Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1653-1653
Author(s):  
Nobuyuki Onai ◽  
Aya Onai ◽  
Markus G. Manz
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Type I Interferon ◽  
Receptor Expression ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions. To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity. In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.

scholarly journals Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

2021 ◽  
Author(s):  
Zixian Liu ◽  
Jinhong Wang ◽  
Miner Xie ◽  
Peng Wu ◽  
Yao Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Pcr Analysis ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Colony Assay ◽  
Megakaryocyte Progenitors ◽  
Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

scholarly journals Interleukin-10 inhibits the osteogenic activity of mouse bone marrow

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2361-2370 ◽  
Author(s):  
P Van Vlasselaer ◽  
B Borremans ◽  
R Van Den Heuvel ◽  
U Van Gorp ◽  
R de Waal Malefyt
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Interleukin 10 ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Mrna Levels ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Bone Proteins

Abstract Murine bone marrow cells synthesize bone proteins, including alkaline phosphatase (ALP), collagen type I, and osteocalcin, and form a mineralized extracellular matrix when cultured in the presence of beta- glycerophosphate and vitamin C. Interleukin-10 (IL-10) suppressed the synthesis of these bone proteins and mineralization without affecting cell proliferation. In addition, mRNA levels for the latter proteins were reduced in IL-10-treated cultures. This inhibitory effect was most outspoken when IL-10 was added before ALP activity peaked, eg, day 15 of culture. No significant effect was observed when IL-10 was added at later time points. This finding suggests that IL-10 acts at osteogenic differentiation stages that precede ALP expression but is ineffective on cells that progressed beyond this maturation stage. Likewise, IL-10 appeared to be unable to block both ALP activity and collagen synthesis in the preosteosteoblastic cell lines MN7 and MC3T3 that constitutively synthesize these proteins. Whereas IL-10 did not alter the number of fibroblast colony-forming cells of the marrow, it significantly reduced their osteogenic differentiation potential. In contrast to control cultures, IL-10-treated stroma was unable to either synthesize osteocalcin or to mineralize when subcultured over a 25-day period in the absence of IL-10. The inhibitory activity of IL-10 coincided with significant changes in stroma morphology. Whereas control cultures contained mainly flat adherent polygonal cells, significant numbers of rounded semiadherent to nonadherent cells were observed in the presence of IL-10. Scanning and transmission electron microscopy showed that, in contrast to control cultures, IL-10-treated stromas completely lacked a mineralized extracellular matrix. Collectively, these data suggest that IL-10 may have important regulatory effects on bone biology because of its capacity to downregulate early steps of osteogenic differentiation.

scholarly journals The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1543-1552 ◽  
Author(s):  
VF Quesniaux ◽  
S Wehrli ◽  
C Steiner ◽  
J Joergensen ◽  
HJ Schuurman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
T Cell ◽  
T Cell Response ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Hematopoietic Recovery ◽  
Stimulating Factor

Abstract The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.

Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4680-4686 ◽  
Author(s):  
Kent W. Christopherson ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mouse Bone Marrow ◽  
Peptidase Activity ◽  
Hematopoietic Stem ◽  
Migratory Response ◽  
Stromal Cell Derived Factor ◽  
Marrow Cells

AbstractCXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

Rapid and Irreversible Alteration of the Ability of Hematopoietic Stem Cells To Execute Both Symmetric and Asymmetric Self-Renewal Divisions by Exposure to Reduced Steel Factor Concentrations with No Effect on Their Survival or Mitogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 684-684
Author(s):  
David G. Kent ◽  
Brad Dykstra ◽  
Connie J. Eaves
Keyword(s):  
Bone Marrow ◽  
Adult Mouse ◽  
Single Cells ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
High Concentrations

Abstract Hematopoietic stem cells (HSCs) are present in the marrow of adult mice at a frequency of 1/104, as measured by limiting dilution transplantation assays for individual cells that produce lymphoid (B and T) as well as myeloid (GM) cells for at least 4 months in irradiated recipients. HSCs thus defined can be reproducibly isolated in the CD45midlin−Rho−SP fraction of adult mouse bone marrow at a purity of >30%. In mice, mutations in c-kit, the receptor for Steel factor (SF) lead to substantial reductions in the adult HSC population. In vitro, SF has been identified as a potent regulator of HSC self-renewal divisions. High concentrations of SF in combination with IL-11 allow adult HSCs to divide with a net 2–4 fold expansion in HSC numbers after 10 days and low concentrations of SF result in loss of HSC activity. To investigate the cellular mechanisms underlying these different outcomes, we cultured 114 CD45midlin−Rho−SP adult mouse bone marrow cells in single cell cultures containing serum-free medium + 20 ng/ml IL-11 and either 300 or 10 ng/ml of SF. Each culture was then examined every 4–6 hr. The kinetics of division of these cells under both conditions was identical with completion of the 1st division occurring between 22–68 hr. During that time none of the input cells died (<1%). After 10 days of culture, during which time all input cells divided at least 5 times (>50 cells), the HSC content of pooled clones (as measured by in vivo transplantation assays) was found to be >10-fold higher in the clones generated under high vs. low SF conditions (p<0.05). To characterize the types of self-renewal divisions undertaken, 9 doublets generated under the high SF condition were harvested between 4 and 8 hr after they underwent their 1st division and then each of the daughters was injected into a separate irradiated mouse. Analysis of the 18 mice showed that for one of the input cells both daughters were HSCs (evidence of a symmetric self-renewal division) and for 3 more, only one of the 2 daughters was an HSC (evidence of an asymmetric self-renewal division). In contrast no daughter HSCs were identified when 6 doublets produced under the low SF condition were assayed. To determine whether the loss of HSC activity under low SF conditions was a pre- or post-mitotic event, additional in vivo HSC assays were performed on cells harvested from individual wells after 8, 16 and 96 hours of incubation. The results revealed no change in the proportion of wells with either low or high concentrations of SF that contained HSCs after 8 hr of incubation (10/36 positive mice injected with starting single cells and 5/17 (low SF) vs. 6/17 (high SF) positive mice injected with 8-hr single cells, respectively). However, a significant difference (p<0.01) was seen after 96 hr (5/35 vs. 2/43 positive mice, respectively) and, after only 16 hr, before a first mitosis was seen under either condition, a decline in HSCs was apparent under the low SF condition (4/15 vs. 1/15 positive mice injected with cells from the high vs. low SF condition). Together, these studies indicate that HSC exposure to different SF concentrations can rapidly and irreversibly alter the ability of HSCs to execute symmetric as well asymmetric self-renewal divisions in vitro.

Candidemia-Induced Emergency Granulopoiesis Consists of Successive Dual Waves Triggered by the Shift From C/EBPalpha to C/EBPbeta Dependency.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3778-3778
Author(s):  
Sakiko Satake ◽  
Hideyo Hirai ◽  
Nobuaki Shime ◽  
Rina Nagao ◽  
Ruriko Tanaka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Steady State ◽  
Early Phase ◽  
Cathepsin G ◽  
Brdu Incorporation ◽  
Mouse Bone Marrow ◽  
Granulocytic Differentiation ◽  
Hematopoietic Stem ◽  
Factor C

Abstract Abstract 3778 Introduction: Granulocyte is a major cellular component in the front line of host defense. The number of granulocytes must be tightly tuned to meet the demand, because both the shortage and the excess of granulocytes can be harmful to the host. During emergency situations such as infections, granulocytes are replenished from peripheral pools and bone marrow production. As the half-life of granulocytes is quite short, granulopoiesis, de novo production of granulocytes in bone marrow, plays an important role during emergency. We have previously shown that granulopoiesis at steady state is largely dependent on a transcription factor, C/EBPalpha, whereas emergency granulopoiesis is dependent on C/EBPbeta (Hirai H, et al. Nature Immunol., 2006). However, the precise developmental stage where the shift from C/EBPalpha dependency to C/EBPbeta dependency takes place is almost unknown. The aim of this study is to dissect the process of granulopoiesis by a novel flow cytometric method and to elucidate the molecular mechanisms involved in the regulation of emergency granulopoiesis. Methods: 4 ≂ 106 cfu Candida albicans were intravenously injected to induce emergency granulopoiesis. Mouse bone marrow cells were harvested and stained with a combination of fluorescent-conjugated antibodies including anti-c-kit, anti-CD34, anti-Ly6G antibodies and markers for other lineages. Then the stained cells were analyzed or sorted by flow cytometry. After eliminating the cells which lost potential to give rise to granulocytes, the remaining cells were dissected into five subpopulations (#1≂ #5) according to the expression levels of c-kit and Ly6G. #1 is c-kithigh Ly6Glow cells, @ #2: c-kitint Ly6Glow, #5: c-kitlow Ly6Ghigh, and the cells residing between #2 and #5 are divided into #3 and #4. Cell number, gene expressions and cell cycle status of each population were analyzed before and after inducing emergency granulopoiesis. @ Results and Discussions: Wright-Giemsa staining and qRT-PCR for granule proteins (cathepsin G, myeloperoxidase, neutrophil elastase2, lactoferrin and MMP9) in each population indicated that lower c-kit expression and higher Ly6G expression correlated well with granulocytic differentiation and that the granulopoiesis progresses from # 1 to #5 in this order both at steady state and during emergencies (Figure 1). Then we applied this method to candidemia-induced emergency granulopoiesis. In vivo BrdU incorporation analysis showed immediate acceleration of the cell cycle in the most immature population (#1) and in one of the intermediate populations (#2). Chronological monitoring of each population after inducing candidemia revealed that rapid increase in mature granulocytes (#5) preceded the replenishment from the most immature population (#1). These results suggested that there are two distinct gwavesh in granulopoiesis at the early phase of infection, a rapid supply (first gwaveh) of granulocytes from relatively mature population (#2≂ #4), and a further and sustained supply (second gwaveh) originated from more immature populations (#1) including hematopoietic stem/progenitor cells (Figure 1). Transcripts of C/EBPalpha were significantly downregulated in #1≂ #4 at the early phase of infection, while those of C/EBPbeta were maintained in all the subpopulation (Figure 2), suggesting that shift from C/EBPalpha dependency to C/EBPbeta dependency took place at multiple developmental steps in granulopoiesis. C/EBPbeta has less inhibitory effects on cell cycle than C/EBPalpha while their abilities to induce granulocytic differentiation are similar (Hirai H, et al. Nature Immunol., 2006). The shift toward C/EBPbeta dependency may trigger the dual waves in emergency granulopoiesis, which demands both differentiation and proliferation of granulocytic precursors. Disclosures: No relevant conflicts of interest to declare.

Combination Therapy with Entinostat (MS-275) and GM-CSF to Enhance Differentiation In Myeloid Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3312-3312 ◽  
Author(s):  
Cho Eunpi ◽  
William Matsui ◽  
Jeanne Kowalski ◽  
Hua-Ling Tsai ◽  
Richard J. Jones ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Combination Therapy ◽  
Research Funding ◽  
Allogeneic Bone ◽  
Differentiation Potential ◽  
Appreciable Change ◽  
Platelet Counts ◽  
Gm Csf

Abstract Abstract 3312 Background: Histone actylases (HAC and histone deacetylases (HDAC) are two important enzymes in epigenetic control that can affect transcription of important regulatory transcription factors. Entinostat is a HDAC inhibitor that has been shown in vivo and in vitro to have anti-proliferative effects on many cancer cell types (Abujamra Leukemia Res 2009). When administered at low concentration to leukemic cell lines, entinostat induced p21-mediated growth arrest and expression of differentiation markers; higher concentrations led to marked increase in reactive oxygen species, mitochondrial damage, caspase activation and apoptosis (Rosato Cancer Res 2003). A Phase I study using entinostat as a single agent in relapsed and refractory leukemia showed in vivo differentiation potential with several patients showing significant increases in their mature granulocyte population and increased acetylation of the CD34+ blast population (Gojo Blood 2006). GM-CSF has been shown to enhance the differentiation potential of various agents such as interferon-alpha, all-trans-retinoic acid, bryostatin, and numerous other anti-neoplastic agents. The effects of combination therapy with GM-CSF and entinostat in patients with high-risk MDS or refractory and/or relapsed AML are presented here. Methods: A Phase II study was conducted to assess the safety and efficacy of combination therapy with GM-CSF and entinostat in patients with high-risk MDS and relapsed or refractory AML who are not eligible for allogeneic bone marrow transplant (BMT). The combination of entinostat and GM-CSF was administered in 6-week (42 day) cycles for at least 2 cycles. Entinostat was originally give at 8 mg/m2 weekly but was eventually adjusted to 4 mg/m2 weekly for the first 4 out of 6 weeks due to toxicity. GM-CSF was given at a single dose of 125 micrograms/m2/day for days 1–35 in the cycles 1, 2, 4 and 6 and days 1–42 in cycles 3 and 5. Patients who tolerated two cycles of 4 mg/m2 were assessed for response through measurements of peripheral blood, bone marrow aspirate and biopsies. Transfusion requirements and adverse events (AE) were recorded on all subjects throughout the study period. Clinical responses for AML and MDS were measured according to International Working Group definitions of complete response (CR), partial response (PR), stable disease (SD), hematologic improvement, and progressive disease (PD). Results: A total of 24 patients met the eligibility criteria for response assessment. Median age was 71 (range 52–84) years and 15 (63%) were male. Of the 19 patients with AML, 8 had relapsed/refractory disease, 7 had AML arising from MDS, 3 had therapy-related AML, and 1 had de novo AML. The remaining 5 patients had a primary diagnosis of MDS. 10 patients (42%) completed 2 or more cycles at the 4 or 6 mg/m2 dose of MS-275. These patients completed a total of 33 cycles, 1 resulting in CR, 4 in PR, 24 in SD, and 4 in PD. In addition to these standard endpoints, improvements were also noted in peripheral neutrophil counts (p<0.019) and platelet counts (p<0.001), without an appreciable change in blast count as a result of treatment (p<0.50). These results were achieved with few toxicities at the noted dosing. A total of 38 cycles at the 4-mg/m2-dose were analyzed for Grade 3 or 4 toxicities, which included febrile neutropenia (n=3), neutropenic infection (n=3), bone pain (n=2), fatigue (n=1), pericardial effusion (n=1), and weakness (n=1). Conclusion: Although treatment with entinostat and GM-CSF did not result in durable remissions, there were notable improvements in absolute neutrophil and platelet counts without negatively impacting the blast percentage. These findings suggests that therapy with entinostat and GM-CSF differentially promotes growth of mature myeloid cells and appears associated with better marrow function by minimizing the need for platelet transfusions. Such strategies may be most effective when applied to patients with low disease burdens or as maintenance therapy for patients with high risk disease in remission. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.

Human CD8+/TCR−/CD56dim/- Facilitating Cells Facilitate Homing and Engraftment of Human HSC in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4093-4093
Author(s):  
Yiming Huang ◽  
Mary J Elliott ◽  
Thomas Miller ◽  
Deborah R Corbin ◽  
Larry D. Bozulic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Mouse Bone Marrow ◽  
Common Procedure ◽  
Peripheral Blood Mononuclear ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Nsg Mice ◽  
Hsc Transplantation

Abstract Abstract 4093 Hematopoietic stem cell (HSC) transplantation has become a common procedure for treatment of hematopoietic malignancies and autoimmune disease. Despite significant advances in HSC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain limitations to application in the clinic. However, these risks can be overcome through less toxic nonmyeloablative conditioning and cell depletion strategies to remove GVHD causing-cells while retaining engraftment enhancing-tolerogeneic cells. We were the first to discover CD8+/TCR− graft facilitating cells (FC) in mouse bone marrow. The addition of as few as 30,000 FC to 10,000 HSC significantly enhances engraftment of HSC in allogeneic recipients without causing GVHD. FC also potently enhance engraftment of limiting numbers of syngeneic HSC. Human CD8+/TCR- FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (mPBMC). In the CD8+/TCR- FC, 48% of cells expressed CD3ε+, 43% were FoxP3+, 43% were CD11c+, 19% were CD19+, and 30% were HLA-DR+. Approximately 55% of FC are also CD56dim/-, and the remaining population is CD56bright. The morphology of human CD8+/TCR− FC with Wright-Giemsa staining under light microscopy suggested that the human FC population is heterogeneous. Here we evaluated if human FC enhance human HSC or progenitor homing to bone marrow of NOD/SCID/IL-2rγnull (NSG) mouse recipients. CD45+CD34+ HSC and CD8+/TCR−/CD56dim/-FC were sorted from mPBMC. NSG recipients were conditioned with 1100 cGy of total body irradiation (TBI). 24 hours after TBI, 100,000 HSC with or without 300,000 CD8+/TCR−/CD56dim/- FC were transplanted into conditioned NSG recipients. Recipients were euthanized 16 hours after transplantation. Bone marrow was harvested from femurs and tibias of recipients and plated in Colony Forming Culture (CFC) Assays. Recipients of HSC plus FC generated significantly more colony formation (colonies = 110) compared with HSC alone (colonies = 65) (P = 0.011), suggesting that CD8+/TCR−/CD56dim/- FC enhanced homing of HSC or progenitors to bone marrow. To test if human CD8+/TCR−/CD56dim/- FC facilitate engraftment of human HSC in NSG mice, 300,000 CD8+/TCR−/CD56dim/- FC were mixed with 100,000 HSC and transplanted into NSG recipient mice conditioned with 325 cGy TBI. Mice that received HSC alone served as controls. At 30 days after transplantation, PBL typing showed that 34% (10 of 29) recipients of HSC alone engrafted. In contrast, 78% of recipients (n = 23) of HSC plus CD8+/TCR−/CD56dim/- FC engrafted, and donor chimerism in PB was 1.1% ± 0.8% and 4.1% ± 1.3% (P <0.05), respectively. At 6 months after transplantation, NSG recipients of HSC + CD8+/TCR−CD56dim/- FC exhibited persistent donor chimerism in PB (9.1% ± 6% vs. 3.8% ± 3.5%) (P <0.05) and significantly higher levels of donor chimerism in spleen (26.3% ± 11.8% vs. 12.3% ± 9.8%) (P <0.05) and BM (11.6% ± 4.8% vs. 2.9% ± 1.3%) (P <0.05) compared to recipients of HSC alone. Our data indicate that CD8+/TCR−/CD56dim/- FC facilitate homing of human HSC or progenitors and enhance engraftment of human HSC in NSG recipient mice. Disclosures: Bozulic: Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1169-1169
Author(s):  
Maren Weisser ◽  
Kerstin B. Kaufmann ◽  
Tomer Itkin ◽  
Linping Chen-Wichmann ◽  
Tsvee Lapidot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Steady State ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Deficient Mice ◽  
Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

hnRNP K: A Tumor Suppressor or Oncogene?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 259-259
Author(s):  
Miguel Gallardo ◽  
Hun Ju Lee ◽  
Carlos E. Bueso-Ramos ◽  
Xiaorui Zhang ◽  
Laura R. Pageon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Conflicts Of Interest ◽  
Chromosome 9 ◽  
Wild Type ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Hnrnp K ◽  
Differentiation And Proliferation

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA and DNA binding protein that regulates critical pathways controlling differentiation and proliferation programs. While alterations in hnRNP K expression are associated with neoplastic malignancies, we currently do not understand how changes in hnRNP K expression contribute to tumor phenotypes in vivo. Previous biochemical and cell line studies demonstrate that hnRNP K transcriptionally regulates p53-dependent activities, suggesting it functions as a potential tumor suppressor. However, hnRNP K has also been shown to positively regulate c-Myc expression, indicating it may behave as an oncogene. The HNRNP K gene maps to a region of chromosome 9 (9q21.32), which is lost in a subset of patients with acute myeloid leukemia (AML). RNA expression analyses of patient samples with AML that harbor 9q21.32 deletions revealed a significant reduction in HNRNP K expression compared to wild type control samples, supporting the notion that hnRNP K acts as a tumor suppressor (Figure 1A). However, patients with AML who do not harbor a 9q21.32 deletion displayed a significant increase in hnRNP K expression (Figure 1A). Thus, to examine the association between altered hnRNP K expression and disease status in patients with AML, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells from 415 de novo AML patient as well as healthy donor controls. Interestingly, we observed a significant correlation between elevated hnRNP K levels and poor outcomes, which supports the idea that hnRNP K has oncogenic potential (Figure 1A). Together, these observations indicate that any change in hnRNP K expression may contribute to the etiology of AML and supports the idea that hnRNP K may potentially act as either a haploinsufficient tumor suppressor or oncogene in AML. To directly interrogate these possibilities in vivo, we generated mouse models that either harbor a deletion of one hnRNP K allele (hnRNP K+/-) or overexpressed hnRNP K (hnRNP KTg) in the hematological compartment. Western blot analyses demonstrated that hnRNP K haploinsufficiency results in a significant reduction in hnRNP K expression while tissue-specific activation of hnRNP K resulted in overexpression of hnRNP K. Similar to our observation in AML patients, either hnRNP K haploinsufficiency or overexpression resulted in similar phenotypes in vitro and in vivo. Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP K+/- and hnRNP KTg mice had significant increases in differentiation and proliferation potential as determined by colony formation assays. In these experiments, we observed a significant increase in the number of total colonies and number of cells per colony in both hnRNP K+/- and hnRNP KTg HSCs as compared to wild type HSCs (Figure 1B). In vivo analyses of the hnRNP K+/- and hnRNP KTg mice revealed a significant increase in myeloid hyperplasia in the peripheral blood and bone marrow, increased tumor formation, genomic instability, and decreased survival compared to wild type mice (Figure 1C). Interestingly, both increased and decreased hnRNP K expression resulted in alterations in similar pathways that regulate differentiation and proliferations potential (e.g.; p53 and c-Myc pathways and alterations in C/EBP expression). Together, these clinical and animal model studies illustrate that either over-expression or under-expression of hnRNP K lead to strikingly similar phenotypes that directly impact the etiology of AML. Furthermore, these data not only implicate that hnRNP K behaves as both a tumor suppressor and oncogene, but also suggest that it functions as a master toggle that dictates the proliferation and differentiation potential of HSCs. We are currently using Whole Transcriptome Shotgun Sequencing (RNA-Seq) and ChIP-Seq to evaluate the mechanisms by which increased and decreased hnRNP K expression impact hematologic malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document