Sulfated Colominic Acid Enhances CXCR4 Function and Promotes Hematopoietic Stem /Progenitor Egress from Bone Marrow.

Abstract Hematopoietic progenitors (HPCs) can be mobilized from bone marrow (BM) compartment to the blood by G-CSF. In this process, CXCR4, a cognitive receptor for chemokine SDF-1/CXCL12, and CD26/dipeptidyl peptidase (DPP) IV play critical roles. Colominic acid (CA) is a polymer of N-acetylneuraminic acid, and it has been reported that sulfated colominic acid (SCA) can inhibit HIV entry, the step which requires CXCR4 and CD26 as co-receptors. Thus, we hypothesized that SCA would modulate HPC egress from BM. First, we injected SCA into C57BL/6 mice (100mg/kg, i.v.) and observed rapid HPC mobilization (assessed by CFU-Cs/mL blood, PBS 42±10, SCA 215±50, n=9–12, p<0.01, peaked at 30 min after injection). CA displayed no mobilization, suggesting that the sulfation is critical for its action. G-CSF-induced HPC mobilization was strongly enhanced by the addition of single dose of SCA (CFU-Cs/ml blood, PBS vs SCA: 225±57 vs 1950±485 in 2 day-G-CSF, n=5, p<0.01; 1950±400 vs 8233±1225 in 4 day-G-CSF, respectively, n=6, p<0.001). To evaluate the stem cell activity in mobilized blood by 2 day-G-CSF+SCA, we mixed the same volume of blood from CD45.2 mice treated with 2 day-G-CSF+SCA and CD45.1 mice treated with 4 day-G-CSF, and injected into lethally irradiated CD45.1 mice. The ratio of CFU-C injected (2 day-G-CSF+SCA: 4 day-G-CSF) was 1.12±0.03 and the ratio of peripheral blood leukocytes derived from engrafted stem cells originated from each donor 12 weeks after transplantation was 2.16±0.54, n=3, p=0.11). These results suggest that 2 day administration of G-CSF together with a single dose of SCA may be sufficient for clinical mobilization and that the addition of SCA in full-term G-CSF administration may overcome poor mobilization cases. To elucidate the mechanism of SCA action on HPCs, we first focused on CD26/DPPIV. Pretreatment of mice with a DPPIV inhibitor Diprotin A strongly inhibited the increase of leukocytes but did not alter HPC mobilization. In addition, SCA-induced mobilization in CD26-deficient mice was similar to that in wild-type (WT) controls (WT vs CD26−/−: 356±80 vs 444±86 CFU-Cs/ml blood, n=4). These results suggest that, in contrast to G-CSF-induced mobilization, CD26 is not important for SCA action. We then next evaluated the functional alteration of CXCR4 by SCA. Flow cytometric analyses revealed that SDF-1 bound more efficiently (2.5 times) to HPC cell line FDCP-mix treated with SCA compared to those treated with PBS (n=3, p<0.01). Strikingly, SCA treatment strongly (4.3 times) enhanced FDCP-mix transwell migration toward SDF-1 (n=8, p<0.05) which was completely blocked by a CXCR4 inhibitor. SDF-1 ELISA with K15C as a capture antibody revealed that non-truncated (active) form of SDF-1 was increased 5 times in serum after SCA injection (n=5, p<0.0001) whereas no change was observed in BM extracellular fluid. Together, we propose that SCA induces HPC mobilization by enhancing CXCR4 function and by altering the gradient of its ligand, active SDF-1, toward circulation.

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CFU-GM content of bone marrow graft correlates with time to hematologic reconstitution following autologous bone marrow transplantation with 4- hydroperoxycyclophosphamide-purged bone marrow

Blood ◽

10.1182/blood.v70.1.271.bloodjournal701271 ◽

1987 ◽

Vol 70 (1) ◽

pp. 271-275

Author(s):

SD Rowley ◽

M Zuehlsdorf ◽

HG Braine ◽

OM Colvin ◽

J Davis ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Autologous Bone Marrow Transplantation ◽

Autologous Bone Marrow ◽

Marrow Graft ◽

Blood Leukocytes ◽

Cell Content ◽

Hematopoietic Stem ◽

Bone Marrow Graft ◽

Autologous Bone

Autologous bone marrow transplants (BMTs) can repopulate the hematologic system of patients treated with marrow-ablative chemotherapy and/or radiotherapy. However, treatment of the bone marrow graft to eliminate residual tumor cells prior to reinfusion can delay the return of peripheral blood elements, presumably from damage to or loss of hematopoietic stem cells responsible for hematologic recovery. To develop a model predictive of hematologic recovery, we studied the progenitor cell contents of 4-hydroperoxycyclophosphamide (100 micrograms/mL)-purged bone marrow grafts of 40 consecutive patients undergoing autologous BMT at this center. Granulocyte-macrophage colonies (CFU-GM) were grown from all grafts after treatment with this chemotherapeutic agent, but erythroid (BFU-E) and mixed (CFU-GEMM) colonies were grown from only 44% and 33% of the grafts respectively. The recovery of CFU-GM after purging ranged from 0.07% to 23%. The logarithm of CFU-GM content of the treated grafts was linearly correlated with the time to recovery of peripheral blood leukocytes (r = -0.80), neutrophils (r = -0.79), reticulocytes (r = -0.60), and platelets (r = -0.66). The CFU-GM content of purged autologous bone marrow grafts may reflect the hematopoietic stem cell content of the grafts and thus predict the rate of hematologic recovery in patients undergoing autologous BMT.

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Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.

Journal of Experimental Medicine ◽

10.1084/jem.181.1.369 ◽

1995 ◽

Vol 181 (1) ◽

pp. 369-374 ◽

Cited By ~ 26

Author(s):

K J Grzegorzewski ◽

K L Komschlies ◽

S E Jacobsen ◽

F W Ruscetti ◽

J R Keller ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Peripheral Blood Leukocytes ◽

Gene Modification ◽

Blood Leukocytes ◽

Hematopoietic Stem ◽

Interleukin 7 ◽

Cell Repopulation

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

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Alterations In the Bone Marrow Microenvironment Contribute to Oxidative Stress and DNA Damage In Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation

Blood ◽

10.1182/blood.v116.21.387.387 ◽

2010 ◽

Vol 116 (21) ◽

pp. 387-387

Author(s):

Ghada M Kunter ◽

Jill Woloszynek ◽

Daniel C. Link

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Dna Damage ◽

Single Dose ◽

Bone Marrow Failure ◽

Bone Marrow Microenvironment ◽

Wild Type ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Increased Risk

Abstract Abstract 387 A shared feature of many bone marrow failure syndromes is their propensity to develop myelodysplasia (MDS) or acute myeloid leukemia (AML). The molecular mechanisms that underlie this susceptibility are largely unknown. Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a marked increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), that truncate the carboxy-terminal tail are associated with the development of MDS/AML in SCN. Transgenic mice carrying a ‘knock-in’ mutation of their Csf3r (termed d715 G-CSFR) reproducing a mutation found in a patient with SCN have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We previously reported that the d715 G-CSFR is able to cooperate with the PML-RARƒÑ oncogene to induce AML in mice. Herein, we summarize data supporting the hypothesis that alterations in the bone marrow microenvironment induced by G-CSF contribute to oxidative DNA damage in hematopoietic stem/progenitors cells (HSPCs) and possibly leukemic transformation. We previously showed that G-CSF treatment is associated with a marked loss of osteoblasts in the bone marrow, thereby potentially disrupting the osteoblast stem cell niche (Semerad, Blood 2005). Of note, patients with SCN chronically treated with G-CSF are prone to develop osteopenia, suggesting that osteoblast suppression by G-CSF also may occur in humans. We first asked whether the d715 G-CSFR was able to mediate this response. Wild-type or d715 G-CSFR were treated with G-CSF for 1–7 days and osteoblast activity in the bone marrow measured by expression of CXCL12 and osteocalcin. Consistent with previous reports, a decrease in osteocalcin and CXCL12 was not apparent until after 3 days of G-CSF treatment and reached a maximum after 7 days. Surprisingly, the magnitude of osteoblast suppression was greater in d715 G-CSFR compared with wild-type mice. The fold-decrease in osteocalcin mRNA from baseline in wild-type mice was 147 ± 70.1 versus 1,513 ± 1091 in d715 G-CSFR mice (p < 0.001). Likewise, a greater fold-decrease in CXCL12 mRNA was observed. We next assessed oxidative stress in c-KIT+ Sca+ lineage− (KSL) progenitors after G-CSF treatment. In both wild-type and d715 G-CSFR KSL cells no increase in reactive oxygen species (ROS) was observed at baseline or 12 hours after a single dose of G-CSF. However, after 7 days of G-CSF, a significant increase (3.4 ± 0.1 fold; p = 0.009) in ROS was observed in d715 G-CSFR but not wild-type KSL cells. To determine whether oxidative stress contributed to DNA damage, histone H2AX phosphorylation (pH2AX) was measured by flow cytometry. No increase in pH2AX was observed after short-term (less than 24 hour) G-CSF treatment. However, a modest but significant (1.9 ± 0.1 fold; p = 0.0007) increase in pH2AX was observed in d715 G-CSFR but not wild-type KSL cells after 7 days of G-CSF. To determine whether increased oxidative stress was casually linked to DNA damage, we co-administered the antioxidant N-acetyl cysteine (NAC) during G-CSF treatment. As expected, induction of ROS in KSL cells was markedly suppressed by NAC administration. Importantly, the increase in pH2AX levels in d715 G-CSFR KSL cells induced by G-CSF was completely blocked by NAC administration. Finally, to determine whether alterations in the bone marrow microenvironment, specifically decreased CXCL12 expression, contributed to DNA damage, we treated mice with AMD3100, a specific antagonist of CXCR4 (the major receptor for CXCL12). Treatment of wild-type or d715 G-CSFR mice with a single dose of G-CSF (3 hour time point) or with AMD3100 alone did not induce H2AXp. However, co-administration of AMD3100 with a single dose of G-CSF induced modest but significant H2AXp in d715 G-CSFR KSL cells (5.74 ± 1.06 fold; P<0.001). Collectively, these data suggest a model in which alterations in the bone marrow microenvironment induced by G-CSF may contribute to genetic instability in HSPCs and ultimately leukemic transformation. The mutant CSF3R may contribute to leukemogenesis through both increased ROS production in HSPCs and increased suppression of osteoblasts. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.363.363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 363-363

Author(s):

Jing Qi ◽

Qi Cai ◽

Sandeep Singh ◽

Ling Li ◽

Hongjun Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Cell Activity ◽

Disease Free Survival ◽

Hematopoietic Stem ◽

The Impact ◽

Acute Myeloid

Abstract The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs. In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. Disclosures No relevant conflicts of interest to declare.

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Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2665 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2665-2671 ◽

Cited By ~ 26

Author(s):

G F Tidmarsh ◽

S Heimfeld ◽

C A Whitlock ◽

I L Weissman ◽

C E Müller-Sieburg

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Cell Activity ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Low Levels ◽

B Lineage ◽

Marrow Cells

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.

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Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Blood ◽

10.1182/blood.v84.2.421.bloodjournal842421 ◽

1994 ◽

Vol 84 (2) ◽

pp. 421-432 ◽

Cited By ~ 4

Author(s):

D DiGiusto ◽

S Chen ◽

J Combs ◽

S Webb ◽

R Namikawa ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hematopoietic Stem ◽

Fetal Bone ◽

Cd34 Antigen

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

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Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset

Blood ◽

10.1182/blood.v80.8.1957.bloodjournal8081957 ◽

1992 ◽

Vol 80 (8) ◽

pp. 1957-1964 ◽

Cited By ~ 2

Author(s):

GJ Spangrude ◽

DM Brooks

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Mouse Strains ◽

Hematopoietic Stem ◽

Stem Cell Activity

Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.

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Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

Blood ◽

10.1182/blood.v85.10.2770.bloodjournal85102770 ◽

1995 ◽

Vol 85 (10) ◽

pp. 2770-2778 ◽

Cited By ~ 35

Author(s):

AH Galy ◽

D Cen ◽

M Travis ◽

S Chen ◽

BP Chen

Keyword(s):

Bone Marrow ◽

T Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Cell Activity ◽

Cell Potential ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Hla Dr ◽

Adult Bone

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

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Abstract 32: Hypercholesterolemia-induced Priming of Hematopoietic Stem and Progenitor Cells Aggravates Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a32 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Tom Seijkens ◽

Marten A. Hoeksema ◽

Linda Beckers ◽

Svenja Meiler ◽

Esther Smeets ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Myeloid Cells ◽

Blood Monocytes ◽

Blood Leukocytes ◽

Hematopoietic Stem ◽

Inflammatory Conditions ◽

Stem And Progenitor Cells ◽

Inflammatory Phenotype

During homeostasis hematopoietic stem and progenitor stem cells (HSPCs) give rise to lymphoid and myeloid cells as well as platelets and erythrocytes. However, during chronic inflammatory conditions hematopoiesis is often skewed towards the myeloid lineage, thereby potentially aggravating the ongoing inflammation. Here we investigated the effects of hypercholesterolemia on HSPCs during atherogenesis. Hypercholesterolemia increased HSPCs, defined as Lin - Sca1 + cKit - , in the bone marrow (BM) of LDLr -/- mice by 253.1%. The number of granulocyte-monocyte progenitors, BM granulocytes and BM monocytes was increased by 18.1%, 34.8% and 13.2%, respectively. In accordance, the myeloid colony forming potential of hypercholesterolemic BM was increased by 25.8%. Peripheral blood monocytes and granulocytes were increased by 203.0% and 161.1%, respectively. Competitive bone marrow transplantations (cBMT) in which we compared the effects of normo- vs. hypercholesterolemia primed HSPCs confirmed that the hypercholesterolemic microenvironment activates HSPCs, as reflected by a 26.5% increased reconstitution of peripheral blood leukocytes 10 weeks after the cBMT. Moreover, hypercholesterolemia-primed, and not normocholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C high monocytes, even under long-term normocholesterolemic conditions in the recipient animals. cBMT demonstrated that hypercholesterolemia-induced activation of HSPCs increased atherosclerosis in LDLr -/- mice by 122.1% and increased CD45.1 + plaque leukocytes by 76.1%. Macrophages differentiated from hypercholesterolemia-primed BM produced increased levels of TNFα (+21.3%), IL6 (+17.4%) and MCP1 (+10.5%) compared to their normocholesterolemic counterparts, demonstrating that hypercholesterolemia-induced priming of HSPCs increased the inflammatory phenotype of their mature offspring. These results demonstrate that hypercholesterolemia-induced priming of HSPCs aggravates atherosclerosis by skewing hematopoiesis towards the pro-inflammatory myeloid lineages. Inhibition of this pro-inflammatory differentiation pathway on HSPC level has the potential to reduce atherosclerosis.

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AMD3100 and G-CSF Synergize To Promote Repopulating Stem Cell Mobilization in Fanca −/− and Fancc −/− Mice.

Blood ◽

10.1182/blood.v108.11.3760.3760 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3760-3760

Author(s):

Anna C. Pulliam ◽

M. Joe Hobson ◽

Fengchun Yang ◽

Hal E. Broxmeyer ◽

D. Wade Clapp

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Chemokine Receptors ◽

Cell Activity ◽

Fold Increase ◽

Phenotypic Analysis ◽

Hematopoietic Stem ◽

Test Cells ◽

Myeloid Progenitors

Abstract Fanconi Anemia (FA) is an autosomal recessive DNA repair disorder clinically characterized by congenital abnormalities, progressive bone marrow (BM) failure and a propensity for malignancies. It is advantageous to bank FA patients’ cells for autologous treatment of pancytopenia, transplantation following chemotherapy or future gene therapy trials. Because the amount of cells able to be collected by BM harvesting is limited and the process is often problematic in children, mobilization of stem cells into the circulation would be an ideal means of collecting repopulating hematopoietic stem and progenitor cells (HSPC). Traditional mobilization protocols utilizing granulocyte colony-stimulating factor (G-CSF) as a mobilizing agent result in poor yields of phenotypic HSPC in FA patients. We have shown that analogous to the human patients with FA, administration of recombinant G-CSF is not sufficient to effectively mobilize myeloid progenitors from the BM of Fancc −/− mice (Broxmeyer et al, JEM, 2005). Antagonists to chemokine receptors, together with G-CSF, can enhance mobilization of murine wild type (WT) HSPC. AMD3100 is a selective antagonist of CXCR4, which is thought to function in the retention of HSPC in the BM. We hypothesized that the combination of AMD3100 and G-CSF would enhance mobilization of HSPC in Fanca −/− and Fancc −/− mice. Peripheral blood was collected to assess the number of myeloid progenitors and HSPC repopulating ability. Myeloid progenitor numbers were assessed using progenitor assays. Either G-CSF or AMD3100 alone resulted in poor mobilization of hematopoietic progenitors. In contrast, administration of AMD3100 and G-CSF together increased the number of colonies of myeloid progenitors up to 18 fold above that of either G-CSF or AMD3100 alone in both Fanca −/− and Fancc −/− mice. The stem cell activity of mobilized peripheral blood cells was evaluated using competitive repopulation assays, in which a common pool of isogenic ‘competitor cells’ and ‘test cells’ from mice of different genotypes or treatment conditions are co-transplanted. Fanca −/− and Fancc −/− cells mobilized with both G-CSF and AMD3100 yielded a 3–4 fold increase in peripheral blood chimerism as compared to the cells mobilized with G-CSF or AMD3100 alone. The latter mice also had a decreased survival. Phenotypic analysis of the peripheral blood of primary recipients confirmed the engraftment of multi-lineage test cells. Transplantation of chimeric bone marrow into secondary recipients maintained the test cell chimerism, confirming that long-term repopulating cells were mobilized. Collectively, these data from two FA murine models demonstrate that the AMD3100 and G-CSF protocol is an effective strategy to mobilize FA deficient repopulating HSPC. This has potential implications for more efficient banking of FA patients’ HSPC for future use or for use in gene transfer protocols.

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