Spleen Colony-forming Units Research Articles

Study on the effects of Guiqi Oral Liquid in promoting recovery of hematopoiesis in acute irradiation injured mice

To explore the effects and possible mechanisms of Guiqi Oral Liquid (GQOL) on the recovery of hematopoiesis in acute irradiation injured mice. The acute irradiation injured mice were randomly divided into 2 groups: the treated group and the control group, and also a normal control group was set up with 6 mice in it receiving no treatment. After the mice in the former two groups were irradiated by 6.0 Gy (60)Co gamma-ray, every one of them was given 0.4 ml GQOL or saline in equal volume through a gastric tube twice a day for 14 days. On the 4th, 8th and 14th day after irradiation, the bone marrow mononuclear cells (BMMNC) and megakaryocytes in bone marrow tissues of the mice were counted, the proportion of hematopoietic tissues (by area) was measured, and the expression of adhesion molecules, CD44 and CD54, in bone marrow were estimated by immunochemistry. The colony forming unit of spleen (CFU-S) in the mice were counted on the 8th day after irradiation. On the 4th, 8th, 14th day after irradiation, the count of BMMNC and megakaryocyte, and the proportion of hematopoietic tissues in the treated group were higher than those in the control group (P < 0.01 or P < 0.05). CD44 and CD54 expression in the treated group were higher than those in the control group on the 4th and 8th day (P < 0.01), but near normal on the 14th day (P < 0.01). On the 8th day, CFU-S count in the treated group was higher than that in the control group (P < 0.01). GQOL can regulate the expression of adhesion molecules, CD44 and CD54, in the bone marrow of the acute irradiation injured mice, which may be one of the mechanisms of GQOL in accelerating the early phase hematopoiesis recovery of mice.

Self-Inactivating Lentiviral Vectors Flanked by a Chromatin Insulator Element Result in Increased, Consistent Expression of Human Beta Globin.

Lentiviral vectors carrying the human β-globin gene (hβ) and hypersensitive sites 2, 3 and 4 of the hβ locus control region (LCR) have revolutionized the field of gene therapy for hemoglobinopathies, resulting in correction in mouse and human models of β-thalassemia. However, their random integration into host chromosome results in highly variable hβ expression, dependent on the flanking host chromatin (chromosomal position effects). Moreover, the recent occurrence of leukemogenesis from activation of a cellular oncogene by the viral enhancer elements calls for safer vector designs, with expression cassettes that can be ‘insulated' from flanking cellular genes. We analyzed the role of the chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) lentiviral vector. The BGM vector carrying the hβ/LCR was compared to an analogous vector BGMI, where the cHS4 was inserted in the SIN deletion to flank the hβ/LCR at both ends upon integration. Both vectors additionally carried the methyl guanine methyl transferase (MGMT) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency in three separate experiments to generate single copy clones, transduced clones identified by PCR (‘Unselected' clones) and analyzed for hβ protein by FACS and mRNA by RNase Protection Assay (RPA). ‘Unselected' BGMI+-MEL clones had a higher proportion of hβ+ cells (68±3%) compared to BGM+ clones (36±9%, n=24; p<0.001). Additionally, the coefficient of variation of hβ expression (CV) in each clone was reduced in BGMI+ clones: 168±20 vs. 327±64 in BGM (P<0.02). RPA showed a 2-fold increase in hβ/total mα-globin mRNA with the BGMI vector [BGMI 45±6% vs. BGM 21±7%, n=24, p<0.01]. Next, the same pool of MEL cells transduced with BGM and BGMI at <5% transduction efficiency were selected with BG/BCNU and 86 single-copy clones isolated (‘Selected' clones). ‘Selected' BGMI+-MEL clones also had a higher proportion of hβ+ cells (BGMI 80±15%, n=54 vs. BGM 72±20%, n=32; p<0.03), with reduced CV (140±2.8 vs. 170±13, P<0.01) and higher hβ-mRNA [BGMI+ 83.6%± 40 vs. 49.6%±25 in BGM+ clones (n=24, p<0.02)]. In vivo studies confirmed MEL cell results: lethally irradiated normal mice were transplanted with BGM and BGMI-transduced thalassemia hematopoietic stem cells (MOI 20). Engraftment and vector copy number in both groups were similar (66±15% vs. 68±10%, and 0.21 vs 0.17 in BGMI and BGM groups, respectively, n=12). While, 18±4% of murine RBC expressed hβ in the BGMI group, only 4±1.5% expressed hβ in the BGM group of mice by FACS (p<0.001). There was a 4-fold increase in chimeric hemoglobin (mα-hβ) in the BGMI mice (13% ± 4 vs. 3±2% in BGM mice) by hemoglobin electrophoresis (p<0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice 12 weeks after transplant showed increased numbers of hβ+ cells in BGMI CFU-S (21±4% vs. 8±4% BGM-CFU-S, n=30, p<0.03) with reduced CV in BGMI CFU-S (698±91 vs. 987±99 in BGM CFU-S, p<0.04). MGMT selection and secondary transplants in BGM and BGMI mice are underway. Taken together, ‘insulated' SIN-hβ/LCR lentiviral vectors increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher hβ expression for gene therapy of β-thalassemia. The enhancer blocking effect of the cHS4, although not tested here, would further improve bio-safety of these lineage-specific, self-inactivated vectors.

Prevention of Apoptosis as a Possible Mechanism behind Improved Cryoprotection of Hematopoietic Cells by Catalase and Trehalose

Our previous in vitro work has shown the usefulness of membrane stabilizers and antioxidants as additives in conventional freezing medium to freeze mouse and human hematopoietic cells. The present work was carried out using murine model to test the in vivo engraftment ability of mouse bone marrow frozen with (test cells) or without (control cells) addition of a combination of trehalose and catalase in the medium containing 10% dimethyl sulfoxide (DMSO). Viability, nucleated cell recovery, and progenitor content of revived cells were measured. Freezing efficacy was tested by in vivo assays like colony forming unit-spleen (CFU-S), pre-CFU-S, and short-term engraftment of frozen marrow in irradiated mice. Long-term engraftment ability of frozen marrow was assessed using a Ly5.1-Ly 5.2 chimera model. Levels of apoptosis and reactive oxygen species (ROS) generated in revived cells were estimated. The former by Annnexin V, TUNEL, and DNA laddering and the latter by DCFH-DA probe. Our results show that the combination of catalase and trehalose with 10% DMSO improves freezing efficacy not only in terms of viability, cell recovery, and progenitor content but also by in vivo assays like CFU-S, pre-CFU-S, and short- and long-term engraftment. Both the level of apoptosis and ROS generation were considerably reduced in test set as compared to control set. We conclude that inclusion of a combination of trehalose and catalase in conventional freezing medium leads to enhanced engraftment potential of cryopreserved mouse bone marrow cells probably by preventing apoptotic cell death. Our observation using animal model may have significant clinical implications.

Mice severely deficient in growth hormone have normal hematopoiesis

Many studies suggest that growth hormone (GH) is important for hematopoietic stem cell (HSC) function. The objective of this study is to determine if the genetic absence of GH reduces hematopoietic function and recovery, by testing various points in hematopoiesis, from numbers and functional abilities of primitive stem cells to the maintenance of normal numbers of differentiated cells. Analyses were conducted on blood and bone marrow to compare GH-deficient C57BL/6J-Ghrhr(lit) / Ghrhr(lit) (lit/lit) mice with their normal (lit/+) littermates. Flow cytometric analysis was used to measure numbers of HSC and progenitor cells based on antigenic markers. Spleen colony-forming units (CFU-S) were examined to determine function of common myeloid progenitor (CMP) cells. Competitive repopulation assays were conducted to test whether normally functional HSCs are produced and supported in the lit/lit hematopoietic environment. The lit/lit mutant mice produced HSC and progenitor cells at least as well as their lit/+ control littermates. In CFU-S assays, the CMP from the lit/lit mice functioned as well as those from the lit/+ controls. Marrow cells from lit/lit mice repopulated irradiated recipients long-term better than did marrow cells from C57BL/6J(+/+) controls; thus, HSC produced in the absence of GH can replenish irradiated recipients. When lit/lit mice were used as irradiated recipients, they supported HSC function as well as lit/+ control recipients did; thus, the lit/lit hematopoietic environment can support normal hematopoiesis.

Anomalous megakaryocytopoiesis in mice with mutations in the c-Myb gene

AbstractMpl-/- mice bearing the Plt3 or Plt4 mutations in the c-Myb gene exhibit thrombopoietin (TPO)–independent supraphysiological platelet production accompanied by excessive megakaryocytopoiesis and defective erythroid and lymphoid cell production. To better define the cellular basis for the thrombocytosis in these mice, we analyzed the production and characteristics of megakaryocytes and their progenitors. Consistent with thrombocytosis arising from hyperactive production, the high platelet counts in mice carrying the c-MybPlt4 allele were not accompanied by any significant alteration in platelet half-life. Megakaryocytes in c-Myb mutant mice displayed reduced modal DNA ploidy and, among the excessive numbers of megakaryocyte progenitor cells, more mature precursors were particularly evident. Megakaryocyte progenitor cells carrying the Plt3 or Plt4 c-Myb mutations, but not granulocyte-macrophage progenitors, exhibited 200-fold enhanced responsiveness to granulocyte-macrophage colony-stimulating factor (GM-CSF), suggesting that altered responses to cytokines may contribute to expanded megakaryocytopoiesis. Mutant preprogenitor (blast colony-forming) cells appeared to have little capacity to form megakaryocyte progenitor cells. In contrast, the spleens of irradiated mice 12 days after transplantation with mutant bone marrow contained abundant megakaryocyte progenitor cells, suggesting that altered c-Myb activity skews differentiation commitment in spleen colony-forming units (CFU-S) in favor of excess megakaryocytopoiesis.

Hematopoietic stem cells from NOD mice exhibit autonomous behavior and a competitive advantage in allogeneic recipients

AbstractType 1 diabetes is a systemic autoimmune disease that can be cured by transplantation of hematopoietic stem cells (HSCs) from disease-resistant donors. Nonobese diabetic (NOD) mice have a number of features that distinguish them as bone marrow transplant recipients that must be understood prior to the clinical application of chimerism to induce tolerance. In the present studies, we characterized NOD HSCs, comparing their engraftment characteristics to HSCs from disease-resistant strains. Strikingly, NOD HSCs are significantly enhanced in engraftment potential compared with HSCs from disease-resistant donors. Unlike HSCs from disease-resistant strains, they do not require graft-facilitating cells to engraft in allogeneic recipients. Additionally, they exhibit a competitive advantage when coadministered with increasing numbers of syngeneic HSCs, produce significantly more spleen colony-forming units (CFU-Ss) in vivo in allogeneic recipients, and more granulocyte macrophage–colony-forming units (CFU-GMs) in vitro compared with HSCs from disease-resistant controls. NOD HSCs also exhibit significantly enhanced chemotaxis to a stromal cell–derived factor 1 (SDF-1) gradient and adhere significantly better on primary stroma. This enhanced engraftment potential maps to the insulin-dependent diabetes locus 9 (Idd9) locus, and as such the tumor necrosis factor (TNF) receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSCs. These findings may have important implications to understand the evolution of autoimmune disease and impact on potential strategies for cure.

Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)

AbstractThe erythrocyte colony-forming unit (CFU-E) is a rare bone marrow (BM) progenitor that generates erythrocyte colonies in 48 hours. The existence of CFU-Es is based on these colonies, but CFU-Es have not been purified prospectively by phenotype. We have separated the “nonstem,” “nonlymphoid” compartment (lineage marker [lin]–c-Kit+Sca-1–IL-7Rα–) into interleukin 3 receptor α negative (IL-3Rα–) and IL-3Rα+ subsets. Within IL-3Rα– but not IL-3Rα+ cells we have identified TER119–CD41–CD71+ erythrocyte-committed progenitors (EPs). EPs generate CFU-E colonies at about 70% efficiency and generate reticulocytes in vivo. Depletion of EPs from BM strongly reduces CFU-E frequencies. EPs lack potential for erythrocyte burst-forming unit, megakaryocyte, granulocyte (G), and monocyte (M) colonies, and for spleen colony-forming units. Chronically suppressed erythropoiesis in interferon consensus sequence-binding protein (ICSBP)–deficient BM is associated with reduced frequencies of both the EP population and CFU-E colonies. During phenylhydrazine-induced acute anemia, numbers of both the EP population and CFU-E colonies increase. Collectively, EPs (lin–c-Kit+Sca-1–IL-7Rα–IL-3Rα–CD41–CD71+) account for most, if not all, CFU-E activity in BM. As a first molecular characterization, we have compared global gene expression in EPs and nonerythroid GM progenitors. These analyses define an erythroid progenitor-specific gene expression pattern. The prospective isolation of EPs is an important step to analyze physiologic and pathologic erythropoiesis.

Contrasting effects of P-selectin and E-selectin on the differentiation of murine hematopoietic progenitor cells

The two endothelial selectins, P- and E-selectin, are critically important for adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed 6 months posttransplant. Bone marrow lineage-negative (Lin(-)) Sca-1(+)c-kit(+) cells were then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoietic potential was assessed using in vitro phenotyping and colony-forming assays and in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. Long-term competitive repopulating HSCs were Lin(-)c-kit(bright) and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of Lin(-)Sca-1(+)c-kit(+) cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S(12) compared to the other cultures. In contrast, E-selectin enhanced the differentiation of Lin(-)Sca-1(+)c-kit(+) cells into cells that expressed the granulocyte maturation marker, Gr-1, accompanied by loss of CFC potential from these cultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S(12) in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of Lin(-)Sca-1(+)c-kit(+) cells, allowing enhanced ex vivo expansion of CFC and CFU-S(12) but not HSCs.

Novel Roles for the Homeobox Gene, HoxA1, and Its Truncated Form, HoxA1-T, in the Regulation of Hematopoietic Stem Cell Self-Renewal.

There is increasing evidence that homeobox (Hox) genes play critical roles in the regulation of hematopoiesis. We found that both forms of the earliest HoxA gene, HoxA1 and its alternatively spliced transcript, HoxA1-T, which lacks the homeobox domain, are expressed in immature populations of hematopoietic stem cells (HSCs) and progenitor cells. In more mature bone marrow cell (BM) populations the levels of HoxA1-T increases relative to HoxA1 with both transcripts absent in mature peripheral blood (PB) cells. Roles for either of these Hox transcripts in hematopoiesis have not yet been described. We overexpressed either HoxA1 or HoxA1-T in BM using a modified GFP-containing MSCV vector (MXIE). Overexpression of either HoxA1 transcript did not affect expression levels of other Hox genes relative to control (empty vector). Overexpression of HoxA1-T significantly reduced the numbers of colony-forming cells (CFCs) produced by 500 GFP+ BM compared to control GFP+ BM (mean±SEM control 73±6.4; HoxA1 57±6.8; HoxA1-T 26.7±5.4; n=6; P<0.01 control vs. HoxA1-T). Interestingly, colonies generated by HoxA1 GFP+ BM contained ~3-fold more cells (mean±SEM: control 22,000±900 cells; HoxA1 61,700±12,000 cells; HoxA1-T 20,600±6,360 cells; n=6; P<0.005 HoxA1 vs. control and HoxA1-T). In each of 2 experiments 10,000 HoxA1 overexpressing BM grew in liquid suspension culture for up to 15 weeks, increasing by ~239-fold weekly. In contrast, control BM proliferated for only 3 weeks, with weekly ~53-fold increases, whereas HoxA1-T BM expired after 2 weeks of culture, with weekly cell increases of only ~7-fold (control v HoxA1 P<0.01; HoxA1 v HoxA1-T P<0.001). There was no difference in the number of day 12 colony-forming unit-spleen (CFU-S) formed from 2500 control or HoxA1 GFP+ BM (11.2±0.47 and 12.6±1.26 respectively). In contrast, 2500 HoxA1-T GFP+ BM produced significantly fewer CFU-S (8.9±0.61) compared to both control and HoxA1 BM (n=4, P<0.02). To assess HSC potential, lethally irradiated CD45.2+ recipients (n=6/group) were injected with 5x106 congenic CD45.1+ BM immediately post-transduction without selection (all groups had similar transduction efficiencies). All recipients had >75% donor cells (CD45.1+, GFP+/−) in their PB at 3 months post-transplant. The average %GFP+ cells in recipients were similar for control (24.5±7.0%) and HoxA1 cells (20.8±2.5%). Strikingly, HSCs overexpressing HoxA1-T had markedly reduced repopulating ability (3.5±0.6% GFP+, P<0.05 HoxA1-T vs. control or HoxA1). Poisson statistics were used to quantitate HSC frequency in secondary transplant recipients. Mice were injected with 5x103, 5x104, 2x105 or 1x106 BM from control or HoxA1 primary recipients together with 2x105 congenic CD45.2+ BM (n=10/group). The frequency of HSCs was markedly higher (~32-fold) in the HoxA1 cells (~1 HSC per 1.8x105 BM) compared to control (~1 HSC per 5.8x106 BM; P<0.001). Although secondary recipients injected with 1x106 HoxA1-T BM showed donor (CD45.1+) contribution (15.3±1.8%), none of the 10 recipients had GFP+ donor cell reconstitution. These data therefore suggest that HoxA1 enhances HSC self-renewal whereas HoxA1-T rapidly promotes HSC differentiation. These novel findings highlight important roles for the two HoxA1 transcripts in the regulation of HSCs, the mechanisms of which are currently being assessed.

Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure

AbstractInfusion of parental lymph node (LN) cells into sublethally irradiated hybrid F1 recipients created a murine model for bone marrow (BM) failure. Affected animals developed fatal pancytopenia within 2 to 3 weeks, accompanied by BM oligoclonal T-cell infiltration and severe marrow hypoplasia indicated by approximately 10-fold declines in total BM cellularity, 15-fold declines in BM Lin-Sca1+c-Kit+cells, 100-fold declines in spleen colony-forming units, and 100-fold declines in hematopoietic progenitor and stem cells as estimated by irradiation protection in vivo. LN cells of both H2b/band H2d/dhaplotypes were effectors. Serum interferon-γ (IFN-γ) concentration increased 2- to 3-fold. Marrow cells were severely apoptotic, with high proportions of Fas+and annexin V+cells. Cotransplantation of 5 × 105BM cells from clinically affected donors and 106BM cells from H2 identical healthy mice could not rescue lethally irradiated recipients. Recipients had significantly lower cellularity in peripheral blood and BM, and cell mixtures failed to produce a stromal feeder layer to support marrow cell growth in vitro. Pathogenic T cells from donors after BM failure appeared capable of destroying hematopoietic progenitor, stem, and stromal cells from fully compatible healthy donors as “innocent bystanders.” This effect can be partially abrogated by anti-IFN-γ antibody. (Blood. 2004;104:1671-1678)

Synergistic effects on erythropoiesis, thrombopoiesis, and stem cell competitiveness in mice deficient in thrombopoietin and steel factor receptors

AbstractThe degree of redundancy between thrombopoietin (Tpo) and steel factor (SF) cytokine pathways in the regulation of hematopoiesis was investigated by generating mice lacking both c-Mpl and fully functional c-Kit receptors. Double-mutant c-Mpl–/–KitWv/Wvmice exhibited reduced viability, making up only 2% of the offspring from c-Mpl–/–KitWv/+intercrosses. The thrombocytopenia and megakaryocytopenia characteristic ofc-Mpl–/–mice was unchanged inc-Mpl–/–KitWv/Wvmice. However, the number of megakaryocytic colony forming units (CFU-Mks) was significantly reduced, particularly in the spleen. WhileKitWv/Wvmice, but notc-Mpl–/–mice, are anemic, the anemia was more severe in double-mutantc-Mpl–/–KitWv/Wvmice, indicating redundancy between Tpo and SF in erythropoiesis. At the primitive cell level,c-Mpl–/–andKitWv/Wvmice have similar phenotypes, including reduced progenitors, colony forming units–spleen (CFU-Ss), and repopulating activities. All of these parameters were exacerbated in double-mutant mice. c-Mpl–/–KitWv/Wvmice had 8-fold fewer clonogenic progenitor cells and at least 28-fold fewer CFU-Ss. c-Mpl–/–mice also demonstrated a reduced threshold requirement for nonmyeloablative transplant repopulation, a trait previously associated only withKitWmice, and the level of nonmyeloablative engraftment was significantly greater inc-Mpl–/–KitWv/Wvdouble mutants. Thus,c-Mpl–/–KitWv/Wvmice reveal nonredundant and synergistic effects of Tpo and SF on primitive hematopoietic cells.

Effect of ligustrazine on the expression of LFA-1, ICAM-1 following bone marrow transplantation in mice.

The effects of ligustrazine on the expression of LFA-1, ICAM-1 in bone marrow tissue and the mechanism promoting hematopoietic reconstitution following bone marrow transplantation (BMT) were investigated. The 150 mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. The normal group received no treatment, while in the saline group and ligustrazine group, the mice were subjected to normal saline (0.2 ml, twice a day) and ligustrazine (0.2 ml, twice a day) respectively through a gastric tube. At the 7th, 14th, 21st and 28th day after BMT, survival rate, colony forming unit of spleen (CFU-S), peripheral blood cells and bone marrow mononuclear cells (BMMNC) were measured, histological changes in bone marrow tissue were observed and the expression level of LFA-1, ICAM-1 was detected. In ligustrazine group CFU-S counts on the 10th day and the peripheral blood WBC, PLT, BMMNC counts, hematopoietic tissue volume as well as the expression level of LFA-1 on the 7th, 14th, 21st, 28th day after BMT were higher than in saline group (P<0.01 or P<0.05). Mature RBC volume and the expression level of ICAM-1 were significantly lower in the ligustrazine group than in the saline group (P<0.01 or P<0.05). In the ligustrazine group, fat tissue volume was higher on the 7th, 14th day after BMT (P<0.01) and was lower on the 21st, 28th day (P<0.01) after BMT than in the saline group. It was concluded that Ligustrazine could improve bone marrow microenvironment and promote hematopoietic reconstitution.

Differential Regulatory and Compensatory Responses in Hematopoiesis/Erythropoiesis in α- and β-Globin Hemizygous Mice

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.

Runx1 is expressed in adult mouse hematopoietic stem cells and differentiating myeloid and lymphoid cells, but not in maturing erythroid cells.

The transcription factor Runx1 marks all functional hematopoietic stem cells (HSCs) in the embryo and is required for their generation. Mutations in Runx1 are found in approximately 25% of acute leukemias and in familial platelet disorder, suggesting a role for Runx1 in adult hematopoiesis as well. A comprehensive analysis of Runx1 expression in adult hematopoiesis is lacking. Here we show that Runx1 is expressed in functional HSCs in the adult mouse, as well as in cells with spleen colony-forming unit (CFU) and culture CFU capacities. Additionally, we document Runx1 expression in all hematopoietic lineages at the single cell level. Runx1 is expressed in the majority of myeloid cells and in a smaller proportion of lymphoid cells. Runx1 expression substantially decreases during erythroid differentiation. We also document effects of reduced Runx1 levels on adult hematopoiesis.

Local environmental factors determine hematopoietic differentiation of neural stem cells.

Stem cells exhibit unique properties and hold high therapeutic promise, but factors influencing their differentiation after transplantation need to be recognized and defined for this promise to be fully met. Here, we demonstrate that endogenous colony-forming unit spleen (CFU-S) colonies are not generated in lethally irradiated mice transplanted with neural stem cells obtained from brain tissue of syngeneic donors. We investigated the proportion of transplanted neural stem cells that contributed to hematopoietic reconstitution and compared the distribution of transplanted cells in nonsplenectomized to that of splenectomized mice following sublethal whole-body irradiation. We also used clonogenic assays, colony assays, and histochemical analyses to explore conditions under which transplanted, beta-galactosidase-tagged neural stem cells underwent hematopoietic differentiation. Our results suggest that neural stem cells do undergo extramedullary hematopoiesis, even while no endogenous hematopoietic colonies develop in the spleen. Furthermore, we found that neural stem cells effectively colonized the bone marrow of splectomized recipients. We conclude that the hematopoietic differentiation of neural stem cells is highly dependent on the extramedullary environment. We also conclude that the bone marrow does not provide an environment supportive of hematopoietic differentiation by neural stem cells.

The myeloprotective effect of medroxyprogesterone acetate in an irradiated animal model.

In this study we evaluated the effect of medroxyprogesterone acetate (MPA) and its major ingredients on protection of the hematopoietic organs against radiation damage. One group of mice was given saline as placebo and the other groups were given MPA. Mice were injected with MPA (10 mg/kg) or saline 10 days before or after a single 8 Gy whole body cobalt irradiation. On day 14 the mice were sacrificed and their bone marrow transplanted to recipient mice. Ten days after the transplantation, spleen colony formation was investigated in mice. Administration of MPA with irradiation increased the formation of the spleen colony. Statistically significant enhancement of the spleen colony formation was found in mice treated with MPA repeatedly, as compared with those treated with placebo (P < 0.001). No significant difference in Spleen Colony Forming Unit (CFU-S) numbers was observed between pre-and post-radiotherapy administration of MPA (P = 0.216). It is an important observation that no significant difference was observed in CFU-S numbers between pre- and post-irradiation administration of MPA.

Hematopoietic cells from mice that are deficient in both Bcrp1/Abcg2 and Mdr1a/1b develop normally but are sensitized to mitoxantrone.

Hematopoietic stem cells (HSCs) express Mdr1a/1b and Bcrp1/Abcg2, which are members of the ATP binding cassette transporter family. Mice lacking both Mdr1-type genes (Mdr1a and Mdr1b) or Bcrp1 had normal hematopoietic development, but it has been unclear whether Mdr1a/1b and Bcrp1 play redundant roles in hematopoiesis. We generated a mouse model lacking both Mdr1a/1b and Bcrp1 expression (M-/-B-/-). The M-/-B-/- mice had normal numbers of peripheral blood cells, bone marrow colony-forming cells (CFCs) and colony-forming units-spleen (CFU-S), and demonstrated normal hematopoietic development. There was a near total elimination of side population (SP) cells in the bone marrow of M-/-B-/- mice compared to M+/+B-/- mice, primarily in the subpopulation lacking other HSC markers, which indicated that Mdr1a/1b was responsible for a small portion of SP cells that were mainly mature cells. Hematopoietic progenitor cells from the bone marrow of M-/-B-/- mice were more sensitive to mitoxantrone in vitro compared to either M-/-B+/+ or M+/+B-/- mice, suggesting that Mdr1a/1b and Bcrp1 may provide additive protection to HSCs against genotoxic agents. These studies demonstrate the lack of functional redundancy between these transporters for HSC development and further clarify their contributing role to the SP phenotype in HSCs and to intrinsic drug resistance within hematopoietic progenitor cells.

Homeostasis and regeneration of the hematopoietic stem cell pool are altered in SHIP-deficient mice

AbstractSH2-containing inositol 5-phosphatase (SHIP) is an important negative regulator of cytokine and immune receptor signaling. SHIP-deficient mice have a number of hematopoietic perturbations, including enhanced cytokine responsiveness. Because cytokines play an important role in the maintenance/expansion of the primitive hematopoietic cell pool, we investigated the possibility that SHIP also regulates the properties of cells in these compartments. Primitive hematopoietic cells were evaluated in SHIP-deficient mice and wild-type littermate controls using the colony-forming unit-spleen (CFU-S) and competitive repopulating unit (CRU) assays for multipotent progenitors and long-term lympho-myeloid repopulating cells, respectively. Absence of SHIP was found to affect homeostasis of CFU-S and CRU compartments. Numbers of primitive cells were increased in extramedullary sites such as the spleen of SHIP-deficient mice, although total body numbers were not significantly changed. In vivo cell cycle status of the CRU compartment was further evaluated using 5-fluorouracil (5-FU). SHIP-deficient CRUs were more sensitive to 5-FU killing, indicating a higher proliferative cell fraction. More strikingly, SHIP was found to regulate the ability of primitive cells to regenerate in vivo, as CRU recovery was approximately 30-fold lower in mice that received transplants of SHIP-deficient cells compared with controls. These results support a major role for SHIP in modulating pathways important in homeostasis and regeneration of hematopoietic stem cells, and emphasize the importance of negative cytokine regulation at the earliest stages of hematopoiesis. (Blood. 2003;102:3541-3547)

Identification of in vitro growth conditions for c-Kit–negative hematopoietic stem cells

AbstractOur laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Linneg), lack c-Kit, and are able to give rise to c-Kit–positive (c-Kitpos) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit–negative (c-Kitneg) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre–colony-forming unit spleen (pre–CFU-s) activity in both the Sca-1neg and Sca-1pos populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1pos population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kitneg cells gave rise to c-Kitpos PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kitneg to c-Kitpos transition.

The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization

AbstractThe directed migration of mature leukocytes to inflammatory sites and the lymphocyte trafficking in vivo are dependent on G protein–coupled receptors and delivered through pertussis toxin (Ptx)–sensitive Gi-protein signaling. In the present study, we explored the in vivo role of G-protein signaling on the redistribution or mobilization of hematopoietic stem/progenitor cells (HPCs). A single injection of Ptx in mice elicits a long-lasting leukocytosis and a progressive increase in circulating colony-forming unit-culture (CFU-C) and colony-forming unit spleen (CFU-S). We found that the prolonged effect is sustained by a continuous slow release of Ptx bound to red blood cells or other cells and is potentially enhanced by an indirect influence on cell proliferation. Plasma levels of certain cytokines (interleukin 6 [IL-6], granulocyte colony-stimulating factor [G-CSF]) increase days after Ptx treatment, but these are unlikely initiators of mobilization. In addition to normal mice, mice genetically deficient in monocyte chemotactic protein 1 (MCP-1), matrix metalloproteinase 9 (MMP-9), G-CSF receptor, β2 integrins, or selectins responded to Ptx treatment, suggesting independence of Ptx-response from the expression of these molecules. Combined treatments of Ptx with anti–very late activation antigen (anti-VLA-4), uncovered potentially important insight in the interplay of chemokines/integrins, and the synergy of Ptx with G-CSF appeared to be dependent on MMP-9. As Ptx-mobilized kit+ cells display virtually no response to stromal-derived factor 1 (SDF-1) in vitro, our data suggest that disruption of CXCR4/SDF-1 signaling may be the underlying mechanism of Ptx-induced mobilization and indirectly reinforce the notion that active signaling through this pathway is required for continuous retention of cells within the bone marrow. Collectively, our data unveil a novel example of mobilization through pharmacologic modulation of signaling.