SDF-1 Acutely Promotes the Physical Association of Megakaryocytes with Vascular Endothelium in the Bone Marrow and Increases the Number of Circulating Platelets.

Abstract Abstract 2306 Thrombocytopenia complicates many diseases and can be a life-threatening consequence of genotoxic treatments including chemotherapy and radiation therapy. It is well established that thrombopoiesis occurs in the bone marrow where mature megakaryocyte (MK) precursor cells associate with sinusoidal endothelial cells and extrude pro-platelets into the vasculature. There has been much interest in elucidating mechanisms that control megakaryopoiesis and in utilizing these pathways to increase platelet output. The leading paradigm of megakaryopoiesis centers on the ability of cytokines, chiefly thrombopoietin (TPO), to promote MK progenitor proliferation and MK precursor maturation. More recently, attention has been focused on the ability of the bone marrow microenvironment to promote MK maturation and platelet formation. The chemokine stromal-derived factor-1 (SDF-1, also known as CXCL12), signaling through its receptor CXCR4, is implicated in the chemotaxis of MKs toward sinusoidal vessels, and in vivo evidence demonstrates that sustained plasma elevation of SDF-1 can increase platelet counts (Avecilla et al. Nature Medicine, 2004). To more specifically determine the short-term effects of SDF-1, we injected mice with a single 400ng intravenous dose of SDF-1 and enumerated the progenitor, precursor, and platelet compartments of the MK lineage. At 24 hours, SDF-1 induced a 30% increase in platelets compared to vehicle control (p<0.05). However, MK progenitors, defined functionally by the formation of acetylcholinesterase-positive colonies in vitro, and MK precursors, enumerated by imaging flow cytometry, were both unchanged (p>0.7 and p>0.5). To quantitatively determine if SDF-1 regulates the physical interactions of MK precursors with sinusoidal endothelium, we developed a double immunohistochemistry assay using Gp1Bβ to distinguish MK precursors and MECA32 to identify vascular endothelial cells. In vehicle-treated mice, 39% of MKs in the marrow localized to the sinusoidal endothelium, and this increased to 53% 24 hours following SDF-1 treatment (p<0.01). Thus, a single dose of SDF-1 acutely increases the number of MKs in the vascular niche as well as peripheral platelet counts. Given these results, we tested whether a single dose of SDF-1 could improve thrombocytopenia in the setting of radiation-induced marrow injury. Mice were treated with SDF-1 4 days after sublethal 4Gy total body irradiation (TBI), when radiosensitive MK progenitors are drastically reduced and radioresistant MK precursors and platelets are just beginning to decline. At 5 days post-TBI (24 hours post-SDF-1), SDF-1 treatment increased the number of circulating platelets by 15% (p<0.01) as well as the percentage of MK precursors in the vascular niche by over 15% (p<0.02) without changing the total number of MK progenitors or precursors in the marrow compared to irradiated vehicle controls (p>0.4 and p>0.7). As the platelet response following SDF-1 was less robust in the setting of TBI injury, we hypothesized that SDF-1-induced thrombopoiesis may improve if the number of MK precursors available to move to the vascular niche is increased. To test this, we administered TPO and SDF-1 at 2 hours and at 4 days, respectively, following TBI. TPO treatment alone resulted in 15% more MK precursors than irradiated vehicle controls at 5 days post-TBI (p<0.02), but did not significantly change the platelet count (p>0.2). In contrast, mice receiving both TPO and SDF-1 had over 20% more platelets than irradiated vehicle controls (p<0.01) and over 12% more platelets than mice receiving TPO alone (p<0.05) with the same increase in MK precursors (p>0.9). Correcting for differences in MK precursor numbers, mice receiving TPO and SDF-1 post-TBI had 1.8-fold more MKs in the vascular niche than irradiated vehicle controls (p<0.03) and 1.4-fold more than mice treated with TPO only (p<0.02). Taken together, we provide quantitative data in support of the concept that SDF-1 acutely promotes increases in the number of MK precursors in the vascular niche as well as peripheral platelet counts, and this effect correlates with the number of MK precursors in the marrow. Therapeutic approaches combining agents that first increase MK number and secondly increase MKs poised for thrombopoiesis by localization within the vascular niche may be a novel strategy to maximally increase peripheral platelet counts. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽

10.1182/blood-2019-122449 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2488-2488 ◽

Cited By ~ 1

Author(s):

José Gabriel Barcia Durán

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fluorescent Microscopy ◽

Hematopoietic Stem ◽

Interleukin Receptors ◽

Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Leukemia-Associated HSC Vascular Niche Is Negatively Regulated By PERK of Unfolded Protein Response (UPR)

Blood ◽

10.1182/blood-2019-127607 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2486-2486

Author(s):

Lan Zhou ◽

Cui Liu ◽

Stanley A Adoro ◽

Lechuang Chen ◽

Diana Ramirez ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Er Stress ◽

Leukemia Cell ◽

Unfolded Protein ◽

Vascular Niche ◽

Activating Transcription Factor ◽

Protein Response

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived from early T cell progenitors. Diffuse infiltration of the bone marrow by T-ALL is associated with worse prognosis. We previously reported that actively proliferating leukemia cells inhibit normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. We found that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. However, the underlying molecular mechanism that leads to the suppression of hematopoiesis and decreased HSPC in the vascular niche is unclear. It has been demonstrated that rapid cellular proliferation associated with oncogenic activity such as MYC in T-ALL leads to a global increase in protein synthesis and an increase in misfolded/unfolded polypeptides in the endoplasmic reticulum (ER), referred to as unfolded protein response (UPR) or ER stress. Elevated ER stress leads to activation of at least three types of ER stress transducers through the release of inhibitory binding by glucose-regulated chaperone protein (GRP78/BIP): the protein kinase RNA-like ER kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcription factor 6 (ATF6). Activation of PERK phosphorylates eIF2 to repress global translation with the exception of a small number of proteins including ATF4 (activating transcription factor-4). ATF4 regulates genes involved in restoring ER homeostasis and genes in apoptosis. Here, we studied the role of UPR in the regulation of HSC niche function in the setting of T-ALL progression. Using in vitro assays in which T-ALL leukemia cells driven by activated Notch1 (ICN1) were co-cultured with endothelial cells (MILE SVEN 1, MS1), and in vivo ICN1-driven T-ALL model, we found that PERK-eIF2a-ATF4 pathway was activated in both MS1 cells and BM endothelial cells isolated from T-ALL mice, while IRE1 and ATF6 pathways were only mildly altered. The activation of PERK was accompanied with the increased expression of Jagged1 and suppressed expression of CXCL12 in both cultured endothelial cells and bone marrow endothelial cells from leukemia mice. PERK inhibitor (GSK2606414) treatment of co-cultured cells largely restored CXCL12 expression, which was also negatively regulated by Jagged1, and accelerated the leukemia cell apoptosis as indicated by the enhanced annexin staining. These findings suggest that PERK is the upstream regulator of Jagged1 and CXCL12 in the endothelial cells; however, the function of cell-autonomous PERK on leukemia cell survival needs to be further clarified. To understand the role of PERK in bone marrow endothelium during leukemia development in vivo, we examined T-ALL leukemia progression and its effect on vascular niche function in VE-CadherinERT2/PERKF/F mice in which Perk was specifically deleted in endothelial cells. Consistent with in vitro findings, T-ALL development induced endothelial PERK-eIF2a-ATF4 activation, while up-regulated Jagged1 and down-regulated CXCL12 were also identified in isolated BM endothelial cells. Compared to the wild type mice, VE-CadherinERT2/PERKF/F mice showed attenuated leukemia progression, increased HSPC (Lin-Sca-1+c-kit+) frequency, and improved survival. Taken together, our findings suggest that PERK activation in BM endothelial cells is a key regulator of the leukemia vascular niche to promote leukemia progression and to suppress normal hematopoiesis. Therefore, targeting PERK may offer an effective strategy in restoring normal HSPC homeostasis and limiting leukemia progression. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Gabp Transcription Factor Is Required for the Development of Mouse Megakaryocytes.

Blood ◽

10.1182/blood.v116.21.2604.2604 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2604-2604

Author(s):

Zhong-Fa Yang ◽

Timothy M Chlon ◽

John Crispino ◽

Alan G. Rosmarin

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Fluorescent Protein ◽

Dna Ploidy ◽

Conflicts Of Interest ◽

Knock Out ◽

Megakaryocytic Differentiation ◽

Platelet Counts ◽

Redundant Role

Abstract Abstract 2604 GABP transcription factor has been implicated in the regulation of genes that are required for normal megakaryocytic differentiation. Megakaryocytes express several related ets factors, including Fli-1, ets2, and GABP, and it has been unclear if any single ets factor plays a non-redundant role in these cells. The tetrameric GABP transcription factor complex contains two molecules of GABPα, which binds DNA, and two molecules of GABPβ, which encodes the transcription activation domain. We created mice with loxP recombination sites which flank exons that encode Gabpa ets-related DNA-binding domains (floxed Gabpa, or Gabpa fl/fl), and bred them to mice that carry Mx1-Cre. In response to injection with the synthetic polynucleotide, pIC, these mice express Cre recombinase and efficiently delete Gabpa; these animals are referred to as knock-out (KO) mice. Control mice, which carry floxed Gabpa but lack Mx1-Cre, were treated identically with pIC. Platelet counts of KO mice declined to less than 50,000 within nine days, while platelet counts in control mice were unaffected. One half of KO mice died within two weeks of Gabpa deletion due to widespread visceral hemorrhage. Histologic examination of the bone marrow and spleen reveals a loss of megakaryocytes in KO mice, compared to control animals. Residual megakaryocytes in KO mice exhibit increased expression of platelet-specific antigens, CD41 and CD42, and a significant increase of DNA ploidy. Because Gabpa KO mice died with a striking loss of megakaryocytes and platelets, yet megakaryocytic differentiation appeared to be unimpaired, we sought to better define the nature of this defect. Bone marrow from Gabpa fl/fl mice was infected with a retrovirus that expresses Cre and green fluorescent protein (GFP), or control virus that expresses only GFP; grown for three days in liquid culture conditions that foster megakaryocytic differentiation; and analyzed for CD41 and CD42 expression, ploidy, and apoptosis. Gabpa was efficiently deleted by the Cre-bearing virus, and Gabpa deletion was associated with increased expression of CD41 and CD42, and increased DNA ploidy. However, Gabpa deletion was also associated with increased megakaryocytic-associated apoptosis, and in vitro megakaryocyte colony formation was dramatically reduced in Gabpα null cells. In summary, deletion of Gabpa in mice is associated with plummeting platelet counts, widespread visceral hemorrhage, and a loss of splenic and bone marrow megakaryocytes. In vitro analysis demonstrates intact megakaryocytic differentiation and a profound loss of megakaryocytic progenitor cells. The increased expression of megakaryocytic antigens and DNA ploidy may indicate that Gabpa deletion enhances megakaryocytic differentiation or, alternatively, it may represent selective loss of more immature megakaryocytic cells following Gabpa disruption. Data that directly test these alternative hypotheses will be presented. In summary, we demonstrate that GABP plays a non-redundant role in megakaryocyte development, that GABP is required for the proliferation of committed megakaryocytic progenitors, but that GABP is not required for the later stages of megakaryocytic maturation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽

10.1182/blood.v122.21.3696.3696 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3696-3696

Author(s):

Lindsay Wray ◽

Christian A Di Buduo ◽

David L. Kaplan ◽

Alessandra Balduini

Keyword(s):

Mechanical Properties ◽

Bone Marrow ◽

Extracellular Matrix ◽

Endothelial Cells ◽

Peripheral Blood ◽

Platelet Production ◽

Vascular Niche ◽

Proplatelet Formation ◽

Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Granulopoiesis in the Control of Hematopoietic Stem Cell Self-Renewal and Niches

Blood ◽

10.1182/blood-2018-99-109517 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-21-SCI-21

Author(s):

Daniel Lucas-Alcaraz

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

B Cell Differentiation ◽

Hematopoietic Stem ◽

Vascular Niche ◽

Reciprocal Transplants ◽

Response To Stress

Abstract The bone marrow (BM) vasculature is a critical component of the hematopoietic stem cell (HSC) niche. While the mechanisms through which the vasculature regulates HSC are intensively studied little is known about how this vascular niche is regulated and its function in supporting other aspects of hematopoiesis. We have recently shown that, after myeloablation, BM neutrophils are both sufficient and necessary for the regeneration of the vascular niche. This crosstalk is dependent of TNFα secretion by the neutrophil and TNFR1/TNFR2 expression in the stroma (Bowers et al., 2018). We now demonstrate that the crosstalk between neutrophils and the vasculature is bidirectional and that bone marrow endothelial cells provide a niche for fast neutrophil production in response to stress. Emergency granulopoiesis (EG) is the process through which the hematopoietic system generates large neutrophil numbers-at the expense of lymphocyte production-in response to inflammation or infection. A key EG driver is IL1. This cytokine can induce a myeloid bias on HSC and this is thought to be the main mechanism for IL1-driven EG. While characterizing the BM response to IL1 treatment we noted a fourfold reduction in erythropoiesis. Since erythroid- and myeloid- lineage cells differentiate from a common myeloid progenitor downstream of the HSC these results indicated that IL1 acted on other cells during EG. Differentiation analyses showed that IL1 blocks B cell differentiation at the common lymphoid progenitor and Pro-B to Pre-B transition stages whereas loss of erythropoiesis is detected at the pre-MegE stage and remains suppressed through terminal reticulocyte differentiation. IL1 signals, exclusively, through the IL1 receptor (IL1R1). Using reciprocal transplants and mixed chimera experiments we demonstrate that IL1R1 expression in hematopoietic cells is completely dispensable for IL1-driven neutrophil production. Instead, IL1 acts on stromal cells to increase neutrophil production and diminish lymphocyte and erythrocyte production. Our analyses revealed that, in the stroma, only LepR+ perivascular cells and endothelial cells express IL1R1. Conditional Il1r1 deletion in LepR+ cells did not abrogate any of the hematopoietic phenotypes. However, conditional Il1r1 deletion in endothelial cells completely abrogated neutrophil production but did not prevent loss of erythroid and lymphoid lineages. Differentiation analyses showed that, after exposure to IL1, endothelial cells force the differentiation of immature, unipotent, neutrophil progenitors into terminally differentiated neutrophils. These studies thus demonstrate that endothelial cells provide a niche that is indispensable for neutrophil production during EG. This endothelial niche also uncouples neutrophil production from suppression of other hematopoietic lineages opening the door to targeting this pathway to selectively regulate neutropoiesis. Finally these data also highlight the bidirectional communication between the vascular niche and bone marrow neutrophils during stress. Bowers E, Slaughter A, Frenette PS, Kuick R, Pello OM, Lucas D. Granulocyte-derived TNFα promotes vascular and hematopoietic regeneration in the bone marrow. Nat Med. 2018;24:95-102 Disclosures No relevant conflicts of interest to declare.

Download Full-text

Endothelial Jak3 expression enhances pro-hematopoietic angiocrine function in mice

Communications Biology ◽

10.1038/s42003-021-01846-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

José Gabriel Barcia Durán ◽

Tyler Lu ◽

Sean Houghton ◽

Fuqiang Geng ◽

Ryan Schreiner ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Cell Types ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Sinusoidal Endothelium ◽

Bone Marrow Vascular Niche ◽

Different Cell Types

AbstractJak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3–/– bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.

Download Full-text

Spatial and Temporal Fluctuations In Marrow SDF-1 Following Radiation Injury Regulate Megakaryocyte-Vascular Niche Interactions and Circulating Platelet Levels

Blood ◽

10.1182/blood.v122.21.568.568 ◽

2013 ◽

Vol 122 (21) ◽

pp. 568-568 ◽

Cited By ~ 1

Author(s):

Lisa M Niswander ◽

Katherine H Fegan ◽

Paul D Kingsley ◽

Kathleen E McGrath ◽

James Palis

Keyword(s):

Bone Marrow ◽

Spatial Distribution ◽

Conflicts Of Interest ◽

Therapeutic Interventions ◽

Post Injury ◽

Hematopoietic Stem ◽

Platelet Production ◽

Vascular Niche ◽

Endosteal Niche

Abstract The development of megakaryocytes (MKs) in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine SDF-1 (CXCL12), signaling through receptor CXCR4, is produced by stromal cell populations throughout the marrow and is implicated in the maturational chemotaxis of MKs to the sinusoids. Understanding the regulation of MK localization has significance not only for optimal platelet production and the development of therapies for thrombocytopenia, but also in light of the recently proposed role for MKs in supporting hematopoietic stem cells (Heazlewood et al. 2013). In the injury setting of lethal total body irradiation (TBI), it was observed that radioresistant mature MKs relocate to the endosteal niche (Dominici et al. 2009, Olson et al. 2013). Complicating the study of marrow niches post-TBI is the vascular dilation that accompanies the drastic loss of marrow cells. Having confirmed that MKs relocate to the endosteum in our model of sublethal radiation-induced thrombocytopenia (4Gy TBI), we asked whether this localization is due to changes in the spatial distribution of the vasculature or to altered microenvironmental SDF-1. In agreement with other TBI models, we find a significant elevation in SDF-1 transcript levels in the marrow at days 1-3 following 4Gy TBI. Radioresistant MKs, which do not decrease in number until after 3 days, have significantly increased CXCR4 surface expression, a finding we also observe following SDF-1 stimulation of MKs both in vitro and in vivo. In situ hybridization was used to localize the spatial distribution of SDF-1 RNA in femoral marrow. At 2 days post-4Gy, a significant SDF-1 gradient develops with 30% higher SDF-1 message adjacent to the endosteum than in the central marrow. However, this gradient is dynamically eliminated 24 hours later at 3 days post-TBI. These shifts in SDF-1 expression are accompanied by parallel changes in the spatial distribution of MKs by immunohistochemistry. At 2 days post-TBI, there is over a 40% increase in MK in the endosteal niche. In contrast, MKs in the endosteal niche decrease by more than 15% at 3 days, coincident with a significant increase in the MKs associated with vascular endothelium. Thus, these data suggest that the spatial distribution of MKs is dependent upon the localization of SDF-1 in the rapidly fluctuating post-injury bone marrow. To determine if SDF-1 functionally contributes to MK niche changes, we stabilized endogenously-produced SDF-1 using Diprotin A, an inhibitor of SDF-1-inactivating protease DPP4. In uninjured marrow, Diprotin A treatment causes over a 30% rise in MK association with vasculature and a 20% increase in circulating platelets 24 hours later, with no change in MK number. Elevation of vascular SDF-1 by intravenous (IV) administration yields similar results. These data indicate that an endogenous SDF-1 gradient toward the vasculature contributes to homeostatic megakaryopoiesis and thrombopoiesis. At 2 days post-TBI, when endosteal SDF-1 message is increased, stabilization with Diprotin A results in a 40% decrease in MKs associated with vasculature and a small but significant decrease in platelets 24 hours later. Further supporting a role for altered SDF-1 gradients, elevating vascular levels with IV SDF-1 at 2 days causes the opposite effect of Diprotin A, with more MKs found in the vascular niche and a rise in peripheral platelet count. In contrast, at 3 days post-TBI, stabilization of endogenous SDF-1 with Diprotin A causes a further 25% increase in MKs in the vascular niche and a 10% rise in circulating platelets, consistent with the rapid loss of the endosteal SDF-1 gradient. Taken together, our data demonstrate that changes in microenvironmental SDF-1 regulate the spatial distribution of MKs in the post-TBI bone marrow. Importantly, the observed SDF-1 changes have functional consequences for platelet production, as the movement of MKs toward the endosteum decreases circulating platelets, while MK association with the vasculature increases circulating platelets. This knowledge will ultimately lead to improved therapeutic strategies to enhance platelet output in the setting of thrombocytopenia and highlights the need to carefully optimize the timing of therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

A Human Bone Marrow-Derived Stromal Cell Population with Hemogenic Potential

Blood ◽

10.1182/blood.v126.23.1201.1201 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1201-1201

Author(s):

Saloomeh Mokhtari ◽

Evan Joseph Colletti ◽

Chad Sanada ◽

Zanetta S. Lamar ◽

Paul J Simmons ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Hemogenic Endothelium ◽

Hematopoietic Stem

Abstract During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Lineage Restricted JAK2V617F Expression and Its Role in Thrombosis and Hemostasis; The Potential Involvement of Endothelial JAK2V617F in Clotting Abnormalities

Blood ◽

10.1182/blood.v118.21.1737.1737 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1737-1737

Author(s):

Ian S Hitchcock ◽

Michelle E Roh ◽

Veena Sangkhae ◽

Leah Etheridge ◽

Rose Chen ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Conflicts Of Interest ◽

Artery Occlusion ◽

Bleeding Diathesis ◽

Flip Flop ◽

Cell Lineages ◽

Platelet Counts ◽

Significant Difference

Abstract Abstract 1737 Myeloproliferative disorders (MPDs) are a heterogeneous group of bone marrow disorders characterized by increases in one or more blood cell lineages. A single, somatic mutation in JAK2 (V617F) is responsible for many of the features of Philadelphia chromosome-negative MPDs (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis). Clinically, the most common cause of death in these patients is arterial thrombosis; however some patients display a bleeding diathesis. Although the incidence and potential causes of dysfunctional hemostasis in patients with MPDs has been studied extensively, the critical regulating factors are unclear and therefore it has been difficult to develop an effective therapeutic regimen for these complications. As the formation of stable thrombi requires interactions between endothelial cells, platelets and leukocytes, we have recently generated mice that express human JAK2V617F in each of these cell lineages by crossing a JAK2V617F/Flip-Flop (FF1) mouse with mice expressing lineage-specific Cre recombinases. These crosses have generated the following mice; 1) Tie2-Cre/FF1, where JAK2V617F is expressed in all hematopoietic and endothelial cells, 2) Pf4-Cre/FF1, where JAK2V617F expression is limited to platelets, and 3) LysM-Cre/FF1, where JAK2V617F expression is limited to leukocytes. Expression of human JAK2V617F was confirmed in megakaryocytes, platelets, leukocytes and endothelial cells (Tie2-Cre/FF1), megakaryocytes and platelets (Pf4-Cre/FF1) and leukocytes (LysM-Cre/FF1) by conventional and real-time PCR. Of the 3 mouse strains, only Tie2-Cre/FF1 exhibited a MPD phenotype. Platelet counts were significantly increased compared to Tie2-Cre controls (at 3 months, Tie2-Cre: 779 (±61)/ml;Tie2-Cre/FF1: 2943 (± 217)/ml) without significant increases in any other cells types. Tie2-Cre/FF1 mice also exhibit greatly increased number of CFU-MKs and bone marrow derived megakaryocytes. Therefore, Tie2-Cre/FF1 mouse exhibits an ET-like phenotype. Although circulating platelet counts did not increase in Pf4-Cre/FF1 mice, we did observe an increase in the number of CFU-MKs in colony assays. Next we determined the roles of the lineage-restricted JAK2V617F expression on hemostasis in vitro and in vivo. Aggregometry on washed platelets showed no significant difference between any group and their controls in response to PAR4 (100–400mM), ADP (2–20mM) or collagen (1–10mg/ml). Additionally, we were unable to show a significant difference in GPIIbIIIa activation or surface expression of P-selectin in response to the same agonists. Despite no clear platelet abnormalities in any of the 3 mouse lineages, we identified significant hemostatic abnormalities in vivo in Tie2-Cre/FF1 mice. Tail bleeding time was significantly increased in Tie2-Cre/FF1 mice compared to Tie2-Cre controls (Tie2-Cre average, 2min 47secs; Tie2-Cre/FF1, 6mins 37secs) while Tie2-Cre/FF1 mice also exhibited an increased occurrence of re-bleeding compared to Tie2-Cre controls. Additionally, we performed FeCl3 carotid artery occlusion assays to better determine in vivo thrombosis. We found that at 10% FeCl3, Tie2-Cre control mice exhibited complete artery occlusion in approximate 6 min. In contrast, Tie2-Cre/FF1 mice failed to show any sign of arterial occlusion throughout the duration of the experiment (30 min). Given the significant increase in platelet numbers in Tie2-Cre/FF1 mice, we next determined if acquired von Willibrand Disease (VWD) could account for prolonged bleeding and reduced clotting; plasma vW Factor levels by ELISA were normal. In contrast to Tie2-Cre/FF1 mice, neither the PF4-Cre/FF1 or LysM-Cre/FF1 mice exhibit dysfunctional thrombosis. These data provide compelling evidence that expression of JAK2V617F in cells other than just platelets or just leukocytes is necessary to generate the hemostatic abnormalities seen in patients with MPDs. Recent findings show that some patients express endothelial JAK2V617F and patients with ET exhibit increased numbers of circulating endothelial progenitors. Thus, our data is consistent with the hypothesis that expression of JAK2V617F in endothelial cells, in addition to hematopoietic cells results in the bleeding diathesis seen in patients with MPDs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text