scholarly journals Differential Effects of RASA3 Mutations on Hematopoiesis are Profoundly Influenced by Genetic Background and Molecular Variant

2020 ◽  
Author(s):  
Raymond F. Robledo ◽  
Steven L. Ciciotte ◽  
Joel H. Graber ◽  
Yue Zhao ◽  
Amy J. Lambert ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Blood Cells ◽  
Genetic Background ◽  
White Blood Cells ◽  
Independent Effect ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Blood Formation ◽  
Molecular Variant

AbstractStudies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5-13.5 due to severe hemorrhage and decreased fetal liver erythropoiesis. Conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-Cre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in mouse models scat (G125V) and hlb381 (H794L) show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. Global transcriptomic studies in scat suggest potential targets to ameliorate disease progression.Author SummaryHematopoiesis is the process by which blood cells are formed. The individual must have a normal complement of red blood cells to prevent anemia, platelets to control bleeding, and white blood cells to maintain immune functions. All blood cells are derived from hematopoietic stem cells that differentiate into progenitor cells that then develop into mature circulating cells. We studied several mouse strains carrying different mutations in RASA3. We show that RASA3 is required at the earliest stages of blood formation, the stem and progenitor cells, and that the complement of genes other than RASA3, or the genetic background of the mutant strain, profoundly alters the overall effect on blood formation. Further, the molecular nature of the mutation in RASA3 also has a profound and independent effect on overall blood formation. One strain, designated scat, suffers cyclic anemia characterized by severe anemic crisis episodes interspersed with remissions where the anemia significantly improves. Comparison of scat crisis and remission hematopoietic stem and progenitor cells reveals striking differences in gene expression. Analyses of these expression differences provide clues to processes that potentially drive improvement of anemia in scat and provide new avenues to pursue in future studies to identify novel therapeutics for anemia.

scholarly journals Differential Effects of TLR1/2 and TLR2/6 Signaling on Normal and Premalignant Hematopoietic Stem and Progenitor Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1806-1806
Author(s):  
Darlene A. Monlish ◽  
Zev J. Greenberg ◽  
Sima T. Bhatt ◽  
Dagmar Ralphs ◽  
John L. Keller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
White Blood Cells ◽  
Hematopoietic Stem ◽  
Specific Effects ◽  
Stem And Progenitor Cells ◽  
Cell Cycling

Abstract Prior studies from our lab and others have demonstrated a role for Toll-like receptor 2 (TLR2) in regulating both normal and premalignant hematopoietic stem and progenitor cells (HSPCs), however the contributions of its binding partners, TLR1 and TLR6, remain unknown. In CD34+ bone marrow cells of patients with myelodysplastic syndrome (MDS), increased TLR2 was associated with lower-risk disease, elevated rates of apoptosis associated with improved prognosis, and enhanced survival. Conversely, increased levels of TLR6, but not TLR1, was associated with higher-risk disease and an increased percentage of bone marrow blasts (Zeng et al., Exp Cell Res 2016 and Wei et al., Leukemia 2013). These data suggest that there may be heterodimer-specific effects of TLR2 signaling on HSPCs influencing disease progression. To elucidate the unique contributions of the heterodimer pairs in MDS pathogenesis and leukemogenesis, we utilized a well-established mouse model of MDS that expresses the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter. The "NHD13" mice recapitulate many of the salient features of human MDS and succumb to cytopenias or leukemia by 14 months of age (Lin et al., Blood 2005). Importantly, we observed significantly increased expression of TLRs 1, 2, and 6 on the c-Kit+, Sca-1+, Lineage- ("KSL") HSPCs of the NHD13 mice, similar to the increased expression of these TLRs on CD34+ cells of MDS patients. To begin to delineate the heterodimeric differences, NHD13 mice were treated chronically with either PAM2CSK4 (PAM2), a TLR2/6-specific agonist, or PAM3CSK4 (PAM3), a TLR1/2-specific agonist, to assess the effects on cytopenias and survival. After five months of treatment, a significant increase was observed in the total number of white blood cells in NHD13 mice treated with PAM2 (p=0.007), but not PAM3 (vs. vehicle (water)-treated controls), a finding that was not recapitulated in wild-type (WT) controls. On the contrary, a significant decrease in the total number of platelets in both NHD13 and WT mice treated with PAM3 was observed as compared to vehicle-treated controls (p=0.024 and p=0.011, respectively). Further supporting the existence of heterodimer-specific differences, death was expedited in NHD13 mice treated with PAM2 as compared to those treated with PAM3 (p=0.019), with a median survival of 243 days vs. 338 for the PAM3-treated cohort. The cause of death, as determined by a hematopathologist based on cytology and blast percentage, was most often due to leukemia. To investigate the potential mechanism through which enhanced TLR2/6 signaling accelerates leukemogenesis and death in NHD13 mice, the HSPCs of premalignant NHD13 mice treated with PAM2 or PAM3 were characterized by flow cytometry and evaluated for cell cycling and cell death. Both the total number and frequency of KSL cells were significantly increased in NHD13 mice treated with PAM2 (p=0.007 and p<0.0001, respectively), but not PAM3, vs. water-treated controls. No significant changes were noted in either cell cycling or apoptosis following agonist treatment. A microarray of bone marrow KSL cells revealed that stimulation of the TLR2/6 pathway is associated with an activated c-Myc signature, suggesting that enhanced signaling through this pathway, but not TLR1/2, may enhance leukemogenesis via Myc activation. Further, the expression levels of six downstream targets of c-Myc, including BAX, APEX1, ODC1, FKBP4, NCL, and HSPD1, were significantly increased in both WT and NHD13 mice following PAM2 treatment. Evaluation of serum cytokines also revealed heterodimer-specific alterations, including increased IL-6 levels in NHD13 mice treated with PAM2, but not PAM3. These data corroborate numerous previous reports linking IL-6 to MDS pathogenesis and transformation to acute myeloid leukemia. Ongoing studies involving mass cytometry, IL-6knockout mice, and pharmacological inhibitors of both IL-6 and c-Myc aim to further elucidate the mechanism through which TLR2/6-specific activation accelerates leukemogenesis and death in the NHD13 mouse model of MDS. These studies hope to inform more targeted therapeutics that could potentially delay MDS progression and reduce off-target effects. Disclosures No relevant conflicts of interest to declare.

scholarly journals Peripheral blood hematopoietic stem cells: Biology, apheresis collection and cryopreservation

2007 ◽  
Vol 60 (1-2) ◽  
pp. 42-47 ◽  
Author(s):  
Ivana Urosevic ◽  
Bela Balint ◽  
Stevan Popovic
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Cell Damage ◽  
Small Population ◽  
Minimal Cell ◽  
Hematopoietic Stem ◽  
Acute Infarction ◽  
Stem And Progenitor Cells ◽  
Processing And Storage

Introduction Hematopoiesis is a continuous, dynamic and highly complex process resulting in production of various mature blood cells from a small population of pluripotent stem and progenitor cells through diverse proliferative and differentiative events. Numerous studies have demonstrated that a complex network of interactive cytokines regulates the survival, maturation, and proliferation of hematopoietic stem and progenitor cells (HSPCs). Application of cell-mediated therapy Massive application of different cell-mediated therapeutic methods has resulted in an increased need for both specific HSPCs and operating procedures providing minimal cell damage during collection, processing and storage in a liquid or frozen state. Therefore, the basic aim of cell harvesting, selection, as well as cryopreservation is to minimize cell damage during these procedures. HSPCs are cells which exhibit extensive self-renewal and proliferative capacity, associated with the capacity to differentiate into all blood cells and other cell lineages (plasticity of HSPC). Thanks to these properties, stem cells can provide complete and permanent restoration of hematopoiesis, which is the basis for clinical employment of HSPC transplantation. In addition, totipotent stem cells can be used for the so called "cell-therapy" in different clinical settings (e.g. myocardial regeneration after acute infarction). Conclusion Despite the fact that HSPC transplantation is already in routine use, some questions related to the optimal blood progenitor/cell collection, selection, storage and clinical use are still unresolved. Therefore, this review only briefly discusses the therapeutic use of HSPCs in different clinical areas and focuses on the recommendations, as well as the specific transfusion policies related to HSPC collection, processing, and cryopreservation with an emphasis on quality control.

scholarly journals Differential effects of RASA3 mutations on hematopoiesis are profoundly influenced by genetic background and molecular variant

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1008857
Author(s):  
Raymond F. Robledo ◽  
Steven L. Ciciotte ◽  
Joel H. Graber ◽  
Yue Zhao ◽  
Amy J. Lambert ◽  
...  
Keyword(s):  
Genetic Background ◽  
Missense Mutations ◽  
Hematopoietic Stem ◽  
Differential Effects ◽  
Conditional Deletion ◽  
Severe Hemorrhage ◽  
Stem And Progenitor Cells ◽  
Molecular Variant ◽  
Vessel Development

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5–13.5 due to severe hemorrhage. Here, conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-iCre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in two mouse models, scat (G125V) and hlb381 (H794L), show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. The mutation effect is mediated at least in part by differential effects on RAS and RAP activation. In addition, we show that the role of RASA3 is conserved during human terminal erythropoiesis, highlighting a potential function for the RASA3-RAS axis in disordered erythropoiesis in humans. Finally, global transcriptomic studies in scat suggest potential targets to ameliorate disease progression.

Generation and Characterization of Transgenic Mice Expressing MplW515L.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3901-3901
Author(s):  
Wanming Zhao ◽  
Shu Xing ◽  
Rufei Gao ◽  
Aref Al-Kali ◽  
Wanting Tina Ho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
B Cells ◽  
Transgenic Mice ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
White Blood Cells ◽  
Hematopoietic Stem

Abstract Abstract 3901 Poster Board III-837 Myeloproliferative neoplasias (MPNs) are a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). JAK2V617F, a gain-of-function mutation of tyrosine kinase JAK2, is found in over 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Attempt to identify other signaling components involving the JAK2 signaling pathways has led to discovery of acquired mutations of Mpl, the receptor of thrombopoietin, in 5-10% patients with PMF and ET. To prove the pathogenesis of Mpl mutants, we have generated transgenic mice expressing the most frequently occurred Mpl mutant designated MplW515L by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. We obtained three lines of MplW515L transgenic mice which all displayed similar hematological abnormalities. As expected, the mice developed ET- and PMF-like phenotypes with much elevated platelet counts, severe splenomegaly/hepatomegaly, and bone marrow/spleen myelofibrosis. Interestingly, these mice also had markedly increased white blood cells in the peripheral blood, majority of which are IgD-positive mature B-cells. Histochemical staining and flow cytometric analyses revealed infiltrations of megkaryocytes and B cells into the spleen, the presence of megkaryocytes and erythroid blast cells in the liver, and infiltrations of the bone marrow with B-cells. Reticulin staining revealed that MplW515L transgenic mice developed profound myelofibrosis in the bone marrow and spleen. In vitro hematopoietic colony assays demonstrated increased numbers of hematopoietic progenitor cells including BFU-E, CFU-GM, CFU-Mk, and CFU-Pre-B in the bone marrow, mobilization of these stem/progenitor cells to peripheral blood and spleen, and their autonomous growth in the absence of growth factors and cytokines. Finally, transplantation of bone marrow cells from MplW515L mice into irradiated normal mice installed the aforementioned phenotypes into the recipient mice, indicating that expression of MplW515L altered the activity of hematopoietic stem cells. Together, our data demonstrated that transgenic expression of MplW515L not only causes PMF- and ET-like phenotypes but also lymphoproliferative disorders. Considering that Mpl is expressed in hematopoietic stem cells and that oncogenic gene mutations are often associated with alteration of gene expression, we believe that MplW515L may be involved in a wider spectrum of human hematological diseases than MPNs. Disclosures: No relevant conflicts of interest to declare.

Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 6-16 ◽  
Author(s):  
E. V. Sotnezova ◽  
E. R. Andreeva ◽  
A. I. Grigoriev ◽  
L. B. Buravkova
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Blood Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cord Blood Cells

Transplantation of umbilical cord blood cells is currently widely used in modern cell therapy. However, the limited number of hematopoietic stem and progenitor cells (HSPCs) and prolonged time of recovery after the transplantation are significant limitations in the use of cord blood. Ex vivo expansion with various cytokine combinations is one of the most common approaches for increasing the number of HSPCs from one cord blood unit. In addition, there are protocols that enable ex vivo amplification of cord blood cells based on native hematopoietic microenvironmental cues, including stromal components and the tissue-relevant oxygen level. The newest techniques for ex vivo expansion of HSPCs are based on data from the elucidation of the molecular mechanisms governing the hematopoietic niche function. Application of these methods has provided an improvement of several important clinical outcomes. Alternative methods of cord blood transplantation enhancement based on optimization of HPSC homing and engraftment in patient tissues have also been successful. The goal of the present review is to analyze recent methodological approaches to cord blood HSPC ex vivo amplification.

scholarly journals Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor

2018 ◽  
Vol 115 (9) ◽  
pp. 2180-2185 ◽  
Author(s):  
Yu-Ting Tan ◽  
Lin Ye ◽  
Fei Xie ◽  
Ashley I. Beyer ◽  
Marcus O. Muench ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Disease Modeling ◽  
Single Factor ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Human Ipsc

Derivation of human hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) offers considerable promise for cell therapy, disease modeling, and drug screening. However, efficient derivation of functional iPSC-derived HSCs with in vivo engraftability and multilineage potential remains challenging. Here, we demonstrate a tractable approach for respecifying iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells (HSPCs) through transient expression of a single transcription factor, MLL-AF4. These induced HSPCs (iHSPCs) derived from iPSCs are able to fully reconstitute the human hematopoietic system in the recipient mice without myeloid bias. iHSPCs are long-term engraftable, but they are also prone to leukemic transformation during the long-term engraftment period. On the contrary, primary HSPCs with the same induction sustain the long-term engraftment without leukemic transformation. These findings demonstrate the feasibility of activating the HSC network in human iPSC-derived blood cells through expression of a single factor and suggest iHSPCs are more genomically instable than primary HSPCs, which merits further attention.

In Vivo Lentiviral Marking Demonstrates Long-Term Myeloid and Lymphoid Lineage Contribution of Individual Hematopoietic Stem Cell Clones to Murine Steady-State Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 813-813
Author(s):  
Oksana Zavidij ◽  
Claudia R Ball ◽  
Sylvia Fessler ◽  
Daniela Belle ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Stem Cell ◽  
Steady State ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cell Clones ◽  
Stem And Progenitor Cells ◽  
One Year

Abstract Abstract 813 Most of the knowledge to date on the in vivo blood forming activity of individual hematopoietic stem and progenitor cells was gained in transplantation experiments of defined cell populations into syngeneic or xenogeneic murine hosts. Consequently, stem and progenitor cells are solely defined by their role in post-transplant reconstitution and very little is known on their clonal activity in steady-state hematopoiesis. To gain new insights into the clonal activity of stem and progenitor cells under steady-state conditions we used a genetic in vivo lentiviral marking strategy and subsequently monitored the clonal activity of marked hematopoietic cells for up to one year by highly sensitive integration site amplification using LAM-PCR. Highly concentrated GFP-expressing lentiviral vectors (LV) were injected intravenously (IV, n=10) or intrafemorally (IF, n=15) into GFP-tolerant B6.Cg-Tg (Krt1-15-EGFP) 2Cot/J (Krt15) mice to directly mark hematopoietic stem and progenitor cells. 5 mice from each of the two cohorts were treated with 5-Fluorouracil (5-FU, 150 mg/kg) to mobilize hematopoietic stem cells prior to LV-marking. The clonality of the transduced myelopoiesis and lymphopoiesis was analyzed by LAM-PCR. A small proportion of all peripheral blood cells in LV-injected mice consistently expressed GFP for up to one year (5-100 GFP+ cells per 20000 PB cells analyzed). Pre-treatment with 5-FU did not affect the percentage or lineage distribution of marked blood cells even when the vector was injected intravenously. Even though the initial percentage of marked cells was similar after IV and IF vector injection (p>0.05) the marking kinetics were different. Whereas the percentage of GFP expressing cells in PB of IF-marked mice remained stable over the whole observation period for up to 1 year, a 2-fold decline of the percentage of marked cells was detected two weeks after IV-marking indicating that predominantly short-lived more mature cells were transduced after IV vector injection. LAM-PCR analyses of sorted cell lineages showed that multiple clones contributed to the marked myeloid and lymphoid long-term hematopoiesis after IF-injection. In summary, our data demonstrate stable marking of steady-state hematopoiesis for up to one year. Our results demonstrate that remarkably stable stem cell clones with myeloid and lymphoid differentiation potential contribute to murine steady-state long-term hematopoiesis. In vivo marking will further allow to directly address the response of individual stem cell clones to hematopoietic stress including chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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