IL3-Receptor Signaling Is Dispensable For The Generation and Maintenance Of Jak2V617F-Induced Myeloproliferative Neoplasm (MPN

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2841-2841
Author(s):  
Therese Vu ◽  
Claudia Bruedigam ◽  
Rebecca Austin ◽  
Catherine Paine-Kuhn ◽  
Hayley S. Ramshaw ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Count ◽  
White Cell Count ◽  
White Cell ◽  
Receptor Signaling ◽  
Short Term ◽  
Hematopoietic Stem

Abstract Myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and primary myelofibrosis are clonal diseases driven by acquired somatic mutations in hematopoietic stem cells (HSCs), the most common of which is JAK2V617F. JAK2V617F leads to cytokine hypersensitivity and activation of JAK-STAT signaling in the presence of an erythropoietin (EPO), thrombopoietin (TPO) or interleukin-3 (IL3) cytokine receptor scaffold (Nature 2005; 434:1144-8, Leukemia 2008; 22:1828-40). Physiological Jak2V617F expression in long-term hematopoietic stem cells (LT-HSCs; lineagelowcKit+Sca1+CD150+CD48-) is necessary and sufficient to generate MPN, and these LT-HSCs contain the sole reservoir of Jak2V617F MPN-initiating stem cells (Cancer Cell 2010; 17:584-96; Blood 2012; 120:166-72). Although Jak2V617F-mediated EPO hypersensitivity drives erythroid expansion and in vitro EPO-independent colony formation, the EPO receptor is not expressed on Jak2V617F HSC populations (Blood 2012; 120:166-72). This suggests that hypersensitivity to IL3 and/or TPO signaling is responsible for driving LT-HSC proliferation and survival in vivo. To determine the respective contributions of IL3 and TPO signaling to Jak2V617F-induced MPN, pStat5 was measured in HSC and progenitor populations from E2ACre+Jak2V617F+/- knockin mice (hereafter Jak2VF) after stimulation with rmIL3 or rmTPO. Committed myeloid progenitors showed robust pStat5 stimulation with rmIL3, but only low level stimulation with rmTPO (TPO 460.25 ± 25.02 MFI vs. IL3 598.25 ± 69.05 MFI, p<0.05, Vehicle 362.25 ± 76.66 MFI). In contrast, LT-HSCs showed strong induction of pStat5 signaling with rmTPO stimulation but less so with rmIL3 stimulation (TPO 571 ± 47.36 MFI vs. IL3 487.5 ± 53.69 MFI, p=0.05, Vehicle 249.5 ± 47.63 MFI). Concordant with these results, TPO signaling appears essential for the generation of Jak2V617F-induced MPN (ASH 2012 Abstract 427). To determine the contribution of IL3 receptor signaling in Jak2V617F-induced MPN, we crossed Jak2VF knockin mice with mice lacking the common beta subunit of the IL3 receptor that is responsible for signal transduction of IL3, IL5 and GM-CSF (Jak2VFIL3Rb-/-). Jak2VFIL3Rb-/- mice developed MPN with similar latency and mortality to Jak2VF controls. There were no differences in peripheral blood white cell count (16.51 ± 1.5×109/L vs. 14.89 ± 2.4×109/L, p=0.39, n=3), haematocrit (67.97 ± 7.85×109/L vs. 61.97 ± 5.73×109/L, p=0.35, n=3) or extramedullary erythropoiesis (spleen weight, 665 ± 107mg vs. 541 ± 106mg, p=0.22, n=3) between Jak2VFIL3Rb-/- and Jak2VF mice respectively. In competitive bone marrow transplantation assays, all recipients of Jak2VFIL3Rb-/- or Jak2VF bone marrow developed MPN with similar diagnostic parameters such as elevated white cell count (14.29 ± 3.41×109/L vs. 18.38 ± 5.78×109/L, p=0.11, n=8), hematocrit (63.4 ± 16.5% vs. 50.5 ± 13%, p=0.16, n=8) and splenomegaly (529 ± 86mg vs. 465 ± 119mg, p=0.23, n=8) in Jak2VFIL3Rb-/- and Jak2VF mice respectively. Jak2VFIL3Rb-/- bone marrow cells initially showed reduced short-term (4 weeks) engraftment and white cell count in recipients compared to the Jak2V617F group (p<0.0005). However after 16 weeks post-transplant there was no difference in chimerism between recipients of Jak2VFIL3Rb-/- or JakVF cells. IL3 was unable to stimulate pStat5 in Jak2VFIL3Rb-/- LT-HSCs or progenitors, but was preserved in Jak2VF LT-HSCs and progenitors. These data show that IL3Rb signaling is dispensable for Jak2V617F-induced MPN and LT-HSC function, however may regulate short-term myeloid progenitor cell expansion. TPO signaling appears preferentially important for Jak2VF LT-HSC pStat5 induction, whereas IL3 is more important for pStat5 induction in progenitor cells. These findings will help to inform strategies aimed at targeting long term Jak2V617F-initiating HSC populations. Disclosures: No relevant conflicts of interest to declare.

Identification of Lin–Sca1+kit+CD34+Flt3– short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2717-2723 ◽  
Author(s):  
Liping Yang ◽  
David Bryder ◽  
Jörgen Adolfsson ◽  
Jens Nygren ◽  
Robert Månsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Transplant Recipients ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Regulatory Pathways ◽  
Stem And Progenitor Cells ◽  
Bone Marrow Ablation

AbstractIn clinical bone marrow transplantation, the severe cytopenias induced by bone marrow ablation translate into high risks of developing fatal infections and bleedings, until transplanted hematopoietic stem and progenitor cells have replaced sufficient myeloerythroid offspring. Although adult long-term hematopoietic stem cells (LT-HSCs) are absolutely required and at the single-cell level sufficient for sustained reconstitution of all blood cell lineages, they have been suggested to be less efficient at rapidly reconstituting the hematopoietic system and rescuing myeloablated recipients. Such a function has been proposed to rather be mediated by less well-defined short-term hematopoietic stem cells (ST-HSCs). Herein, we demonstrate that Lin–Sca1+kithiCD34+ short-term reconstituting cells contain 2 phenotypically and functionally distinct subpopulations: Lin–Sca1+kithiCD34+flt3– cells fulfilling all criteria of ST-HSCs, capable of rapidly reconstituting myelopoiesis, rescuing myeloablated mice, and generating Lin–Sca1+kithiCD34+flt3+ cells, responsible primarily for rapid lymphoid reconstitution. Representing the first commitment steps from Lin–Sca1+kithi CD34–flt3– LT-HSCs, their identification will greatly facilitate delineation of regulatory pathways controlling HSC fate decisions and identification of human ST-HSCs responsible for rapid reconstitution following HSC transplantations.

Bone Marrow Osteoblast Ablation Enhances Short-Term Hematopoietic Stem Cells Without Altering Long-Term Hematopoietic Stem Cell Populations and Accelerates Leukemia Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 586-586
Author(s):  
Marisa Bowers ◽  
YinWei Ho ◽  
Ravi Bhatia
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Lower Extremity ◽  
Hematopoietic Stem Cells ◽  
Myelogenous Leukemia ◽  
Short Term ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) within the bone marrow (BM) microenvironment reside in close proximity to endosteal osteoblasts (OBs). Although OBs have been considered to provide a HSC niche, other studies suggest that perivascular mesenchymal cells or endothelial cells may be the primary HSC niches, and the specific role of OBs in regulation of HSCs requires further clarification. Moreover, the role of OBs in regulating leukemic stem cells (LSC) is even less well studied. To address these questions, we used a conditional OB ablation mouse model (Col2.3Δtk) in which a truncated version of the herpes simplex virus thymidine kinase (Δtk) is expressed under an OB-specific promoter. In these mice, daily intraperitoneal (IP) administration of ganciclovir (GCV) leads to production of a toxic DNA base analogue in OBs, resulting in their death. We crossed Col2.3Δtk mice with Col2.3GFP mice that specifically express GFP in OBs to facilitate assessment of OB ablation. We confirmed that 4 weeks of GCV administration resulted in ablation of endosteal OBs in this model using both immunofluorescence microscopy and flow cytometry analysis. OB ablation was associated with reduced BM cellularity (Δtk+ 3.7e7±3.0e6, Δtk- 4.8e7±3.8e6 per 4 lower extremity bones, p=0.04), but did not alter spleen (SP) cellularity (Δtk+ 5.1e7±5.3e6, Δtk- 6.3e7±7.4e6 cells per SP, p=0.19). OB ablation was also associated with significantly increased numbers of cells with long-term HSC (LTHSC) phenotype (Lin-Kit+Sca-1+Flt3-CD150+CD48-) in both the BM (Δtk+ 6490±1315, Δtk- 4236±922 per 4 lower extremity bones; p=0.03) and SP (Δtk+ 980±473, Δtk- 96±40 per SP; p=0.04). Significant increases in common myeloid progenitor (CMP) (Δtk+ 145114±43608, Δtk- 82200±26754; p=0.002) and granulocyte/monocyte progenitor (GMP) (Δtk+ 51411±17349, Δtk- 20206±9279, p=0.003, p=0.02) numbers were seen in SP of OB-ablated mice, whereas significant alterations in other hematopoietic populations in BM, SP or PB were not seen. We performed limiting-dilution competitive repopulation assays to determine the functional LTHSC potential of BM cells from OB-ablated and control mice. OB-ablated mice demonstrated a higher frequency of short-term repopulating cells compared to LTHSCs from non-ablated mice (5 weeks: Δtk+ 1 in 4,941; Δtk- 1 in 17,351 BM cells) but similar long-term engraftment (15 weeks: Δtk+ 1 in 22,853; Δtk- 1 in 23,137 BM cells). Transplantation of BM cells from primary transplant recipients into secondary recipients demonstrated similar long-term engraftment potential after second transplant. These results suggest that despite increased numbers of phenotypic LTHSCs in OB-ablated mice, the long-term repopulating and self-renewing capacity of BM cells remains unchanged in OB-ablated mice, but on the other hand there is an increase in functional short-term repopulating capacity. Next, to examine the role of OBs in regulation of Chronic Myelogenous Leukemia (CML) stem cells, we crossed the Col2.3GFPΔtk mice with an inducible transgenic BCR-ABL mouse model of CML (ScltTA-BCR/ABL). In these mice withdrawal of tetracycline results in induction of BCR-ABL expression in HSCs and development of a CML-like myeloproliferative disorder. GCV administration to achieve OB ablation was initiated one week prior to BCR-ABL induction by tetracycline withdrawal, and was continued for the duration of the experiment. CML development was monitored by checking blood counts every 2 weeks after induction and mice were followed for survival. We observed significantly accelerated development of CML in OB-ablated versus non-ablated mice, with 50% of the OB-ablated mice dying within 47 days of CML induction, whereas >50% of the non-ablated mice survived to day 73 (p=0.017). Collectively, these studies suggest that BM OBs are not essential for maintenance of long-term repopulating and self-renewing HSCs, but regulate the expansion of short-term HSCs in the BM. Our studies also indicate an important and previously unrecognized role for OBs in regulating the leukemogenicity of CML LSCs. Disclosures: No relevant conflicts of interest to declare.

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

1987 ◽  
Vol 5 (3) ◽  
pp. 231-241 ◽  
Author(s):  
Vincent S. Gallicchio ◽  
Thomas D. Watts ◽  
George P. Casale ◽  
Philip M. Bartholomew
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Short Term ◽  
Hematopoietic Stem

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Robo4 Plays a Role in Bone Marrow Homing and Mobilization, but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e50849 ◽  
Author(s):  
Yuko Goto-Koshino ◽  
Yumi Fukuchi ◽  
Fumi Shibata ◽  
Daichi Abe ◽  
Kana Kuroda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1442-1449 ◽  
Author(s):  
CM Verfaillie ◽  
JS Miller
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Antigen Expression ◽  
Hematopoietic Stem ◽  
Interleukin 3 ◽  
Inflammatory Protein ◽  
Hla Dr ◽  
Macrophage Inflammatory Protein

Abstract Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

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