Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

Download Full-text

Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

Blood ◽

10.1182/blood.v78.7.1706.1706 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1706-1712 ◽

Cited By ~ 195

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Tyrosine Kinase Receptor ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

Download Full-text

Pharmacologic Control of HOXB4-Mediated Expansion of Murine Hematopoietic Stem Cells; Evidence for a Restricted Time Interval for Expansion.

Blood ◽

10.1182/blood.v104.11.1689.1689 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1689-1689 ◽

Cited By ~ 1

Author(s):

Yan Shou ◽

Lilia Stepanova ◽

Brian Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Time Interval ◽

Hematopoietic Stem ◽

Marrow Cells

Overexpression of the homebox transcription factor HOXB4 can enhance self-renewal of murine hematopoietic stem cells (HSCs) and thereby result in an increased number of HSCs in vivo. In mice transplanted with bone marrow cells transduced with a retroviral vector expressing HOXB4, HSC expansion stopped after HSC numbers regenerated to a normal level. Furthermore, when transduced bone marrow cells from primary transplant recipients were transplanted into secondary recipients, HSCs failed to recover to normal numbers (G. Sauvageau et al, Genes and Dev, 9:1753, 1995). One possible explanation for these results is that HSC expansion could be limited to an early time interval in the primary transplant recipient. In order to determine if a time-window exists for HOXB4-mediated HSC expansion, and to develop a method to control HSC expansion for gene therapy applications, we generated a retroviral vector expressing a HOXB4 protein that was fused to a variant estrogen responsive binding element (ERT2). This HOXB4-ERT2 protein allowed HOXB4 function to be regulated with 4-hydroxytamoxifen (TAM). Murine bone marrow cells were transduced with the MSCV- HOXB4-ERT2-GFP vector and transplanted into lethally irradiated recipients. A 3 week course of daily TAM treatment was started either immediately after transplant, or in a second cohort, 12 weeks after transplant. When TAM treatment was administered for the first 3 weeks after transplant, there was a 7-fold increase in the percentage of GFP positive peripheral blood leukocytes compared to the cohort transplanted with the same cells but not receiving TAM treatment (15% +/−8, n=7, VERSUS 2 % +/− 2, n=9). In contrast, an identical 3-week course of TAM treatment beginning at 12 weeks post-transplant had no effect on the proportion of GFP+ cells in the peripheral blood (3% +/−2, n=5 VERSUS 2% +/−2, n=4). Bone marrow cells from mice in each of these cohorts were harvested at 21 weeks after transplant, and infused into secondary recipients. The proportion of GFP+ blood cells noted in the primary recipients that were treated with TAM for weeks 1 through 3 was maintained in untreated secondary recipients, confirming that early TAM treatment had resulted in amplification at the level of HSCs. The other half of these secondary recipients was treated immediately after transplant with the same 3 week course of daily TAM treatment. TAM treatment in secondary recipients did not lead to a further increase in the proportion of GFP+ blood cells compared to values in the untreated secondary recipients (9% +/−7, n=9 VERSUS 10% +/−3, n=6). These results show that the early 3 week time interval for HSC expansion was not reset with secondary transplantation and suggest that there is a HSC intrinsic mechanism that limits HOXB4-mediated expansion based on past replication history. This model would explain the physiologic limitation on HSC expansion that has been noted with wildtype HOXB4 vectors. Experiments are now in progress to further elucidate this putative mechanism, including further refinement of the time limits for expansion and microarray analysis of downstream target genes at different time points relative to transplantation.

Download Full-text

Erythropoietin Signaling Inhibits Long Term Marrow Reconstitution.

Blood ◽

10.1182/blood.v108.11.3194.3194 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3194-3194

Author(s):

George L. Chen ◽

Kotung Chang ◽

Xiaosong Huang ◽

Gerald J. Spangrude ◽

Josef T. Prchal

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Total Bone Marrow ◽

Marrow Cells

Abstract Murine hematopoietic stem cells (HSC) transfected with a gain-of-function human erythropoietic receptor (EPOR) transgene were reported to have a competitive advantage over wild type mouse hematopoietic stem cells in a bone marrow transplantation (BMT) model (Kirby, Blood95(12): 3710, 2000). However, EPOR transgenes may not be normally expressed in early progenitor/stem cells. Moreover, whether Epo/EpoR signaling plays a role in hematopoietic stem cell engraftment is unknown. Our lab previously created mouse models harboring the wild type human EPOR (wthEPOR) or the mutant human gain-of-function EPOR (mthEPOR) gene knocked into the mouse EPOR locus (Divoky, PNAS 98(3): 986, 2001). This animal model has augmented Epo signaling in all tissues that express EpoR, thus the wthEPOR mice are anemic while the mthEPOR mice are polycythemic. We compared the relative engraftment efficiency of mthEPOR vs. wthEPOR HSCs in a competitive bone marrow transplantation (BMT) assay using C57/Bl6 congenic mice. Bone marrow from wthEPOR (CD45.1) and mthEPOR (CD45.2) mice were co-transplanted (1:1) into lethally irradiated (137Cs > 11Gy split) normal recipients (CD45.1/CD45.2). At 7 months after transplantation, peripheral blood chimerism demonstrated skewing towards wthEPOR rather than mthEPOR origin in the granulocyte, macrophage, T cell, and B cell compartments (Data Table). Bone marrow chimerism paralleled peripheral blood chimerism (not shown). Examination of the stem cell compartment by Hoechst 33342 staining demonstrated similar skewing towards wthEPOR origin (Data Table). Because unequal numbers of HSC may result in skewed chimerism, we examined the relative proportions of HSC to total bone marrow cells. In wthEPOR mice, the Flt3− Rh123low subset of cKit+Sca1+ cells (KLS-FS) cells represented 0.011±0.003% of total bone marrow cells while in mthEPOR mice these cells represented 0.023±0.006% of total bone marrow cells (p=0.025). Since equal numbers of wthEPOR and mthEPOR total bone marrow cells were co-transplanted, relatively fewer wthEPOR HSC than mthEPOR HSC were transferred. Taken with the above chimerism data showing skewing towards wthEPOR, these results suggest that wthEPOR HSCs have a significant engraftment advantage over mthEPOR HSCs. Furthermore, enhanced Epo/EpoR signaling may interfere with the long term repopulation of hematopoietic progenitors. Hematopoietic stem cells undergo self renewal or differentiation/proliferation; in the presence of erythropoietin, a cytokine with proliferative and differentiating properties, it may be that self renewal is suppressed leading ultimately to the observed skewed chimerism. These data suggest that erythropoietin administration to patients during and immediately after marrow transplantation may be detrimental and should be used judiciously. Peripheral Blood and Marrow Chimerism Compartment wthEPOR (CD45.1) mthEPOR (CD45.2) Endogenous control (CD45.1/CD45.2) All p values for wthEPOR vs mthEPOR < 0.01 Neutrophil (blood) 72.7% 18.8% 8.5% Macrophage (blood) 76.8% 14.7% 8.5% T cell (blood) 78.6% 9.3% 12.2% B cell (blood) 72.8% 17.7% 9.5% HSC (marrow) 66% 15.1% 18.9%

Download Full-text

MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Blood ◽

10.1182/blood.v118.21.551.551 ◽

2011 ◽

Vol 118 (21) ◽

pp. 551-551

Author(s):

Zeenath Unnisa ◽

Jason P Clark ◽

Elizabeth Wojtowicz ◽

Lino Tessarollo ◽

Neal G. Copeland ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Bone Marrow Analysis ◽

Marrow Cells

Abstract Abstract 551 Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Role Of Sf3b1 On Hematopoiesis

Blood ◽

10.1182/blood.v122.21.600.600 ◽

2013 ◽

Vol 122 (21) ◽

pp. 600-600

Author(s):

Manabu Matsunawa ◽

Ryo Yamamoto ◽

Masashi Sanada ◽

Aiko Sato ◽

Yusuke Shiozawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Transplantation ◽

Hematopoietic Stem Cells ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Ring Sideroblasts ◽

Marrow Cells

Abstract Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CblY371H Transgene Combined with Hematopoietic Deletion of the Endogenous c-Cbl Gene Results in GM-CSF Hypersensitivity and Leukocytosis

Blood ◽

10.1182/blood.v126.23.3672.3672 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3672-3672

Author(s):

Kenneth Lieuw ◽

Jayasree Krishnamurthy ◽

Mignon L. Loh

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Embryonic Lethality ◽

Hematopoietic Stem ◽

Endogenous Gene ◽

Gm Csf ◽

Marrow Cells

Abstract Juvenile Myelomonocytic Leukemia (JMML) is a mixed myeloproliferative/myelodysplastic disease that is rapidly fatal with infiltration of myeloid cells into multiple organs. About 15% of JMML patient samples contain a mutation in c-Cbl, and germline mutation results in the predisposition for developing JMML. The c-Cbl gene encodes a multifunctional adaptor protein that contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif that contains E3 ligase activity, and a C-terminal ubiquitin-associated domain. The TKB domain is involved in adaptor functions of the protein, whereas the ubiquitin ligase domain results in mono-ubiquitination of receptors which promotes lysosomal mediated degradation of activated receptors. Interestingly, a hotspot for mutations at residue 371 exists in JMML patients, where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H. This residue belongs in the linker region of the CBL protein, and it was previously observed that Tyr-371 plays key roles in activating the ubiquitin ligase activity of the protein. In vitro, CblY371H mutation does indeed destroy its ligase function, resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene is silenced. How mutant Cbl gives rise to JMML, however, and how it acts in concert with other genes in the pathogenesis of JMML requires further study. To address these questions, we overexpressed the oncogenic CblY371H mutation using transgenic mice. As expected, overexpression of CblY371H by itself in wildtype mice had no apparent phenotype. Therefore, Cbl transgenic mice were bred to Cbl heterozygous knockout mice (Cbl+/-) followed by further breeding in an attempt to generate Cbl transgenic mice with the endogenous Cbl gene inactivated (CblY371H; Cbl-/-). Surprisingly, unlike Cbl null mice, which are viable, overexpression of mutant Cbl allele in Cbl null mice caused embryonic lethality between 11.5 dpc and 12.5 dpc. In order to circumvent the developmental effects of expressing the mutant Cbl protein, we used a conditional Cbl knockout mouse to tissue specifically delete the endogenous Cbl gene. We chose the MMTV-Cre strain, which expresses Cre recombinase in only 10% of hematopoietic stem cells (CD34-; Lin-; Sca-1+; c-Kit+). With subsequent breeding with the CblY371H transgenic mice, we were able to bypass the embryonic lethality and produce mice with the correct genotype (MMTV-Cre;CblY371H;Cblfl/fl). These mice look normal but develop significant leukocytosis and show GM-CSF hypersensitivity even though only 10% of hematopoietic stem cells are affected. These mice, however, appear unaffected by the leukocytosis, and show no obvious difference with its littermates up to one year of age. We conclude that mutant CblY371H by itself is not sufficient for the development of JMML in this model and requires additional cooperating events. Whether further aging of these mice will result in JMML remains to be seen. In conclusion, we have developed a mouse model overexpressing the CblY371H protein ubiquitously, which causes deleterious development when it is the only c-Cbl protein available. This confirms the important role of c-Cbl activity during development. In hematopoietic cells, the overexpression of CblY371H results in leukocytosis and GM-CSF hypersensitivity when the endogenous gene is inactivated. We are currently investigating the cooperating events that are required for the development of JMML in this mouse model. Figure 1. Phenotype of CblY371H Transgenic Mice A and B. Embryonic lethality of Cbl transgenic mice. The embryos look normal on day 10 of development but by day 12.5, no homozygous embryos are found. C and D. There is significant leukocytosis when the CblY371H transgene is combined with inactivation of the endogenous gene only in hematopoietic stem cells using the MMTV Cre. Figure 1. Phenotype of CblY371H Transgenic Mice A and B. Embryonic lethality of Cbl transgenic mice. The embryos look normal on day 10 of development but by day 12.5, no homozygous embryos are found. C and D. There is significant leukocytosis when the CblY371H transgene is combined with inactivation of the endogenous gene only in hematopoietic stem cells using the MMTV Cre. Figure 2. GM-CSF Hypersensitivity of Bone Marrow Cells from Triple Transgenic Mice A. Western Blot of bone marrow cells stimulated with GM-CSF. Panel A shows time course after stimulation of bone marrow cells from conditional Cbl mice (Cblfl/fl) that have the endogenous Cbl gene deleted using the MMTV-Cre transgene. B-D.Quantitation of several blots showing GM-CSF hypersensitivity. When normalized to the nontransgenic mice at time point zero, there is increased activity of downstream signaling pathways with and without GM-CSF. Figure 2. GM-CSF Hypersensitivity of Bone Marrow Cells from Triple Transgenic Mice A. Western Blot of bone marrow cells stimulated with GM-CSF. Panel A shows time course after stimulation of bone marrow cells from conditional Cbl mice (Cblfl/fl) that have the endogenous Cbl gene deleted using the MMTV-Cre transgene. B-D. Quantitation of several blots showing GM-CSF hypersensitivity. When normalized to the nontransgenic mice at time point zero, there is increased activity of downstream signaling pathways with and without GM-CSF. Disclosures No relevant conflicts of interest to declare.

Download Full-text

In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v112.11.3861.3861 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3861-3861

Author(s):

Jun Ooehara ◽

Hina Takano ◽

Shin-ichiro Takayanagi ◽

Hiromitsu Nakauchi ◽

Hideo Ema

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Culture Conditions ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Lymphoid Progenitors ◽

Marrow Cells

Abstract Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.

Download Full-text

The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.4694.4694 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4694-4694

Author(s):

Hong Xu ◽

Ziqiang Zhu ◽

Yiming Huang ◽

Suzanne T. Ildstad

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

Dose Titration ◽

Great Promise ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

Download Full-text

Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

Journal of Experimental Medicine ◽

10.1084/jem.175.1.175 ◽

1992 ◽

Vol 175 (1) ◽

pp. 175-184 ◽

Cited By ~ 250

Author(s):

N Uchida ◽

I L Weissman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hematopoietic Stem ◽

Stem Cell Activity ◽

Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

Download Full-text

Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice

Blood ◽

10.1182/blood-2009-06-229443 ◽

2010 ◽

Vol 115 (7) ◽

pp. 1406-1415 ◽

Cited By ~ 184

Author(s):

Michael G. Kharas ◽

Rachel Okabe ◽

Jared J. Ganis ◽

Maricel Gozo ◽

Tulasi Khandan ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Akt Activation ◽

Marrow Cells

Abstract Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT–expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT–expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

Download Full-text