VEGF mRNA expression was significantly increased. Id1−/− BM showed decreased number of mesenchymal stem/progenitor cells. Thus, Id1 does not play a role in maintaining HSC, but is involved in regulating hematopoietic progenitor niche. Funded by NCI contract No. N01-CO-12400.
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Kumar, Sachin, and Hartmut Geiger. "HSC Niche Biology and HSC Expansion Ex Vivo." Trends in Molecular Medicine 23, no. 9 (September 2017): 799–819. http://dx.doi.org/10.1016/j.molmed.2017.07.003.
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Sugiyama, Daisuke, Tomoko Inoue-Yokoo, Stuart T. Fraser, Kasem Kulkeaw, Chiyo Mizuochi, and Yuka Horio. "Embryonic Regulation of the Mouse Hematopoietic Niche." Scientific World JOURNAL 11 (2011): 1770–80. http://dx.doi.org/10.1100/2011/598097.
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Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called “niche cells” surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells.
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Santamato, Angela, Emilia Fransvea, Francesco Dituri, Alessandra Caligiuri, Michele Quaranta, Tomoaki Niimi, Massimo Pinzani, Salvatore Antonaci, and Gianluigi Giannelli. "Hepatic stellate cells stimulate HCC cell migration via laminin-5 production." Clinical Science 121, no. 4 (April 28, 2011): 159–68. http://dx.doi.org/10.1042/cs20110002.
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Activated HSCs (hepatic stellate cells) are the main source of extracellular matrix proteins present in cirrhotic liver on which HCC (hepatocellular carcinoma) commonly develops. HCC cells behave differently according to differences in the surrounding microenvironment. In the present study, we have investigated a mechanism whereby HSCs modulate the migratory activity of HCC cells. We used primary cultures of human HSCs to investigate their effect on Hep3B, Alexander, HLE and HLF HCC cells. The expression of Ln-5 (laminin-5) was documented at transcript and protein levels both in vitro and in vivo. HCC cells strongly adhere, migrate and spread in the presence of HSC-conditioned medium and of co-culture. HSCs produce and secrete Ln-5 in the CM (conditioned medium). The electrophoretic pattern of secreted Ln-5 is consistent with that of a migratory substrate, showing the presence of the γ2x fragment. Blocking antibodies against Ln-5 inhibit HCC migration in the presence of HSC-CM. HCC cells migrate very poorly in the presence of Ln-5 immunodepleted HSC-CM. HCC migration in the presence of HSCs is dependent on the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway, but not the PI3K (phosphoinositide 3-kinase)/Akt pathway. HSC-CM, as well as Ln-5, activates the MEK/ERK but not the PI3K/Akt pathway. In human HCC tissues, Ln-5 is mainly distributed along α-SMA (smooth muscle actin)-positive cells, whereas in peritumoural tissues, Ln-5 is absent. HSCs stimulate HCC migration via the production and secretion of Ln-5.
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Winkler, Ingrid G., Bianca Nowlan, Valerie Barbier, and Jean-Pierre Levesque. "Absence of E-Selectin at the Vascular Niche Delays Hematopoietic Stem Cell Turn-Over." Blood 110, no. 11 (November 16, 2007): 609. http://dx.doi.org/10.1182/blood.v110.11.609.609.
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Abstract Hematopoietic stem cells (HSC) reside in specialized niches in the bone marrow (BM), that regulate their survival, proliferation and differentiation. Two types of HSC niches have been reported: endosteal niches in close contact with osteoblasts, and endothelial niches near vascular sinuses. Whether these niches have distinct functions in controlling HSC fate remains unknown. One difference between these two niches is the constitutive expression of E-selectin and P-selectin by BM endothelial cells. E- and P-selectin are two cell adhesion molecules that modulate hematopoietic progenitor cell (HPC) survival, proliferation and differentiation in vitro. We now show that deletion of E-selectin, but not P-selectin, delays HSC turn-over in the BM in vivo. Mice lacking either E-selectin (E−/ −), P-selectin (P−/ −) or both (PE−/ −) were given bromo-deoxyuridine (BrdU) in their drinking water for up to 14 days. Lineage-negative c-KIT+ Sca-1+ CD34− (LKS34) cells were sorted from the BM and stained for BrdU incorporation into genomic DNA. Although it took only 3.6 days for 50% of LKS34 cells from wild-type (WT) and P−/ − mice to incorporate BrdU, 9 days were required for 50% BrdU incorporation in LKS34 cells from E−/ − and PE−/ − double KO mice. Thus, HSC cycling time is 2.5 times slower in the absence of E-selectin. To confirm these findings, LKS cells were stained with rhodamine123, a vital dye that is retained by metabolically active cells but effluxed from quiescent HSC. A higher proportion of LKS cells from E−/ − mice were rhodamine dull (34±2%) than WT LKS (23±1%; p=0.037) confirming that a greater proportion of HSC from E−/ − mice are quiescent. To further support these findings, we determined the effect of E-selectin deletion on HSC recovery following cytotoxic stress with a single dose of 5-fluorouracil (5FU 150mg/kg). As KIT is strongly down-regulated in the BM of 5FU-treated mice, we examined frequency and BrdU incorporation in Lin− Sca1+ CD41− CD48− CD150+ long-term reconstituting HSC. We found HSC recovery to be enhanced in E−/ − mice with a 5-fold increase in HSC numbers per femur compared to WT mice at day 7 post-5FU. Despite the more rapid recovery of E−/ − HSC, BrdU incorporation remained significantly lower in E−/ − HSC on days 3 and 7 post-5FU suggesting the decreased HSC turn-over in the absence of E-selectin protects them from the cytotoxic effect of 5FU. To determine whether this effect was mediated by the two described E-selectin receptors PSGL-1 and/or CD44, BrdU incorporation experiments were repeated with mice lacking both the PSGL-1 and CD44 genes. LKS cell turnover in these mice was identical to that of WT suggesting that the effect is mediated by a distinct unknown receptor(s) on HSC. The fact that a novel E-selectin receptor on HSC/HPC is involved was confirmed both using flow cytometry with selectin-IgM chimeras as well as cell adhesion assays using plastic-adsorbed selectin-IgG chimeras. In both assays, 90-95% of LKS cells from CD44−/ − PSGL-1−/ − double KO mice bound E-selectin whereas adhesion to P-selectin was completely lost. Taken together our findings suggest that E-selectin, whose constitutive expression is restricted to BM endothelial cells, plays an important role in the regulation HSC turnover in vivo, endothelial niches, where E-selectin is expressed, support more rapid HSC turn-over within the BM, and this effect is mediated by unknown E-selectin receptors distinct from PSGL-1 or CD44.
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Welner, Robert, Giovanni Amabile, Deepak Bararia, Akos Czibere, Henry Yang, and Daniel Tenen. "Normal stem and progenitor cell sociology within the leukemic microenvironment (HEM3P.300)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 51.21. http://dx.doi.org/10.4049/jimmunol.192.supp.51.21.
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Abstract Specialized bone marrow (BM) microenvironment niches are crucial for hematopoietic stem and progenitor cell (HSC/HPC) maintenance. We are just starting to learn how the integrity of the niche changes with leukemia, however, the impact on normal HSC/HPC behavior and functionality is yet to be addressed. Therefore, we started by studying the kinetics and differentiation of normal HSC/HPC in mice with Chronic Myeloid Leukemia (CML). Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic environment imposed distinct effects on HPC predisposing them toward the myeloid lineage, similar to that of the leukemic population. Meanwhile, the leukemic-exposed normal HSC remained functional on transplantation. Analysis of the microenvironment identified several cytokines were dysregulated in the leukemia, and we found IL-6 to be responsible for many of these bystander responses. These results were similarly validated using BM obtained from CML patient samples. Co-culture of CML BM and human CD34+ HPC resulted in selective proliferation and altered differentiation of the normal primitive progenitors compared to mixed cultures using normal BM. Therefore, our results show how hematologic malignancy can influence the bone marrow, in turn causing disturbances in normal blood cell progenitors. Knowledge of such bystander effects could suggest new therapeutic interventions for cancer prevention and novel therapeutic approach for leukemia patients.
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Vávrová, Jiřina, and Martina Řezáčová. "Apoptosis and Senescence – Main Mechanisms of Accelerated Aging of Haematopoietic Cells after Irradiation." Acta Veterinaria Brno 78, no. 2 (2009): 205–17. http://dx.doi.org/10.2754/avb200978020205.
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Haematopoietic stem cell (HSC) is one of the most radiosensitive cells in organism. In mice it is characterized as lin-Sca-1+CD117+ cell. This review discusses the role of HSC subpopulations in recovery of haematopoiesis after irradiation, and molecular mechanisms of reaction of HSC to damage induced by genotoxic stress, mainly to double strand breaks (DSB) of DNA. Various proteins are accumulated on the site of break, e.g. 53BP1 and γH2AX. Repair of the damage and related signalling is executed by many proteins, such as ATM, ATR, and DNA-PK kinases, MRN complex and proteins of homologous recombination and non-homologous end joining. Repetitive irradiation by low doses of ionizing radiation causes in HSC accumulation of proteins into so-called “ionizing radiation inducing foci” (detectable by γH2AX) and decreases repair capacity of HCS. Furthermore, two possible molecular mechanisms of HSC reaction to radiationinduced DNA damage are discussed – apoptosis and senescence. While majority of differentiated haematopoietic cells, leukaemic cells, and haematopoietic progenitors die after irradiation by apoptosis, in HSC also senescence was detected. It also seems that decrease in proliferative capacity of HSC related to old age is caused by accumulation of DNA damage induced by oxygen radicals in the pool of quiescent stem cells.
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Hadland, Brandon, Barbara Varnum-Finney, Stacey Dozono, Tessa Dignum, Cynthia Nourigat-Mckay, Dana Jackson, Shahin Rafii, Cole Trapnell, and Irwin D. Bernstein. "Integrated Single Cell Transcriptomics Defines an Engineered Niche Supporting Hematopoietic Stem Cell Development Ex Vivo." Blood 134, Supplement_1 (November 13, 2019): 3699. http://dx.doi.org/10.1182/blood-2019-126109.
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During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.
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Tjwa, Marc, Nicolai Sidenius, Koen Theunissen, Rute Moura, Lieve Moons, Stefan Janssens, Francesco Blasi, Desire Collen, and Peter Carmeliet. "Novel Role of Plasmin in Mobilization and of uPAR in Retention of Hematopoietic Stem/Progenitor Cells in the Bone Marrow Niche: Therapeutic Implications." Blood 104, no. 11 (November 16, 2004): 117. http://dx.doi.org/10.1182/blood.v104.11.117.117.
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Abstract Hematopoietic stem/progenitor cells (HSC/HPCs) are retained in the bone marrow (BM) niche via receptor-ligand interactions and mobilized from the BM after proteolytic degradation of these retention complexes. Yet, the proteinases and retention signals involved remain incompletely identified. Here, we studied the role of the plasminogen proteinase system with its plasminogen activators PA) tPA and uPA, and active plasmin (Pli) in chemo- and G-CSF-induced mobilization of HPCs and HSCs. Therefore, 5-fluorouracil (5-FU) or G-CSF were administered to mice lacking plasminogen (Plg−/−), tPA (tPA−/−), uPA (uPA−/−), both activators (tPA−/−uPA−/−), uPAR (uPAR−/−), PAI-1 (PAI-1−/−), or α2-antiplasmin (α2-AP−/−). 5-FU treatment in WT mice elevated Pli activity in BM plasma 5-fold, killed 10% of WT mice, and increased number/proliferation of HSC/HPCs in the surviving mice, with full hematopoietic recovery after 3 weeks. In contrast, up to 75% of 5-FU-treated Plg−/−and tPA−/−uPA−/− demised in the early phase of recovery, with reduced number/proliferation of HSC/HPCs and delayed hematopoietic recovery. Following G-CSF, deficiency of Plg or inhibition of Pli by tranexamic acid reduced HPC expansion and HSC translocation in the BM, resulting in impaired HPC/HSC mobilization, by up to 55% and 75%, respectively. Further analysis using uPA−/− and tPA−/− mice revealed that uPA was critical for 5-FU-induced mobilization, whereas tPA was crucial for G-CSF-induced mobilization. In addition, analysis of mice lacking MMP-2, -3, -9, and -12 revealed that 5-FU- and G-CSF-induced mobilization required predominantly MMP-9 and -3, respectively. MMP-3 and MMP-9 activities upon mobilization were reduced in Plg−/− mice, suggesting that Pli activates these MMPs. In the absence of Plg, degradation of fibronectin in the BM and production of soluble Kit ligand (but not SDF1 α) were impaired, indicating additional downstream targets of Pli. uPAR is a membrane-anchored receptor for uPA, which is cleaved into a soluble form (suPAR) by Pli and other proteinases. Interestingly, uPAR was expressed on BM-derived HPC/HSCs, and uPAR deficiency reduced their retention within the BM niche in vitro and in vivo. Furthermore, uPAR−/− mice showed poor HSC/HPC mobilization in response to 5-FU and G-CSF, while suPAR administration in WT mice amplified G-CSF-induced mobilization. Moreover, suPAR levels in the BM were increased in WT but not in Plg−/− mice during mobilization, indicating, all together, that uPAR might be a novel retention signal for HSC/HPCs in the BM niche. Finally, increased Pli activity in PAI-1−/− and α2-AP−/− mice, and WT mice treated with tenecteplase (i.e. recombinant tPA variant used for clinical thrombolysis) enhanced G-CSF-induced mobilization. Importantly, initial results suggest that thrombolytic treatment of individuals after acute myocardial infarction also seemed to stimulate HPC mobilization, extrapolating our findings to man. In conclusion, these genetic and pharmacological data reveal, for the first time, a novel role for uPAR as a retention signal for HSC/HPCs in the BM and suggest that strategies to increase PA or Pli activity might offer novel therapeutic opportunities for HSC/HPC mobilization.
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Chua, Hui Lin, Vikram Anand, Carol Sampson, Ngoc-Thinh Nguyen, Artur Plett, Evan West, and Christie M. Orschell. "Kinetics of Self-Renewal and Differentiation Divisions of Transplanted Hematopoietic Stem and Progenitor Cells Early After Transplantation Into Lethally-Irradiated Recipients." Blood 114, no. 22 (November 20, 2009): 2443. http://dx.doi.org/10.1182/blood.v114.22.2443.2443.
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Abstract Abstract 2443 Poster Board II-420 Transplanted hematopoietic progenitor cells (HPC) and stem cells (HSC) provide short- and long-term hematopoietic support, respectively, in myeloablated recipients after transplantation. Despite the reliance on these cells for successful clinical engraftment and reconstitution of transplant recipients, little is known regarding their proliferation kinetics in vivo during the period of engraftment, or how this relates to the vast literature describing steady state hematopoiesis. We have previously established methodology that can track donor HSC and HPC in mice after transplantation using retention and loss of CFSE fluorescence to identify CFSEbright and CFSEdim cells, respectively. Cells identified as CFSEbright on d5 post-transplantation were shown to be exclusively enriched for donor long-term repopulating potential, thus comprising all the HSC within the donor cell population. In the current study, we used this methodology to examine the long-term repopulating potential and progenitor activity of CFSEbright, CFSEmid, and CFSEdim cells isolated from primary recipients on days 3, 5, 7, and 10 after transplantation of low density bone marrow (LDBM) cells. We aimed to determine when HSC activity moved from CFSEbright cells into the CFSEmid, as a means of estimating the time point at which donor HSC undergo self-renewal divisions in recipient BM. Likewise, using clonogenic assays, HPC content of the three CFSE fractions was followed to determine the kinetics and nature of proliferation of donor progenitor cells. As expected, the percentage and absolute number of CFSEbright and CFSEmid cells decreased by day 10 to approximately 1-10% of day 3 values, while CFSEdim cells increased ∼200-fold to comprise >95% of donor cells by day 10 (n=14-16 mice/time point). Interestingly, when the HPC content of the various CFSE populations was examined, all HPC activity at day 3 post-transplant resided in the CFSEmid cells, suggesting that HPC divide rapidly upon transplantation and leave the CFSEbright pool within 1-2 days. Progenitor activity began to appear in the CFSEdim population by d5 post-transplant, increasing 5- to 20-fold in absolute number by day 10, roughly paralleling the increase in absolute number of CFSEdim cells during this same time frame. While the frequency of total HPC in CFSEmid and CFSEdim populations was similar to that of steady state LDBM, 5- to 15-fold more of these progenitors were identified as CFU-GEMM and HPP-CFC compared to steady state BM, suggesting that engrafting cells expand their primitive HPC content at a faster rate than steady state BM. In contrast to the rapid proliferation kinetics of engrafting HPC, results of competitive transplantation studies of the various CFSE fractions suggest that long-term multi-lineage engraftment potential moves from the CFSE-bright to the CFSE-mid population around day 7-8 post-transplantation. Using the number of transplanted CFSE graft cells and their 6mo chimerism values to determine an enrichment factor for HSC potential, we estimate that d5 CFSEbright cells are 28-fold more enriched for HSC activity than steady state LDBM, while d7 CFSEmid cells are 8-fold more enriched. These data suggest that within the first 7 days post-transplant, 1 in 3.5 cell divisions of CFSEbright cells are self-renewal in nature. In contrast, using the same formula, CFSEdim cells were found to possess ∼1% of the HSC activity of steady state LDBM when analyzed up to 1 month post-transplantation, suggesting that CFSEdim cells are functionally weakened at these early time points post-transplant, and thus unable to provide significant chimerism in secondary recipients. Taken together, these data suggest that donor HSC undergo self-renewal divisions at approximately 1 week post-transplant and at a much higher rate than during steady state hematopoiesis. In addition, transplanted HPC were found to proliferate between 1-2 days post-transplant, and appear to give rise to a pool of progenitors 5 to 15-fold more enriched for primitive HPC than that present in steady state LDBM. These results add to our understanding of HSC/HPC engraftment and the kinetics of self-renewal and differentiation divisions in vivo, and may have clinical implications in designing methodologies to optimize hematopoietic engraftment and reconstitution. Disclosures: No relevant conflicts of interest to declare.
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Shank, Kaitlyn, Yusup Shin, Carson Wills, Nicole Cunningham, Alevtina Domashenko, Russell Garrett, Jenni A. Punt, and Stephen G. Emerson. "IFNs Upregulate Sca-1 and Block Proliferation in Murine Hematopoietic Stem and Progenitor Cells,." Blood 118, no. 21 (November 18, 2011): 3394. http://dx.doi.org/10.1182/blood.v118.21.3394.3394.
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Abstract Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.
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Fang, Jing, Lyndsey Bolanos, Juana Serrano-Lopez, Susanne Christie, Jose A. Cancelas, and Daniel T. Starczynowski. "TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis." Blood 128, no. 22 (December 2, 2016): 568. http://dx.doi.org/10.1182/blood.v128.22.568.568.
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Abstract Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function. To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis. To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells). Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. Disclosures No relevant conflicts of interest to declare.
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Felker, Sydney, Archana Shrestha, and Punam Malik. "Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells." Blood 136, Supplement 1 (November 5, 2020): 22–23. http://dx.doi.org/10.1182/blood-2020-138529.
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Gene therapy/editing of CD34+ HSPC ex vivo, followed by their transplantation, can cure a variety of hematologic diseases. However, a substantial loss of HSPC occurs from collection to transplant. Losses occur during processing for HSPC enrichment, ex vivo genetic manipulation and culture, formulation, and testing prior to transplant. Further, HSPC are lost to peripheral organs during homing when delivered intravenously (IV), reducing the effective gm HSPC dose; a loss compounded by the lack of helper cells that aid in the homing and engraftment process which are removed during enrichment. Direct BM delivery of gm HSPC can overcome some of these limitations. This has been tried previously, with non-enriched whole cord blood (CB) and non-gm HSPC, with conflicting results. We hypothesized that BM delivery of a limited dose of gm adult HSPC would improve long-term repopulation over that of IV delivery by bypassing HSPC loss during homing. Using bioluminescent imaging, we determined that CB HSPC transduced with a luciferase lentiviral vector (LV) delivered by intra-femoral (IF) injection localized to the injected femur, validating our injection method. Next, we delivered mobilized peripheral blood (MPB) HSPC transduced with a GFP LV into irradiated NOD.LtSz-scid IL2rg -/- (NSG) mice via IV or IF injection in limiting dilution. Total human engraftment (hCD45+ cells), transduced human engraftment (hCD45+GFP+ cells), and multi-lineage engraftment were measured in the BM at 3- and 6-months post-transplant. HSPC gave rise to a bi-lineage (B-myeloid) graft at 3 months, suggesting hematopoietic progenitor cell (HPC) engraftment, and a multi-lineage graft (hCD33+, hCD19+, hCD3+, and hCD34+ cells) at 6 months, suggesting engraftment from a long-term repopulating cell or hematopoietic stem cell (HSC). At 3 months, IF delivery of HSPC resulted in significantly higher total and transduced human cell engraftment, measured in the non-injected femur (Table 1). The engraftment was bi-lineage. At 6 months, IF delivery of HSPC no longer significantly increased engraftment over IV delivery (Table 1). However, a multi-lineage graft was present, indicating full hematopoietic repopulation. There was no significant difference in the lineage output between either delivery method at 3 or 6 months. These data suggest that HPC homed and engrafted more efficiently than HSC, when delivered IF. Alternatively, IF delivery altered the BM microenvironment, allowing preferential homing of HPC. However, CD34- cells injected IF, to simulate pressure and passage of cells through the BM with IF delivery, followed by IV delivery of CD34+ cells (sham IF with IV HSPC delivery) resulted in similar homing patterns to CD34+ cells delivered IV (p=0.1, Figure 1A), suggesting that differences between IV and IF delivery were likely due to cell-intrinsic rather than cell-extrinsic differences between HPC and HSC. To study the mechanism of preferential engraftment of HPC over HSC with IF delivery, we analyzed expression of the major homing receptors CXCR4 and VLA-4 on HPC and HSC. CXCR4 (Figure 1B) and VLA-4 were both expressed at significantly higher levels on HPC than on HSC (CXCR4 p<0.01; VLA-4 p<0.05) and their expression increased with increasing culture time and with HSPC cycling. However, VLA-4 expression was significantly increased in GFP+ (MFI 65313 ± 4750) compared to GFP- (MFI 48969 ± 2099; p<0.01) HSPC. CXCR4 expression was similar in both GFP+ (MFI 4261 ± 189) and GFP- (MFI 5245 ± 1186) HSPC, mimicking the in vivo engraftment pattern of GFP+ and GFP- cells, suggesting that CXCR4 may be the molecule responsible for enhancing HPC homing and engraftment with BM delivery. An initial experiment shows that when we remove the high CXCR4 expressing CD34+38+ HPC and deliver HSC-enriched CD34+38- cells IV or IF, IF delivery results in higher long-term engraftment (additional experiments ongoing, Figure 1C, D). These data support the hypothesis that cell-intrinsic differences in the homing behavior of HSC and HPC is likely due to their differential expression of CXCR4. Studies underway on blockade of CXCR4 or VLA-4 on gm HPC and/or gm HSC followed by their IF or IV delivery will be presented. Overall, we show IV delivery of gm HSPC is comparable to BM delivery. However, as HSC-enriched cells become clinically available for genetic therapies, BM delivery of enriched gm HSC may result in superior engraftment. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.
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Scheller, Marina, Frank Schwoebel, Doerte Vossmeyer, and Achim Leutz. "Rapid and Efficient Mobilization of Murine Hematopoietic Stem and Progenitor Cells with Nox-A12, a New Spiegelmers®-Based CXCR4/SDF-1(CXCL12) Antagonist." Blood 118, no. 21 (November 18, 2011): 2995. http://dx.doi.org/10.1182/blood.v118.21.2995.2995.
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Abstract Abstract 2995 Mobilization of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) is important in many hematological therapies. However, up to 30% of the patients respond poorly to standard granulocyte colony-stimulating factor (G-CSF) treatment, highlighting the need for more effective mobilizing strategies. The CXCR4/stromalcell-derived factor 1 (SDF-1) axis plays a crucial role in the interaction between HSCs and the marrow niche and is involved in HSC mobilization. NOX-A12 is a structured mirror-image RNA oligonucleotide, a so-called Spiegelmer®, that was identified to bind SDF-1 thereby inhibiting its activity with subnanomolar IC50. HSC/HPC mobilization by NOX-A12 was examined in the mouse. Single NOX-A12 administration induced reversible mobilization of HSC/HPC populations within a few hours. NOX-A12 synergized with G-CSF to strongly enhance HSC/HPC mobilization. In particular, the progenitor compartment mobilized by single NOX-A12 administration contained more differentiated short-term HSCs (ST-HSCs), and combined administration of NOX-A12 and G-CSF mobilized a significantly higher proportion of primitive and more potent murine long-term repopulating cells that successfully engrafted primary and secondary lethally-irradiated recipients. These results characterize NOX-A12 as a potent HSCs/HPCs mobilizing therapeutic in mammals and suggest its clinical potential. Disclosures: No relevant conflicts of interest to declare.
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Ang, Siok Hoon, Benjamin Haaland, Minn Minn Myint Thu, Sai Sakktee Krishna, Wallace Chen, Siok Gek Jacqueline Hwang, Puay Hoon Tan, et al. "P16 and cyclin D1 (CYD1) as prognostic markers in hypopharyngeal (HSC) and oropharyngeal squamous cell carcinoma (OSC)." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 5580. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.5580.
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5580 Background: In head and neck cancer (HNC), dysregulation of cell cycle proteins p16 and CYD1 are common. Variable associations of p16 over- and under- expression and CYD1 overexpression with overall survival (OS) have been described in different HNC sites. We evaluated the relationship of p16 and CYD1 expression with clinical characteristics and OS in OSC and HSC. Methods: p16 and CYD1 expression was evaluated by immunohistochemistry in 77 HSC and 103 OSC patients (pts) and recorded as p16N (0% tumor cells stained), p16L (5-69%) and p16H (≥70%), CYD1-(<10%) and CYD1+ (≥10%). OS between groups was evaluated by Kaplan-Meier method and compared by log rank test. Hazard ratio (HR) for death was estimated using multivariable Cox models. Results: Pts were predominantly Chinese (83.6% v 85.4%) with locally advanced HNC (91.4% v 92.2%). Compared to OSC, HSC pts were older (median age 67 v 61 yrs), more likely male (89.3% v 74.0%), current or ex-smokers (83.3% v 63.6%) with higher comorbidity-age combined risk score (ageCCI), less likely p16H (6.5% v 30.1%)(all p<0.001) and had similar CYD+. p16H pts were younger (median age 58 (p16H) v 65 (p16L) v 66 (p16N) yrs, p=0.002), more likely non-smoker (51.4% v 23.4% v 13% p<0.001) with lowest ageCCI (p<0.001). Clinical characteristics did not differ by CYD1 status. At median f/u of 50mths, median OS was 33 mths. Median OS was poor in HSC compared to OSC (23.9 v 72.1, p<0.001). Multivariate analysis showed associations of N2/N3 disease (HR 1.57, p=0.036), ageCCI (HR 1.22 per 1 pt increase, p<0.001), p16 (p16H: ref; p16L: HR 2.34, p=0.045; p16N: HR 2.74, p=0.013) and CYD1+ (HR 1.94, p=0.015) with death, independent of gender, smoking and site. Association of p16 with OS was seen mainly in OSC (median OS p16H: not reached (NR), p16L: 62, p16N: 22 mths, p<0.001) compared with median OS (HSC) (27 v 28 v 21, p=0.609). Similarly the association of CYD1 with OS was mainly in OSC (median OS CYD1-: NR v 23 mths, p<0.001 v HSC: 25 v 25 mths, p=0.19). Conclusions: In OSC, p16 expression correlates with OS, with p16N associated with worst OS. CYD1 has an independent association with OS. Poorer OS in HSC may be due to adverse clinical characteristics. Assessment of p16 and CYD1 status in HSC did not predict for OS.
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Ramdas, Baskar, Joydeep Ghosh, Raghuveer Singh Mali, Zollman Amy, Nadia Carlesso, Malgorzata Kamocka M, Dunn W. Kenneth, Lawrance Quilliam, and Reuben Kapur. "Rap1 Gtpases Regulate the Retention and Engraftment of Hematopoietic Stem and Progenitor Cells." Blood 132, Supplement 1 (November 29, 2018): 326. http://dx.doi.org/10.1182/blood-2018-99-117197.
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Abstract Signaling molecules that control the homing and mobilization of hematopoietic stem and progenitor cells (HSC/Ps) are poorly understood. Rap1, a small-molecular-weight GTP-binding protein belongs to the Ras-like superfamily of GTPases and regulates several signal transduction cascades. Rap1 cycles between a GDP-bound inactive and a GTP-bound active form and exists in two isoforms - Rap1a and Rap1b, which have been implicated in the regulation of actin based functions in non-hematopoietic cells. Although Rap1 has been involved in regulating several hematologic disorders including chronic lymphocytic leukemia, myeloproliferative stem cell disorders, polycythemia vera and sickle cell anemia, its role in the development and function of HSC/Ps has not been investigated. We have generated a mouse model in which both Rap1a and Rap1b isoforms were conditionally deleted in HSC/Ps individually or in combination (double knockout; DKO). Our results demonstrate that deletion of both isoforms of Rap1 results in profound mobilization of primitive hematopoietic stem cells in peripheral blood. In the bone marrow, Rap1ab deficiency shows increased frequency of LSK cells, HPC-1 (LSK CD150-CD48+), HPC-2 (LSK CD150+CD48+) along with an increase in granulocyte-macrophage progenitor cell (GMP) population. Furthermore, spleen size and cellularity were significantly enhanced in DKO mice relative to controls. We hypothesized that Rap1 plays an essential role in regulating the retention of HSC/Ps in the bone marrow (BM) and that loss of Rap1 might inhibit the interaction of HSC/Ps with the BM niche cells, leading to egress of HSC/Ps and thus creating empty space(s) in the marrow for enhanced engraftment of donor derived cells when transplanted under non-myeloablative conditions. To test this, we performed BM transplantation using Rap1ab DKO mice as recipients and WT GFP expressing HSC/Ps as donors in the absence of any myeloablative conditioning. Our long-term engrfatment results showed significantly greater donor derived reconstitution of GFP positive cells in peripheral blood of DKO recipients compared to WT controls (WT: 19.2% vs DKO: 82.18% n=3, *p<0.05), suggesting that loss of Rap1ab creates functional open niche(s) in the BM due to mobilization of endogenous HSC/Ps. To better understand the mechanism behind this observation and to determine whether the GFP donor cells localize closer to the endosteal or vascular niche, we transplanted GFP positive cells into unconditioned (non-myeloablative) WT and Rap1ab DKO mice as described above. We measured the median distance of engrafted GFP cells from the bone surface and vasculature as a measure of proximity utilizing intravital microscopy. DKO recipients, transplanted with WT HSC/Ps preferentially localized to the vascular niche compared to control WT recipients (WT: 8µm vs DKO: 3 µm) and compared to osteoblastic niche, which was comparable in the two recipients, suggesting that GFP+ donor HSC/Ps preferentially localize and engraft near vascular niches providing indirect evidence to suggest that loss of Rap1ab leads to egress of hematopoietic cells from the vascular niche as opposed to osteoblastic niche. We next assessed the potential of Rap1ab deficient cells to engraft in a lethally irradiated host in a competitive repopulation assay. Rap1ab DKO HSC/Ps showed a defect in engraftment as well as multi-lineage reconstitution when transplanted into lethally irradiated hosts compared to WT controls. The defect in engraftment was largely due to impaired homing of DKO HSC/Ps. To assess which specific isoform of Rap1 is essential for mobilization and engraftment/homing of HSC/Ps, we induced deletion in Rap1a and Rap1b separately (single knock out mice) and assessed these mice for peripheral blood cell counts. We found no significant changes in the peripheral WBC counts in single Rap1a KO mice relative to controls; and only a modest increase in single Rap1b KO mice; suggesting that mobilization of HSC/Ps was relatively unperturbed in these mice and requires the loss of both isoforms of Rap1. In contrast, engraftment of HSC/Ps derived from the single KOs of Rap1a and Rap1b was impaired to the same extent as DKO HSC/Ps. These data suggest that loss of single Rap1 isoform contributes similarly to the engraftment of HSC/Ps, whereas the combined loss of both isoforms is required for efficient mobilization of HSC/Ps. Disclosures No relevant conflicts of interest to declare.
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Elbasha, Nuri Mohamed. "Reinforced HSC Beams." Key Engineering Materials 629-630 (October 2014): 544–50. http://dx.doi.org/10.4028/www.scientific.net/kem.629-630.544.
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The primary long and short term advantages of high strength concrete are, low creep and shrinkage, higher stiffness, higher elastic modulus, higher tensile strength, higher durability (resistance to chemical attacks) and higher shear resistance. In addition, high strength concrete reduces the size of the member, which in turn reduces the form size, concrete volume, construction time, labor costs and dead load. Reducing the dead load reduces the number and size of the beams, columns and foundations. Thus there is a positive impact on reduction of maintenance and repair costs and an increase in rentable space. Other, yet to be discovered advantages may also exist. High strength concrete has definite advantages over normal strength concrete. The ductility of over reinforced HSC beams is enhanced through the application of helical reinforcement located in the compression region. The pitch of helix is an important parameter controlling the level of strength and ductility enhancement. This paper presents an experimental investigation of the effect of helices on the behavior of over reinforced high strength concrete beams through testing ten helically confined full scale beams. The helix pitches were 25, 50, 75, 100 and 160 mm. Beams’ cross section was 200×300 mm, and with a length of 4 m and a clear span of 3.6 m subjected to four point loading. The main results indicate that helix effectiveness is negligible when the helical pitch is 160 mm (helix diameter). The experimental program in this study proved that the HSC, HSS and helical confinement construct a reinforced concrete beam. This beam has the ability to resist weathering action and chemical attack while maintaining its desired engineering properties. In near future Reinforced High Strength Concrete Beam with Helical Confinement will be considered as a durable and sustainable Reinforced Concrete Beam.
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Yang, Qi, and Avinash Bhandoola. "Decoding HSC heterogeneity." Blood 119, no. 21 (May 24, 2012): 4819–20. http://dx.doi.org/10.1182/blood-2012-03-417238.
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Amor, R. Ben. "Die Hochgeschwindigkeitsbearbeitung (HSC)." wt Werkstattstechnik online 91, no. 4 (2001): 239. http://dx.doi.org/10.37544/1436-4980-2001-4-239.
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Cavazzana-Calvo, Marina, Emmanuel Payen, Olivier Negre, Salima Hacein-Bey-Abina, and Philippe Leboulch. "HSC Transplantation for Hemoglobinopathies: Allogeneic or Autologous Gene-Modified HSC?" Blood 118, no. 21 (November 18, 2011): SCI—46—SCI—46. http://dx.doi.org/10.1182/blood.v118.21.sci-46.sci-46.
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Abstract Abstract SCI-46 The hematopoietic stem cell (HSC) transplantation pioneered by the Pesaro group is now applied wildly worldwide. The European Group for Blood and Marrow Transplantation (EBMT) has established the hemoglobinopathy registry, which now contains detailed epidemiological data on over 3000 patients. Today, allogeneic transplantation remains the only definitive curative therapy for thalassemia and other hemoglobinopathies. The development of iron chelation did not change this position. However, this has not settled the debate on how this curative but potentially lethal treatment stands vis-à-vis a medical noncurative therapy for adult and advanced disease patients. Nevertheless, despite the substantial improvements, a number of problems remain unsolved, such as the absence of an HLA genoidentical donor for all the diseased patients especially when they come from Outreach countries and the long-term morbidity due to HSCT (i.e., infectious complications, acute and chronic graft-versus-host disease, sterility). In this context and despite the unsolved debate, the development of gene therapy is highly justified. Sadelain and Leboulch pioneered the use of lentiviruses in human globin gene therapy, showing that anemia in mice affected with diseases mimicking the human pathological conditions can be abbreviated significantly by using lentiviruses containing a normal human β-globin gene. These pre-clinical studies set the basis for the current human clinical trial in Paris and for the upcoming trials. In our gene therapy trial, the human β-globin gene sequence has been modified by mutating a single amino acid at position 87 of the β-globin sequence. The major reason for using the b87 globin gene in lentiglobin in patients with β-thalassemia is that Hbb87 expression can easily be distinguished from that of normal Hb (HbA). In this trial, the patients received a full dose of Busulfex in the aim to obtain a myeloablation allowing the successful intake of gene modified autologous HSC. Two patients with thalassemia have been treated to date. One out of the two patients obtained a clinical benefit and is stably transfusion independent more than 3 years after the last transfusion. He currently has approximately 9 to 10 g percent of Hb in his blood, approximately one third being Hbb87, one third human HbF, and one third HbE. The patient is healthy up to now, with a full-time job. Integration site profile data will be discussed in detail during the meeting. Additional patients affected with thalassemia are currently being recruited in the Paris trial. Disclosures: No relevant conflicts of interest to declare.
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Hu, Xiaoxia, Hongmei Shen, Hui Yu, Feng Xu, Jianmin Wang, and Tao Cheng. "Limited Regeneration but Functional Preservation of Hematopoietic Stem Cells in the Mice Developing Leukemia via Constitutive Expression of Notch1." Blood 110, no. 11 (November 16, 2007): 204. http://dx.doi.org/10.1182/blood.v110.11.204.204.
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Abstract Leukemia development is a complex process involving both intrinsic and extrinsic factors. While many environmental factors have been studied, the impact of leukemic environment on normal hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) has not been definitively investigated. In this study, we have formally addressed this important issue by examining the potential functional alterations of HSC and HPC in the mice bearing Notch1-induced T acute lymphoblastic leukemia (T-ALL). The MSCV retrovirus vector containing cDNA encoding oncogenic intracellular domain of Notch1 (ICN1) pseudotyped with VSV-G was used to infect Lin−Sca-1+ cells in order to induce leukemic development. Normal hematopoietic cells from the B6.SJL strain (CD45.1+) were co-transplanted with Notch1 transduced Lin−Sca-1+ cells (CD45.2+) into lethally irradiated recipients. In this robust leukemia model with 100% penetrance, the normal hematopoietic cell compartment marked by CD45.1 in the leukemic marrow was sorted for phenotypic analyses and functional assays at different time points. Same numbers of the normal hematopoietic cells without Notch1-transduced cells were transplanted into the irradiated recipients as controls. As expected, progressive hematopoietic suppression was observed at both HSC and HPC levels in the leukemic mice. The frequency of HSC enriched population (Lin−c-Kit+Sca-1+, LKS) in the leukemic group was 7 times lower than that in the control at the 4th week of leukemogensis. When normalized to the bone marrow cellularity, the absolute yield of each population was 246 times lower in the leukemic group than that in the control group. These data were highly consistent with significantly lower yields of colony forming unit (CFU) and cobblestone area forming cell (CAFC). To measure the long-term engraftment of HSCs from leukemic environment, we performed the competitive bone marrow transplantation (cBMT), in which equal numbers of CD45.1+ cells isolated from leukemic or control mice and competitor cells (CD45.1/.2) at the 2nd week of leukemogenesis were co-transplanted into lethally irradiated C57BL/6J recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells isolated from the leukemic mice was 3 times more than that of the control group. Moreover, HSCs from the leukemic environment remained functional in serial transplant recipients. Finally, to explore the underlying molecular mechanisms for the enhanced function of normal HSC in the cBMT model, we examined a number of cell cycle and self-renewal regulators in HSC and HPC from leukemic marrow and control group at the time of harvest prior to transplantation by qRT-PCR. There was a significant decrease in p18 expression when compared with the control, whereas p21 expression was significantly increased. Notch1, Gfi1 and c-myc signalings were also elevated in the HSCs from leukemic environment. In summary, our current work provides the first definitive evidence for the reversible inhibition of normal HSC growth by the leukemic environment, thereby having important implications for HSC transplantation as well as leukemogenesis.
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Pel, Melissa van, Matthieu Monge, Michiel Siebelt, Marina M. Aleksinskaya, Hetty C. de Boer, Jacques Duijs, Coen van Solingen, et al. "Chronic Kidney Disease Induces a Reduction In Hematopoietic Stem –and Progenitor Cell Frequencies." Blood 116, no. 21 (November 19, 2010): 3857. http://dx.doi.org/10.1182/blood.v116.21.3857.3857.
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Abstract Abstract 3857 The hematopoietic microenvironment (niche) plays a key role in the maintenance of hematopoietic stem cells (HSC). The bone lining-osteoblast has been identified as a crucial component of the stem cell niche and regulates the number of HSC in the niche via a variety of membrane-bound and secreted molecules. Chronic Kidney Disease (CKD) is marked by a specific Mineral Bone Disease (CKD-MBD), due to a sustained parathyroid hormone (PTH) release. This chronical hyperparathyroidy results in increased bone turnover due to increased osteoblast activity. We have established a mouse model of CKD to study the relationship between disturbed bone metabolism and hematopoiesis. C57Bl/6 mice underwent surgically-induced CKD (thermocauterisation-nephrectomy). Twelve weeks after CKD-induction, bone structure was analyzed by micro-CT scan to confirm CKD-MBD. Indeed, all mice showed the features of CKD including significantly increased urea levels, increased trabecular bone volume, and a decrease in span incurvation and cortical thickness. In addition, CKD mice developed anemia. Subsequently, bone marrow cells (BMC) were harvested from CKD mice and sham-operated controls. Long-term HSC and short-term hematopoietic progenitor cells (HPC) were analyzed phenotypically by flowcytometry and functionally by cobblestone area forming cell (CAFC) assays and colony assays (CFU-C). The frequencies of long-term repopulating LinnegSca1posc-KitHI (LSK) CD135negCD34neg HSC were significantly decreased in CKD mice compared to sham-operated controls (0.0028% ± 0.001 vs 0.0049% ± 0.001, p<0.05; n=5 per group), whereas the frequencies of HPC (LSK CD135negCD34pos) and multipotent progenitors (MPP; LSK CD135posCD34pos) remained stable. Also, total LSK cell number correlated positively with trabecular bone volume. However, CAFC analysis showed a decrease in both HSC and HPC frequencies (0.5 ± 0.1 vs 1.4 ± 0.7 CAFC week 5 per 105 BMC, p<0.05; 0.6 ± 0.002 vs 1.5 ± 0.2 CAFC week 3 per 105 BMC, p<0.05 for CKD vs controls respectively; n=4 per group), indicating a functional defect in their repopulating capacity. The colony-forming capacity of BMC obtained from CKD mice and controls was similar (4.4 × 104 ± 1.5 × 104 vs 4.6 × 104 ± 1.7 × 104 CFU-C per femur for CKD vs controls respectively; n=5). In contrast, a 3.6-fold increase in the frequency of peripheral blood CFU-C was found in CKD mice compared to sham-operated controls (42.7 ± 2.6 vs 11.8 ± 1.6 CFU-C per ml for CKD vs controls respectively; n=5). CKD mice showed a 2.8-fold reduction in G-CSF-induced HSC/HPC mobilization compared to controls (355 ± 281 vs 1005 ± 476 CFU-C per ml peripheral blood for CKD vs controls respectively; n=5 per group). Analysis of BM extracellular fluid showed a 2.3-fold reduction in elastase activity and an increase of the protease inhibitor a1-antitrypsin in CKD mice compared to controls. No differences in MMP-9 activity were observed between the groups. Together, our data show that in CKD 1) LSK CD135negCD34neg HSC frequencies are significantly decreased 2) LSK CD135negCD34pos HPC and LSK CD135posCD34pos MPP remain at similar levels 3) CAFC-week 3 HPC and CAFC week 5 HSC are significantly decreased 4) BMC CFU-C remain at similar levels, while peripheral blood CFU-C are increased and 5) G-CSF-induced stem cell mobilization is impaired in CKD. We hypothesize that the observed changes in the hematopoietic compartment are due to increase osteoblast activity. Disclosures: Massy: Amgen: Honoraria, Research Funding; SHIRE: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding; Baxter: Research Funding; INEOS: Honoraria, Research Funding.
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Broxmeyer, Hal E., Maegan L. Capitano, Liang Zhao, Scott Cooper, and Charles S. Abrams. "Role for Megakaryocyte Phosphatidylinositol Transfer Proteins-Alpha and -Beta in TGF Beta-Mediated Regulation of Hematopoietic Homeostasis." Blood 126, no. 23 (December 3, 2015): 780. http://dx.doi.org/10.1182/blood.v126.23.780.780.
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Abstract There are still unknowns regarding homeostatic regulation of hematopoietic stem (HSC) and progenitor (HPC) cells. Deciphering these processes are important for understanding and treating hematopoietic diseases. Phosphatidylinositol is a rare membrane structure lipid, but is critical for cellular signaling upon phosphorylation by lipid kinases to generate phosphoinositide. While phosphoinositide pathways contribute to events linked to the cytoskeleton, little is known of these pathways in regulating hematopoiesis. Critical to this pathway are phosphatidylinositol transfer proteins (PITPs) that in vitro enhance transfer of aqueous insoluble phosphatidylinositol from one membrane to another. Class I PITP proteins PITP α and β are highly conserved, small, and ubiquitously expressed in mammalian cells. To test the hypothesis that phosphatidylinositol signaling contributes to hematopoiesis, we generated conditional knock out mice that lack either PITPα single isoform (PITPαfl/fl PF4Cre+) or both PITPα and PITPβ (PITPαfl/fl βfl/fl PF4Cre+) specifically in their platelets and megakaryocytes, and observed a bone marrow (BM) HSC/HPC phenotype. BM from these mice and their littermate controls were evaluated for absolute numbers of nucleated cells, HSC, and HPC. Cells were analyzed by rigorous phenotyping for long-term (LT)-HSC, short-term (ST)-HSC, multipotential (MPP), common myeloid (CMP), and granulocyte macrophage (GMP) progenitors. They were also assessed for functional HPC by colony assays in vitro for multi-cytokine (Epo, GM-CSF, IL-3, SCF, hemin) stimulated granulocyte macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitors, and for their cycling status using a high specific activity tritiated thymidine kill assay. PITPα-/-, and to a greater extent PITPα/β-/-, progenitor cells demonstrated significant decreases in LT-HSC and ST-HSC per femur. While there were no significant changes in numbers of MPP, CMP, and GMP in the PITPα and PITPα/β-/- BM compared to controls, there were significant decreases of approximately 50% in numbers of CFU-GM, BFU-E, and CFU-GEMM per femur. PITC-/- HPC were in a slow or non-cycling state compared to the rapid cell cycle (40-57% in S-phase) of control HPC. Thus PITPα-/- and PITPα/β -/- BM cells were associated with decreased HSC and functional HPC numbers. To evaluate mechanisms for this phenotype, we focused on BM megakaryocytes, as they have been implicated in microenvironmental regulation of hematopoiesis, and PITPα and PITPα/β activities are associated with megakaryocyte/platelet function. BM derived TPO-culture expanded megakaryocytes were allowed to condition medium for 48 hours, and conditioned medium (CM) from PITPα-/-, PITPα/β-/-, and control BM megakaryocytes were assayed for effects on colony formation by multicytokine stimulated BM cells derived from normal mice. CM from PITPα-/- and PITPα/β-/- megakaryocytes, but not from control mice, significantly suppressed colony formation by CFU-GM, BFU-E and CFU-GEMM (by ~50%). Limiting dilution analysis of the CM demonstrated that PITPα/β-/- cells had more potent suppressor activity than PITPα-/- cells. Bioplex analysis of the CM from PITPα -/- and PITPα/β -/- megakaryocytes demonstrated significantly higher levels of cytokines/chemokines with known myelosuppressive activities (including: TNF-α, VEGF, LIF, IP-10, ENA-78, MDC, MIG, and MIP-1α). However, ELISA analysis of TGF-β1, demonstrated minimal protein in BM flushes from control mice, but large amounts of TGF-β (>350 pg/ml) in BM flushes from the PITPα/β -/- mice. CM from PITPα and α/β-/- megakaryocytes also contained highly elevated TGF-β protein. Thus, we hypothesized that the effect of PITP -/- on the suppression of HPC colony formation was mediated by TGF-β. The myelosuppressive CM derived from PITPα and PITPα/β -/- megakaryocytes was completely neutralized by a monoclonal TGF-β antibody. This demonstrates that PITPα and PITP α/β-/- megakaryocytes produce elevated TGF-β that at least in part, and possibly in synergy with other myelosuppressive cytokines/chemokines, decreases numbers of HSC and functional HPC. Our studies demonstrate a link between PITPα and α/β and TGF-β levels with significant effects on HSCs and HPCs, thus demonstrating involvement of the phosphoinositide pathway in homeostatic regulation of hematopoiesis. Disclosures No relevant conflicts of interest to declare.
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Broxmeyer, Hal E., Ferdinand Kappas, Nirit Mor-Vaknin, Maureen Legendre, John Kinzfogl, Scott Cooper, Giao Hangoc, and David M. Markovitz. "DEK Regulates Hematopoietic Stem Engraftment and Progenitor Cell Proliferation." Blood 118, no. 21 (November 18, 2011): 1275. http://dx.doi.org/10.1182/blood.v118.21.1275.1275.
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Abstract Abstract 1275 Hematopoiesis is regulated by cell-cell and cytokine-cell interactions on hematopoietic stem (HSCs) and progenitor (HPCs) cells. In our continuing efforts to elucidate players involved in regulation of HSC and HPC growth, we focused on DEK, an abundant and unusual protein found in multicellular organisms. DEK has two DNA binding modules and has some affinity for specific DNA sequences, but primarily recognizes and binds to superhelical and cruciform DNA and induces positive supercoiling. DEK manifests multiple cellular activities, which include transcriptional repression and activation, mRNA processing, and chromatin architectural functions. We recently demonstrated that DEK modulates global heterochromatin integrity in vivo. Interestingly, DEK, can leave the cell and act as a chemoattractant for CD8+T cells and natural killer cells. Being intrigued that a nuclear protein was able to be secreted by hematopoietic cells, and act on other hematopoietic cells, we hypothesized that DEK might play a role in HSC/HPC function and hematopoiesis. In order to determine if DEK had an effect on steady state hematopoiesis, BM and spleen cells from DEK −/− mice were compared to that of wildtype (WT) control mice for absolute numbers and cycling status of HPC. Absolute numbers of CFU-GM, BFU-E, and CFU-GEMM per femur and per spleen were increased in DEK −/− mice. These effects were consistent with significantly increased percentages of HPCs in S-Phase of the cell cycle in DEK −/− BM and spleen, suggesting that DEK acts as a negative regulator of HPC proliferation in vivo. To confirm this, recombinant human DEK was tested for effects on HPC proliferation using unseparated mouse BM and low density human CB cells. DEK, dose-dependently suppressed colony formation by mouse BM CFU-GM stimulated by either IL-3 or GM-CSF, each alone; it did not influence colony formation stimulated by M-CSF alone. However, it dose-dependently inhibited CFU-GM colony formation by either IL-3, GM-CSF, or M-CSF when these cytokines were combined with the potent co-stimulating cytokine SCF. In fact, inhibition by DEK was greater on CFU-GM stimulated by the combination of IL-3, GM-CSF or M-CSF, each in the presence of SCF, compared to CFU-GM stimulated by IL-3, GM-CSF or M-CSF each alone in terms of percent inhibition, as well as the amount of DEK required to inhibit colony formation. Similar results were noted for HPCs present in human CB. This suggests that immature subsets of HPCs are more sensitive in vitro to the suppressive effects of DEK, than are the more mature HPCs. To determine if the DEK effects were directly or indirectly manifesting on the HPCs, DEK was assessed for effects on single isolated CD34+ cord blood cells, each in a single well stimulated by EPO, GM-CSF, IL-3, and SCF. DEK significantly decreased the number of wells containing a CFU-GM-, BFU-E-, or CFU-GEM- colony, demonstrating that DEK initiates it's suppressive effect directly on HPC. Using a mouse competitive repopulating HSC assay in vivo, allows assessment of the short- and longer-term repopulating HSC, and transplantation of BM cells from primary to secondary lethally-irradiated recipients in a non-competitive assay describes the longer-term repopulating HSC, and can offer information on the self-renewal capacity of this population of HSCs. While there was no difference in the HSC repopulating capacity of DEK −/− and WT shorter-term repopulating cells (months 1 and 2 for blood chimerism), there was a significant decrease in DEK −/− compared to WT BM cell repopulation at month 4 in the blood, and month 6 in the BM. This decreased repopulation of DEK −/− compared to WT HSC was even more apparent in secondary mouse recipients suggesting that DEK played a positive role in engraftment of longer-term repopulating HSCs, and perhaps in the self-renewal capacity of mouse BM HSCs. These studies demonstrate a here-to-fore unknown role for DEK in the regulation of HPCs, HSCs and hematopoiesis. DEK could have separate effects on HPC and HSC, as suggested by the direct acting effects of DEK on single HPC. Alternatively, DEK may alone, or in addition allow HSC to favor a self-renewal, vs. a differentiation pathway to HPC. Thus, DEK has potent effects on HSCs, HPCs, and hematopoiesis, and may be of potential clinical value for enhancing HSC activity/proliferation in vivo, or in an ex-vivo situation. Disclosures: No relevant conflicts of interest to declare.
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Li, Xinlei, Ruju Chen, Sherri Kemper, and David R. Brigstock. "Dynamic Changes in Function and Proteomic Composition of Extracellular Vesicles from Hepatic Stellate Cells during Cellular Activation." Cells 9, no. 2 (January 25, 2020): 290. http://dx.doi.org/10.3390/cells9020290.
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During chronic liver injury, hepatic stellate cells (HSC) undergo activation and are the principal cellular source of collagenous scar. In this study, we found that activation of mouse HSC (mHSC) was associated with a 4.5-fold increase in extracellular vesicle (EV) production and that fibrogenic gene expression (CCN2, Col1a1) was suppressed in Passage 1 (P1; activated) mHSC exposed to EVs from Day 4 (D4; relatively quiescent) mHSC but not to EVs from P1 mHSC. Conversely, gene expression (CCN2, Col1a1, αSMA) in D4 mHSC was stimulated by EVs from P1 mHSC but not by EVs from D4 mHSC. EVs from Day 4 mHSC contained only 46 proteins in which histones and keratins predominated, while EVs from P1 mHSC contained 337 proteins and these were principally associated with extracellular spaces or matrix, proteasome, collagens, vesicular transport, metabolic enzymes, ribosomes and chaperones. EVs from the activated LX-2 human HSC (hHSC) line also promoted fibrogenic gene expression in D4 mHSC in vitro and contained 524 proteins, many of which shared identity or had functional overlap with those in P1 mHSC EVs. The activation-associated changes in production, function and protein content of EVs from HSC likely contribute to the regulation of HSC function in vivo and to the fine-tuning of fibrogenic pathways in the liver.
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Rezzoug, Francine, Yiming Huang, Michael K. Tanner, Marcin Wysoczynski, Carrie L. Shannie, Mariusz Z. Ratajczak, Isabelle J. Fugie-Vivier, and Suzanne T. Ildstad. "TNFα Mediated Facilitating Cell Enhancement of Hematopoietic Stem Cell Function in Vivo and In Vitro Involves Bcl-3." Blood 104, no. 11 (November 16, 2004): 2174. http://dx.doi.org/10.1182/blood.v104.11.2174.2174.
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Abstract Approaches to enhance engraftment of hematopoietic stem cells (HSC) are of primary interest in BM transplantation. CD8+TCR− facilitating cells (FC) improve HSC engraftment in allogeneic recipients without causing graft versus host disease. FC also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients, suggesting that FC act directly on HSC. We therefore analyzed the mechanism for FC-mediated effects on HSC. We found that FC increased the ability of purified HSC to generate colonies in both the CFC and CAFC/LTC-IC assays, confirming a direct effect of FC on different HSC compartments. Co-incubation of HSC with FC for 18 or 40 hours significantly increased the survival of HSC and their subsequent ability to generate CFC at these same time points. We determined that the anti-apoptotic effect of FC on HSC was associated with the up regulation of anti-apoptotic Bcl-3 transcripts. We postulated here that the effect of FC on HSC was due to cytokine secretion. As FC produce TNFα after CpG ODN stimulation and TNFα has various activities on HSC, we evaluated the role of TNFα on FC function. FC were sorted from TNFα deficient mice and the facilitative activity of FC on HSC engraftment was assessed. FC from TNFα deficient mice were impaired in facilitating HSC engraftment in both the syngeneic and allogeneic models, suggesting a role for TNFα in FC function. Notably, TNFα transcripts were present in FC by 16 hours of co-incubation of FC + HSC and FC produce TNFα (surface and intra-cellular) when in contact with HSC. Furthermore, when TNFα was blocked (using anti-TNFα mAb), FC from wild type mice lost the ability to increase HSC clonogenicity (from 38.2±13.6 CFC/1000 HSC in HSC alone to 65.7 ± 22 for HSC + FC and 38.2 ± 19.6 for FC pre-incubated 1 hour with anti-TNFα mAb before incubation with HSC). Moreover, anti-TNFα mAb also blocked the ability of FC to up-regulate Bcl-3 transcripts in HSC. In conclusion, FC act directly on HSC via several mechanisms to maintain the balance between proliferation/differentiation/survival of HSC. One central mechanism implicates TNFα production by FC, which may protect HSC from undergoing apoptosis by up-regulating anti-apoptotic transcripts (e.g. Bcl-3). These findings may have great impact for the use of accessory cells in HSC transplantation, especially when numbers of HSC are limiting.
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Tjwa, Marc, Lieve Moons, Koen Theunissen, Rute Moura, Francesco Blasi, Mieke Dewerchin, Nicolai Sidenius, Desire Collen, and Peter Carmeliet. "Role of Plasmin and uPAR in Bone Marrow HSC/HPC Retention and Mobilization." Blood 106, no. 11 (November 16, 2005): 472. http://dx.doi.org/10.1182/blood.v106.11.472.472.
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Abstract We previously identified the plasmin protease family as a critical determinant of the mobilization of hematopoietic stem and progenitor cells (HSC/HPC), but the role of the urokinase receptor uPAR remained unclear. uPAR is a membrane-anchored glycoprotein, which not only localizes its ligand urokinase (uPA) to the cell surface via its GPI-anchor but also regulates β1-integrin dependent cell adhesion and migration. Following 5-FU myeloablation or G-CSF treatment, mice lacking uPAR (uPAR−/−) had impaired hematopoietic recovery and HSC/HPC mobilization as compared to wild type (WT) mice. However, this phenotype was not mimicked in mice lacking uPA, suggesting a role of uPAR in mobilization independent of uPA-mediated proteolysis. The impaired mobilization in uPAR−/− mice was reversed upon pre-transplantation with WT BM cells (BMC), suggesting functional expression of uPAR on transplantable BMCs. Conversely, loss or inhibition of uPAR on transplanted BMCs impaired homing to the BM but not to the spleen, and compromised survival of myeloablated WT recipients. In vitro experiments revealed that loss or inhibition of uPAR impaired BMC adhesion to stromal cells and fibronectin. Anti-α4-β1 antibodies blocked adhesion of WT but not uPAR−/− BMCs. Thus, uPAR appears to regulate BM homing and α4-β1 dependent retention of transplantable BMCs, possibly HSC/HPCs. If uPAR mediates retention of HSC/HPCs, then this signal should be inactivated upon mobilization. Indeed, in 5-FU or G-CSF-treated WT mice, we found increased uPAR cleavage, and elevated levels of soluble uPAR (suPAR) in BM plasma. These processes failed to occur in mice lacking plasminogen, suggesting that plasmin cleaves uPAR during mobilization. Cleavage of uPAR appeared critical as the inactivation of the retention signals membrane-bound Kit ligand and SDF-1α was normal in uPAR−/− mice. Moreover, the generated suPAR may also affect the BM, as administration of recombinant suPAR in WT mice enhanced hematopoietic recovery and HSC/HPC mobilization after 5-FU or G-CSF. In vitro and transplantation experiments revealed that suPAR blocked α4-β1 dependent adhesion. Thus, in steady state, membrane-anchored uPAR appears to function as a BM retention signal for transplantable BMCs, possibly HSC/HPCs. In conditions of mobilization, the uPAR retention signal is cleaved, which weakens α4-β1 dependent adhesion and allows mobilization out of the BM. Soluble uPAR may then additionally amplify mobilization, in part by further attenuating α4-β1 dependent adhesion to the BM. Currently, we are investigating the role of uPAR on subsets of HSC/HPCs, and in the different BM niches. We are also performing long-term competitive repopulation experiments to further delineate the therapeutic potential of uPAR.
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Trinh, Thao, Scott Cooper, Arafat Aljoufi, Edward F. Srour, and Hal E. Broxmeyer. "Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells." Blood 134, Supplement_1 (November 13, 2019): 3712. http://dx.doi.org/10.1182/blood-2019-128659.
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Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)+ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells. Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr+HSCs (28.5% Lepr+LT-HSC and 17.2% Lepr+ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr+HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr+ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (-/-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2+) Lepr - Lineage-Sca1+cKit+ (LSK) vs. Lepr+LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1+) cells into lethally irradiated CD45.2+/CD45.1+ F1 recipients. A consistently higher engraftment capacity of Lepr+LSK cells was manifested in comparison to Lepr - LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr+HSCs and Lepr+MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr - populations, suggesting that homing differences may not explain increased engraftment of Lepr+ LSK. At month 6, Lepr+LSK, but not Lepr - cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr -LSK cells, Lepr+LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr+LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr+LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr -LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production. For beginning efforts at mechanistic insight, we hypothesized that Lepr+ HSCs and Lepr+MPP may be different than Lepr - cells in mitochondrial activity. Compared to Lepr - cells, Lepr+HSC and Lepr+MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay). In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans. Disclosures No relevant conflicts of interest to declare.
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Grignani, Francesco, Mauro Valtieri, Marco Gabbianelli, Vania Gelmetti, Rosanna Botta, Luisella Luchetti, Barbara Masella, et al. "PML/RARα fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype." Blood 96, no. 4 (August 15, 2000): 1531–37. http://dx.doi.org/10.1182/blood.v96.4.1531.
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Abstract The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RARα fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RARα mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RARα alters the early hematopoietic program through N-CoR–dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells.
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Grignani, Francesco, Mauro Valtieri, Marco Gabbianelli, Vania Gelmetti, Rosanna Botta, Luisella Luchetti, Barbara Masella, et al. "PML/RARα fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype." Blood 96, no. 4 (August 15, 2000): 1531–37. http://dx.doi.org/10.1182/blood.v96.4.1531.h8001531_1531_1537.
Full textAbstract:
The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RARα fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RARα mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RARα alters the early hematopoietic program through N-CoR–dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells.
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Ildstad, Suzanne T., Francine Rezzoug, Yiming Huang, Marcin Wysoczynski, Carrie L. Schanie, Mariusz Z. Ratajczak, and Isabelle J. Fugier-Vivier. "CD8+/TCR− Graft Facilitating Cells Enhance HSC Engraftment and Survival Via TNF-α Mediated Prevention of Apoptosis." Blood 106, no. 11 (November 16, 2005): 1352. http://dx.doi.org/10.1182/blood.v106.11.1352.1352.
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Abstract The use of accessory cells to enhance hematopoietic stem cell (HSC) engraftment could have a significant therapeutic impact, especially when stem cell numbers are limited. The bone marrow (BM) microenvironment is involved in regulation of HSC, allowing production of mature blood cells while maintaining HSC self renewal. To date, the precise identity of specific cells in the microenvironment that exert this regulatory effect on HSC has not been defined. We recently reported that CD8+/TCR− facilitating cells (FC), a subpopulation of BM cells containing predominantly B220+/CD11c+/CD11b− tolerogenic precursor-plasmacytoid dendritic cells, enhance HSC engraftment in allogeneic recipients. Additionally, FC significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. In the present studies, we investigated the mechanism of FC-mediated enhancement of HSC engraftment. We show for the first time that FC significantly increase HSC survival in vitro and exert an anti-apoptotic effect on HSC via TNF-α. Co-culture of FC with HSC induces production of physiologically relevant low levels of TNF-α by FC. FC from TNF-α−/− mice are impaired in function in vitro and in facilitating HSC engraftment in vivo. Furthermore, neutralization of TNF-α on FC using anti-TNF antibody results in loss of FC function in vitro, confirming a major role for TNF-α in FC function. Notably, co-culture of FC with HSC prevents HSC apoptosis and is associated with significant upregulation of the anti-apoptotic I-κB family member Bcl-3 in HSC. Blocking of TNF-α on FC abrogates the anti-apoptotic effect of FC on HSC and prevents upregulation of Bc1-3 in HSC. Taken together, these findings demonstrate that TNF-α-induced in FC affects highly primitive HSC and identify Bcl-3 as a possible pathway for TNF-α in regulating HSC survival.
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Varga, Gergely I. B., Gábor Csordás, Gyöngyi Cinege, Ferenc Jankovics, Rita Sinka, Éva Kurucz, István Andó, and Viktor Honti. "Headcase is a Repressor of Lamellocyte Fate in Drosophila melanogaster." Genes 10, no. 3 (March 5, 2019): 173. http://dx.doi.org/10.3390/genes10030173.
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Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.
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Christopherson, Kent W., Sherene E. Uralil, Shannon M. Kidd, Nehal K. Porecha, and Ryan C. Zabriskie. "Cytokine Induced Changes in the Hematopoietic Stem Cell Trafficking Regulatory Peptidase CD26." Blood 106, no. 11 (November 16, 2005): 5220. http://dx.doi.org/10.1182/blood.v106.11.5220.5220.
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Abstract Hematopoietic stem cell transplantation (HSCT) serves as a successful treatment option for patients with malignant or non-malignant severe hematologic diseases. However, large numbers of transplantable donor cells are needed and patient survival is compromised when donor cell numbers are limited, as is the case when umbilical cord blood (CB) donor cells are utilized as a donor source for transplantation into adult patients. Given that CB is readily available, has a lower histocompatibility requirement, and has a reduced risk of graft vs. host disease (GVHD) there are advantages to utilizing CB for HSCT, in particular when an appropriate matched donor is not available. However, in the majority of cases potential recipients have been confined to children because in adults, the amount of CB collected appears to be a limiting factor. Given the promise of CB for HSCT there is significant potential and application for improvements in the efficiency of Hematopoietic Stem Cell (HSC) trafficking to the bone marrow (BM) in both scenarios. To facilitate our long term goal of improving HSCT efficiency, we have recently delineated a novel method by where the inhibition of peptidase CD26 on donor HSC and Progenitor Cells (HPC) increases the number of donor HSC/HPC trafficking to recipient’s BM, resulting in a significant increase in HSC transplant efficiency. CD26 (DPPIV/dipeptidylpeptidase IV) is a membrane bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. Natural substrates of CD26 protease activity include the pancreatic polypeptide family, the glucagon family, and the chemokine family. Since the removal of N-terminal amino acids from chemokines frequently result in significant changes in functional activity, proteolytic cleavage of chemokines has significant implications with respect to their ability to act on cells. We have previously shown that that suppression of CD26 activity, by the use of CD26 inhibitors or the use of CD26−/− donor cells, increases the efficiency of HSC/HPC settlement and growth in the transplanted recipient. This translates into an increase in overall recipient survival in mice. We report here the results of experiments that examine the ability of several cytokines to alter CD26 on the surface of CD34+CD38− phenotypically defined HSC/HPC from CB. HSC isolated from CB were treated with varying dilutions of either Stem Cell Factor (SCF/steel factor/kit ligand), Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), Erythropoietin (EPO), or Trombopoietin (TPO) during culture in IMDM, 20% FBS, 37°C, 5%CO2 for 18 hours and then analyzed for changes in CD26. We observed significant dose dependent increases in the percentage of cells expressing CD26 and the amount of CD26 being expressed on each cell in samples treated with G-CSF, GM-CSF, and SCF as measured by multi-varient flow cytometric analysis. More modest changes in CD26 expression were observed during EPO and TPO treatment. Differential expression of CXCR4 was also noted. We feel these observations have important clinical implications with respect treatment of donor cells prior to transplantation and treatment of patients post HSCT.
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Mantel, Charlie, Steven Messina-Graham, Akira Moh, Scott Cooper, Giao Hangoc, Xin-Yuan Fu, and Hal E. Broxmeyer. "Mouse hematopoietic cell–targeted STAT3 deletion: stem/progenitor cell defects, mitochondrial dysfunction, ROS overproduction, and a rapid aging–like phenotype." Blood 120, no. 13 (September 27, 2012): 2589–99. http://dx.doi.org/10.1182/blood-2012-01-404004.
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Abstract Nuclear transcription factor Stat3 is important for proper regulation of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) proliferation, survival, and cytokine signaling responses. A new, noncanonical role for Stat3 in mitochondrial function has been discovered recently. However, there is little information on the role(s) of mitochondrial Stat3 in HSC/HPC function, especially potential effects of Stat3/mitochondrial dysregulation in human diseases. We investigated hematopoietic cell–targeted deletion of the STAT3 gene in HSCs/HPCs with a focus on mitochondrial function. We found that STAT3−/− mice, which have a very shortened lifespan, dysfunctional/dysregulated mitochondrial function and excessive reactive oxygen species production in HSCs/HPCs that coincides with pronounced defects in function. These animals have a blood phenotype with similarities to premature aging and to human diseases of myelodysplastic syndrome and myeloproliferative neoplasms such as erythroid dysplasia, anemia, excessive myeloproliferation, and lymphomyeloid ratio shifts. We show herein that the lifespan of STAT3−/− animals is lengthened by treatment with a reactive oxygen species scavenger, which lessened the severity of the blood phenotype. These data suggest a need for more detailed studies of role(s) of Stat3 in HSC/HPC mitochondrial function in human diseases and raise the idea that mitochondrial Stat3 could be used as a potential therapeutic target.