scholarly journals EPCR/PAR1 Signaling Navigates Long-Term Repopulating Hematopoietic Stem Cell Bone Marrow Homing to Thrombomodulin-Enriched Blood Vessels

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 33-33 ◽  
Author(s):  
Shiri Gur Cohen ◽  
Tomer Itkin ◽  
Sagarika Chakrabarty ◽  
Claudine Graf ◽  
Orit Kollet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Protein C ◽  
No Production ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Epcr Shedding

Abstract Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) is an active and essential first step in clinical stem cell transplantation. EPCR is expressed by murine BM LT-HSCs endowed with the highest repopulation potential and its ligand, activated protein C (aPC), has anticoagulant and anti-sepsis effects in EPCR+/PAR1+ endothelial cells. We recently found that signaling cascades, traditionally viewed as coagulation and inflammation related, also independently control EPCR+ LT-HSC BM retention and recruitment to the blood via distinct PAR1 mediated pathways. EPCR/PAR1 signaling retains LT-HSCs in the BM by restricting nitric oxide (NO) production and Cdc42 activity, promoting VLA4 affinity and adhesion. Conversely, thrombin/PAR1 signaling overcome EPCR+ LT-HSC BM retention by initiating NO production, leading to TACE-‎mediated EPCR shedding, CXCR4 and PAR1 upregulation and parallel CXCL12 secretion by PAR1+ BM stromal cells, enhancing stem cell migration and mobilization. Since EPCR shedding is essential for BM LT-HSC recruitment, we tested EPCR role in LT-HSC BM homing. EPCR+ LT-HSC exhibited reduced in vitro migration towards CXCL12 and enhanced CXCL12-dependent adhesion to fibronectin. Unexpectedly, transplanted EPCR+ LT-HSCs preferentially homed ‎to the host BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing antibody treatment, which blocks binding to aPC, leading to attenuated EPCR+ LT-HSC homing to the BM but not to the spleen. Importantly, short term aPC pretreatment inhibited NO production and dramatically increased EPCR+ LT-HSC BM homing. Since EPCR navigates LT-HSC to the BM, we studied the role of EPCR signaling in LT-HSC BM repopulation. Mimicking EPCR signaling by in vivo NO inhibition induced preferential expansion of blood and bone-forming stem cells and gave rise to higher donor type EPCR+ LT-HSCs in competitive repopulation assays. Similarly, repeated treatment with aPC expanded BM EPCR+ stem cells and increased competitive LT-repopulation. Importantly, loss of EPCR function reduced HSC long-term repopulation ability while maintaining their short-term repopulation activity. BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with normal stem cells in competitive long-term repopulation assays. Consistent with inferior HSC BM repopulation, Procrlow mice exhibited reduced numbers of BM LT-HSC with reduced adhesion capacity. Additionally, these mice displayed increased HSC frequencies in the blood circulation and the spleen, which were pharmacologically corrected by inhibiting NO generation with L-NAME treatment. BM retention is essential for quiescent HSC protection from chemotherapy. Mice treated with NO donor SNAP, or with blocking EPCR antibody as well as Fr2-/-mice lacking PAR1 expression, were more susceptible to hematological failure and mortality induced by 5-FU treatment compared to control mice. Together, these results indicate a functional aPC/EPCR/PAR1 signaling pathway, regulating EPCR+ LT-HSC BM homing, adhesion and long-term repopulation potential. The thrombin-thrombomodulin (TM) complex converts protein C to its activated form aPC, facilitating high affinity binding to its receptor EPCR. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by a unique BM endothelial cell (BMEC) subpopulation, but not in the spleen. Moreover, EPCR+ LT-HSCs were found adjacent to TM+/aPC+ BMECs, imposing their adhesion and retention. Interestingly, similar to BMECs, BM EPCR+ LT-HSC also express surface TM, implying the possibility of autocrine aPC generation. Herein we define EPCR as a guidance molecule, navigating slow migrating LT-HSC in the blood flow specifically to TM+ BMEC supporting niches, maintaining NOlow stem cell retention, long-term blood production and protection from myelotoxic insult. Conversely, thrombin/PAR1 signaling oppositely increase NO generation and EPCR shedding allowing increased CXCR4-dependent LT-HSC migration and mobilization. Harnessing EPCR signaling may improve clinical stem cell transplantation, increasing LT-HSC specific BM homing and repopulation by aPC pretreatment, as well as potentially to overcome malignant stem cell chemotherapy resistance. Disclosures No relevant conflicts of interest to declare.

EPCR Guides Hematopoietic Stem Cells Homing to the Bone Marrow Independently of Niche Clearance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4538-4538
Author(s):  
Shiri Gur-Cohen ◽  
Francesca Avemaria ◽  
Orit Kollet ◽  
Seymen Avci ◽  
Tomer Itkin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Protein C ◽  
No Production ◽  
Platelet Factor ◽  
Hematopoietic Stem

Abstract Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) are active and essential first steps during embryonic development and in clinical stem cell transplantation. Rare, BM LT-HSCs endowed with the highest self-renewal and durable repopulation potential, functionally express the anticoagulant endothelial protein C receptor (EPCR) and PAR1. In addition to coagulation and inflammation, EPCR-PAR1 signaling independently controls a BM LT-HSC retention-release switch via regulation of nitric oxide (NO) production within LT-HSCs. EPCR+ LT-HSCs are maintained in thrombomodulin+ (TM) periarterial BM microenvironments via production of activated protein C (aPC), the major ligand for EPCR. Restriction of NO production by aPC-EPCR-PAR1 signaling, activates VLA4-mediated adhesion, anchoring EPCR+ LT-HSCs to the BM and protecting them from chemotherapy insult, sparing hematological failure and premature death (Gur-Cohen S. et al, Nat Med 2015). We report that transplanted EPCR+ LT-HSCs preferentially homed ‎to and were retained in the BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing treatment that block aPC binding and attenuate EPCR+ LT-HSC BM homing. Furthermore, short term aPC in vitro pretreatment dramatically augmented EPCR+ LT-HSC BM homing, lodgment and long-term repopulation. PAR1 deficient stem cells were irresponsive to treatment with aPC and displayed reduced BM homing efficiency, all pointing to the aPC-EPCR-PAR1 axis as a crucial mediator of BM LT-HSC homing. Additionally, aPC pretreated EPCR+ LT-HSCs had a striking advantage to competitively home to the BM. Consistently, BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with wild type stem cells in competitive repopulation assays. Importantly, the competitive homing results strongly imply that the BM available niches for newly arrived EPCR+ LT-HSCs are limited. Indeed, aPC pretreated EPCR+ LT-HSCs BM homing reached a plateau, as increasing the transplanted cell dose above 5x106 BM mononuclear cells, did not yield higher donor EPCR+ LT-HSC homing. These results reveal that there is a limited BM space for newly arrived transplanted EPCR+ stem cells to non-irradiated hosts. Importantly, we found that EPCR+ LT-HSCs can engraft the BM of non-conditioned mice with high efficiency, while remaining in a dormant, non-cycling state. Furthermore, the dormant homed EPCR+ LT-HSCs were later awakened and activated solely by treating the engrafted hosts with a low dose 5-FU chemotherapy, or with NO donor SNAP, revealing that preconditioning and clearance of occupied BM HSC niches are not required. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by unique BM arterioles, and not in the spleen. BM homed EPCR+ LT-HSCs were found adjacent to TM+ arterioles, imposing their retention. Homed BM EPCR+ LT-HSCs highly express full-length TM with intact lectin-like domain, and the BM TM+ endothelium was found to be enriched with a Glycocalyx layer, in particular with Heparan Sulfate Proteoglycan-2 (HSPG-2). HSGP-2 might specifically interact with the lectin-like domain of TM-expressingLT-HSCs, providing BM specific recognition and accelerated homing. Intriguingly, stabilizing TM function by in vitro pretreatment with platelet factor-4 (PF4) bypassed BM-derived cues and increased EPCR+/TM+ LT-HSC homing also to the spleen, suggesting a supportive role for PF4, highly secreted by BM megakaryocytes, in guiding EPCR+/TM+ LT-HSCs to the BM. Herein we define EPCR as a guidance molecule, navigating LT-HSC specifically to BM TM+ aPC-secreting blood vessels, allowing stem cell retention and protection from DNA damaging agents. The BM harbors a limited number of available stem cell niches for newly arrived transplanted EPCR+/TM+ LT-HSCs, and in vitro aPC pretreatment dramatically augments EPCR+/TM+ LT-HSC BM homing. Our findings provide new mechanistic insights and identify key players concerning LT-HSC homing specifically to the BM, leading to better repopulation following transplantation. This up-to-date approach and new knowledge may potentially lead to improved BM transplantation protocols and to prevent chemotherapy resistance of EPCR-expressing cancer stem cell mediated relapse. Disclosures Ruf: Iconic Therapeutics: Consultancy.

EPCR Limits Nitric Oxide Levels, Mediating Human and Murine Stem Cell Adhesion and Retention In The Bone Marrow, By Conjugating PAR1 and CXCR4 Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 795-795
Author(s):  
Shiri Gur Cohen ◽  
Tomer Itkin ◽  
Aya Ludin ◽  
Sagarika Chakrabarty ◽  
Orit Kollet ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Protein C ◽  
Cxcr4 Signaling ◽  
Cell Mobilization ◽  
Epcr Shedding

Abstract Long term repopulating hematopoietic stem cells (LTR-HSC) in the murine bone marrow (BM) highly express endothelial protein C receptor (EPCR), yet the function of EPCR in HSC is incompletely defined. EPCR is expressed primarily on endothelial cells and has anti coagulation and anti inflammatory roles. While physiological stress due to injury or bleeding is a strong inducer of HSC mobilization and leukocyte production, a role for the coagulation protease thrombin, and its major receptor PAR1 in regulation of HSC has not yet been identified. We hypothesized that thrombin plays a role in HSC mobilization in the context of injury and that, conversely, signaling involving EPCR and its ligand activated protein C (aPC) play a regulatory role in HSC maintenance. Herein, we report that murine BM EPCRhigh stem cells display enhanced CXCL12 mediated adhesion and reduced migration capacitie, while motile circulating HSC in the murine blood and spleen lack high EPCR expression. Mechanistically, we found that EPCR is a negative regulator of nitric oxide (NO) levels. EPCRhigh stem cells display low intracellular NO levels, low motility, and increased adhesion to BM stroma. Furthermore, EPCRlow transgenic mouse cells displayed reduced stem cell adhesion to BM stroma and increased motility, manifested by reduced EPCRlow HSC in the BM and their corresponding increased levels in the blood. In vitro stimulation with the EPCR ligand, aPC, which we found to be physiologically expressed adjacent to small murine BM blood vessels, augmented EPCRhigh HSC adhesion and further limited their intracellular NO content by increasing eNOS phosphorylation at Thr495 in BM HSC, causing reduced production of NO. Conversely, administration of the pro-coagulant protease thrombin to mice induced PAR1 mediated EPCR shedding from BM HSC, followed by CXCR4 upregulation on HSC, and PAR1-mediated CXCL12 secretion by BM stromal cells. Together, these events lead to loss of retention and rapid stem cell mobilization to the blood. Interestingly, shedding of EPCR was found to be mediated by elevation of intracellular NO content, leading to EPCR co-localization with Caveolin. Correspondingly, thrombin failed to induce EPCR shedding and mobilization in eNOS and PAR1 deficient mice. Additionally, we found that BM LTR-HSC functionally express the metalloproteinase TACE (ADAM17) on the cell membrane, and that in- vitro inhibition of TACE activity by a newly developed selective inhibitor, reduces thrombin- mediated EPCR shedding, suggesting the involvement of TACE in EPCR shedding and HSC mobilization. Moreover, EPCR shedding was also CXCR4 dependent, revealing a crosstalk between EPCR, PAR1 and CXCR4. HSPC mobilized by thrombin possessed increased long-term repopulation capability following transplantation into lethally irradiated recipient mice and re-synthesis of EPCR by donor HSC in the engrafted host BM. In addition, EPCR expression was re-induced on circulating stem cells following in vitro treatment with eNOS inhibitor. Interestingly, bypassing eNOS by directly injecting NO donor, induced EPCR shedding, CXCR4 upregulation and rapid HSPC mobilization in both wild type and eNOS KO mice. Importantly, we found that similar to mice, EPCR was selectively and highly expressed by primitive human BM CD34+CD38- HSC, but not in the blood circulation of clinical G-CSF mobilized stem cells or in motile cord blood stem cells. Human BM CD34+/CD38- HSC are functionally EPCRhigh cells, maintaining low levels of intracellular NO which mediates their increased adhesion, while EPCR shedding was important for their migration and mobilization. In the functional pre-clinical NOD/SCID mouse model, G-CSF mobilization induced EPCR shedding, up-regulation of PAR1 and CXCR4 on human stem and progenitor cells, while NO signaling inhibition blocked G-CSF induced mobilization and increased both murine and human EPCRhigh stem cell accumulation in the murine BM. Our results define functional roles for EPCR, on both human and murine HSC, and suggest that regulation of EPCR expression is linked to NO, PAR1 and CXCR4 signaling as a pivotal mechanism determining HSC localization and function. Our study reveals that activation of coagulation in the context of cell injury controls stem cells retention and motility, and suggests that targeting this system may be useful in improving clinical stem cell mobilization and transplantation protocols. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment

Blood ◽  
2018 ◽  
Vol 132 (7) ◽  
pp. 735-749 ◽  
Author(s):  
Simranpreet Kaur ◽  
Liza J. Raggatt ◽  
Susan M. Millard ◽  
Andy C. Wu ◽  
Lena Batoon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Key Points ◽  
Bone Marrow Engraftment

Key Points Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation. Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Maintenance of Earlier Hematopoietic Stem Cell (HSC) Phenotype and Improved NOD/SCID Engraftment for UCB Expanded Short-Term in Cytokines over a Human Mesenchymal Stem Cell (huMSC) Platform.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2846-2846
Author(s):  
M. Kozik ◽  
J. Banks ◽  
L. Fanning ◽  
M. Finney ◽  
Y. Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Flow Analysis ◽  
Scid Mice ◽  
Mouse Bone Marrow ◽  
Short Term ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer: Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M) UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

Haplo-Identical Allogeneic Stem Cell Transplantation Using GCSF-Mobilized Marrow Plus Blood Stem Cells from Parent Donors for Children with High-Risk Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5084-5084
Author(s):  
Quanyi Lu ◽  
Xiaoqing Niu ◽  
Peng Zhang ◽  
Delong Liu
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
High Risk ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells

Abstract Increasing number of patients in China have difficulty of finding sibling donors due to limited number of siblings. We therefore explored the feasibility using haploidentical parent donors for allogeneic hematopoietic stem cell transplantation. Eight leukemia patients were studied in our hospital. These included 2 CML-BC, 2 MDS-RAEB, 3 relapsed ALL and 1 relapsed AML. The median age was 12 (7–17). GCSF- mobilized bone marrow and peripheral blood stem cells were collected from parents (1 to 3 locus mismatched). The conditioning regimen consisted of fludarabine (30mg/m2/d x5), bulsulfan (4mg/kg/d x3) and cyclophosphamide (50mg/kg/d x2). Cyclosporin A, mycophenolate mofetil, methotrexate, and ATG were used for GVHD prophylaxis. The total number of CD34+ cell in the grafts ranged between 5–10 x 106/kg. The median follow- up was 13 months (6–24). One patient failed to engraft, the other 7 patients achieved full donor chimerism at day 28. The incidence of acute GVHD (grade II-IV) was 57.1% (4 of 7). The incidence of chronic GVHD of limited stage occurred in the same 4 patients. One patient died of lung complication at 17th month, another patient with CML-BC relapsed 10 months after transplantation. The rest 6 patients are alive without disease. These results suggested that parents could be considered as stem cell donors in the absence of alternative donors for young patients with high-risk diseases. GCSF-primed bone marrow plus peripheral blood stem cells might be beneficial to reduce the risk of GVHD for leukemia children in China. More patients are needed to further study this approach.

G-CSF Treatment Induces Hematopoietic Stem Cell Quiescence but Loss of Repopulating Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1206-1206
Author(s):  
Joshua N. Borgerding ◽  
Priya Gopalan ◽  
Matthew Christopher ◽  
Daniel C. Link ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Inflammatory Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
A Cell

Abstract Abstract 1206 There is accumulating evidence that systemic signals, such as inflammatory cytokines, can affect hematopoietic stem cell (HSC) function. Granulocyte colony stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced in response to infection or inflammation. Additionally, G-CSF is the most commonly used agent for HSC mobilization prior to stem cell transplantation. Recently there has been a renewed interest in the use of “G-CSF primed bone marrow” for stem cell transplantation, so understanding the affect of G-CSF on bone marrow HSCs is clinically relevant. Because the G-CSF receptor is expressed on HSCs, and G-CSF creates biologically relevant modifications to the bone marrow microenvironment, we hypothesized that increased signaling through G-CSF may alter the repopulating and/or self-renewal properties of HSCs. Due to G-CSF's role as an HSC mobilizing agent, we predicted that the number of HSCs in the bone marrow would be reduced after 7 days of G-CSF treatment. Surprisingly, we observe that stem cell numbers markedly increase, regardless of which HSC-enriched population is analyzed. C-kit+lineage−sca+CD34− (KLS-34−), KLS CD41lowCD150+CD48− (KLS-SLAM), and KLS-SLAM CD34− increase by 6.97±2.25 fold, 1.79±0.29 fold, and 2.08±0.39 fold, respectively. To assess HSC repopulating activity, we conducted competitive bone marrow transplants. Donor mice were treated with or without G-CSF for 7 days, and bone marrow was transplanted in a 1:1 ratio with marrow from untreated competitors into lethally irradiated congenic recipients. Compared to untreated HSCs, we found that G-CSF treated cells have significantly impaired long-term repopulating and self-renewal activity in transplanted mice. In fact, on a per cell basis, the long-term repopulating activity of KLS-CD34− cells from G-CSF treated mice was reduced approximately 13 fold. The loss of repopulating activity per HSC was confirmed by transplanting purified HSCs. Homing experiments indicate that this loss of function is not caused by an inability to home from the peripheral blood to the bone marrow niche. As HSC quiescence has been positively associated with repopulating activity, we analyzed the cell cycle status over time of KLS-SLAM cells treated with G-CSF. This analysis revealed that after a brief period of enhanced cycling (69.8±5.0% G0 at baseline; down to 55.9±4.1% G0after 24 hours of G-CSF), treated cells become more quiescent (86.8±2.8% G0) than untreated HSCs. A similar increase in HSC quiescence was seen in KLS-34− cells. Thus our data show that G-CSF treatment is associated with HSC cycling alterations and function impairment. Because G-CSF is associated with modifications to the bone marrow microenvironment, and the microenvironment is known to regulate HSCs at steady state, we asked whether the G-CSF induced repopulating defect was due to a cell intrinsic or extrinsic (secondary to alterations in the microenvironment) mechanism. To do this, we repeated the competitive transplantation experiments using chimeric mice with a mixture of wild-type and G-CSF receptor knockout (Csf3r−/−) bone marrow cells. We find that only the repopulating activity of HSCs expressing the G-CSF receptor is affected by G-CSF, suggesting a cell-intrinsic mechanism. To identify targets of G-CSF signaling that may mediate loss of stem cell function, we performed RNA expression profiling of sorted KSL-SLAM cells from mice treated for 36 hours or seven days with or without G-CSF. The profiling data show that G-CSF treatment is associated with activation of inflammatory signaling in HSCs. Studies are in progress to test the hypothesis that activation of specific inflammatory signaling pathways mediates the inhibitory effect of G-CSF on HSC function. In summary, G-CSF signaling in HSCs, although associated with increased HSC quiescence, leads to a marked loss of long-term repopulating activity. These data suggest that long-term engraftment after transplantation of G-CSF-primed bone marrow may be reduced and requires careful follow-up. Disclosures: No relevant conflicts of interest to declare.

Cycling Marrow Stem Cells Are Lost with Purification.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2308-2308
Author(s):  
Laura R Goldberg ◽  
Mark S Dooner ◽  
Mandy Pereira ◽  
Michael DelTatto ◽  
Elaine Papa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tritiated Thymidine ◽  
Hematopoietic Stem ◽  
Cell Purification ◽  
Marrow Stem Cell ◽  
Marrow Cells

Abstract Abstract 2308 Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document