AML1/ETO and PML/RAR α Can Immortalize Committed Myeloid Progenitor Cells In-Vitro but Not Expand Them In-Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2544-2544
Author(s):  
Naoki Hosen ◽  
Emmanuelle Passegue ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Myeloid Progenitors

Abstract For most leukemia the target cells of transforming mutations are still unknown. Here, we studied the developmental origin of t(8;21)-acute myeloid leukemia (AML), t(15;17)-acute promyelocytic leukemia (APL), and t(9;22)-chronic myeloid leukemia (CML). Purified mouse hematopoietic stem cells (HSCs) and various committed myeloid progenitor cells were retrovirally transduced with AML1/ETO, PML/RARα, or p210 BCR/ABL and subjected to in-vitro serial replating assay and in-vivo transplantation. Myeloid progenitors were efficiently immortalized in-vitro by AML1/ETO or PML/RARα as assayed in serial replating assays. However, following transplantation into lethally irradiated mice, neither AML1/ETO- nor PML/RARα-transduced myeloid progenitors were expanded in-vivo, although cells carrying the fusion gene DNA persisted well beyond their non-transduced control progenitors. In addition, 10 months after the transplant with transduced myeloid progenitor cells, PML/RARα but not AML1/ETO mRNA expression was still detected in committed myeloid progenitors, although PML/RARα-expressing cells were still not expanded. This finding demonstrates the ability of PML/RARα to increase the lifespan of committed myeloid progenitor cells both in-vitro and in-vivo and suggest that t(15;17)-APL could possibly evolve from persisting progenitor-derived cells. In contrast, BCR/ABL-expressing myeloid progenitors disappeared within 3 months post transplantation. Analysis of mice transplanted with transduced-HSCs demonstrated that AML1/ETO induced the accumulation of the most immature subset of HSCs (Lin-/c-kit+/Sca-1+/Flk-2-), while in contrast PML/RARα induced HSCs disappearance in most (5 out of 6) cases. In addition, we demonstrate that p210 BCR/ABL could induce the development of a CML-like disease from transduced HSCs (6 out of 16 cases), which is compatible with previous report. Together, these results suggest the existence of novel pre-leukemic stem cells (pre-LSCs) entities. Slowly expanding pre-LSCs could be generated from HSCs transformed by the expression of AML1/ETO. Persisting pre-LSCs could be generated from myeloid progenitors transformed by the expression of PML/RARα. These pre-LSC populations could mediate the early phases of t(8;21)-AML and t(15;17)-APL pathogenesis and could represent novel key targets for anti-leukemia therapies.

scholarly journals Involvement of Interleukin (IL) 8 receptor in negative regulation of myeloid progenitor cells in vivo: evidence from mice lacking the murine IL-8 receptor homologue.

1996 ◽  
Vol 184 (5) ◽  
pp. 1825-1832 ◽  
Author(s):  
H E Broxmeyer ◽  
S Cooper ◽  
G Cacalano ◽  
N L Hague ◽  
E Bailish ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Negative Regulation ◽  
Platelet Factor ◽  
Germ Free ◽  
Myeloid Progenitor ◽  
Large Expansion ◽  
Myeloid Progenitor Cells ◽  
Myeloid Progenitors

Expansion of mature neutrophils has been observed in mice lacking the murine interleukin (IL) 8 receptor homolog [mIL-8Rh(-/-)], and human (hu) IL-8 suppresses proliferation of primitive myeloid cells in vitro and in vivo. To evaluate involvement and relevance of murine IL-8 receptor homolog (mIL-8Rh) in negative regulation of myelopoiesis, we studied mIL-8Rh(-/-) and (+/+) mice raised in a normal or germ-free environment. Immature myeloid progenitors from mIL-8Rh(+/+) mice bred under normal or germ-free conditions were significantly suppressed in vitro by recombinant huIL-8, macrophage inflammatory protein (MIP)-1 alpha, platelet factor (PF) 4, interferon inducible protein (IP) 10, monocyte chemotactic peptide (MCP) 1, and H-ferritin. In contrast, progenitors from mIL-8Rh(-/-) mice were insensitive to inhibition by IL-8, but not to these other chemokines and H-ferritin. Mouse MIP-2, a ligand for mIL-8Rh, suppressed progenitors from normal but not mIL-8Rh(-/-) mice. Under normal environmental conditions, enhanced numbers of myeloid progenitors were found in femur, spleen, and blood of mIL-8Rh(-/-) compared with mIL-8Rh(+/+) mice. Numbers of myeloid progenitors were greatly decreased in mIL-8Rh(-/-)and (+/+) mice in germ-free conditions, and were either not significantly enhanced in mIL-8Rh(-/-) mice compared with (+/+) mice or were only moderately so. Differences in progenitors/organ between a germ-free and normal environment were greater for the mIL-8Rh(-/-) mice. These results document selective insensitivity of myeloid progenitor cells from mIL-8Rh(-/-) mice to inhibition by huIL-8 and mouse MIP-2 and a large expansion of myeloid progenitors in these mice, the latter effect being environmentally inducible. This provides strong support for a negative myeloid regulatory role played by the mIL-8Rh in vivo, whose active ligand may be MIP-2.

scholarly journals Sensitization of Hematopoietic Stem and Progenitor Cells to Trimetrexate Using Nucleoside Transport Inhibitors

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3546-3554 ◽  
Author(s):  
James A. Allay ◽  
H. Trent Spencer ◽  
Sarah L. Wilkinson ◽  
Judith A. Belt ◽  
Raymond L. Blakley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Nucleoside Transport ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor Cells ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors ◽  
Myeloid Progenitors

Abstract Antifolates such as methotrexate (MTX) and trimetrexate (TMTX) are widely used in the treatment of cancer and nonmalignant disorders. Transient, yet sometimes severe myelosuppression is an important limitation to the use of these drugs. It has previously been shown that clonogenic myeloid progenitors and colony-forming units-spleen are resistant to antifolates, suggesting that myelotoxicity occurs late in hematopoietic development. The goal of this study was to define the mechanisms by which primitive hematopoietic cells resist the toxic effects of antifolate drugs. To test the hypothesis that myeloid progenitors may salvage extracellular nucleotide precursors to resist TMTX toxicity, a defined liquid culture system was developed to measure TMTX toxicity in expanding progenitor populations. These in vitro experiments showed that both human and murine progenitors can resist TMTX toxicity by importing thymidine and hypoxanthine from the serum. As predicted from these findings, several drugs that block thymidine transport sensitized progenitors to TMTX in vitro, although to differing degrees. These nucleoside transport inhibitors were used to test whether progenitors and hematopoietic stem cells (HSCs) could be sensitized to TMTX in vivo. Treatment of mice with TMTX and nitrobenzylmercaptopurineriboside phosphate (NBMPR-P), a potent transport inhibitor, caused significant depletions of clonogenic progenitors within the bone marrow (20-fold) and spleen (6-fold). Furthermore, NBMPR-P administration dramatically sensitized HSCs to TMTX, with dual-treated mice showing a greater than 90% reduction in bone marrow repopulating activity. These studies demonstrate that both myeloid progenitor cells and HSCs resist TMTX by using nucleotide salvage mechanisms and that these pathways can be pharmacologically blocked in vivo using nucleoside transport inhibitors. These results have important implications regarding the use of transport inhibitors for cancer therapy and for using variants of dihydrofolate reductase for in vivo selection of genetically modified HSCs.

scholarly journals ANTIGENIC DIFFERENCES BETWEEN HEMOPOIETIC STEM CELLS AND MYELOID PROGENITORS

1974 ◽  
Vol 139 (6) ◽  
pp. 1621-1627 ◽  
Author(s):  
Gerrit J. Van den Engh ◽  
Edward S. Golub
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Myeloid Progenitors

Bone marrow contains pluripotent stem cells which give rise to colonies when injected into irradiated syngenic hosts as well as more differentiated progenitor cells of the myeloid cell which are able to form colonies in vitro. Antisera against brain is known to contain antistem cell antibody. The present experiments were designed to determine if the myeloid progenitor cell still expresses the stem cell antigen. Bone marrow cells were treated with antibrain antiserum plus complement and then survival of stem cells was determined by injection into irradiated hosts. Survival of myeloid progenitor cells was determined by culturing the cells in vitro. It was found that stem cells were eliminated by the antiserum but that myeloid progenitors were not. Inefficient in vitro lysis was ruled out as the reason for this difference since in vitro colonies were not reduced when the cells were treated with anti-immunoglobulin or after passage through an irradiated host. In the differentiation from stem cell to myeloid progenitor there is an associated surface antigen change.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

scholarly journals The influence in vivo of murine colony-stimulating factor-1 on myeloid progenitor cells in mice recovering from sublethal dosages of cyclophosphamide

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 913-918 ◽  
Author(s):  
HE Broxmeyer ◽  
DE Williams ◽  
S Cooper ◽  
A Waheed ◽  
RK Shadduck
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Colony Stimulating Factor ◽  
Myeloid Progenitor ◽  
Accessory Cells ◽  
Myeloid Progenitor Cells ◽  
In Vivo Administration ◽  
Stimulating Factor

Abstract Pure murine colony-stimulating factor-1 (CSF-1) was assessed for its effects in vivo in mice pretreated seven days earlier with a sublethal dosage of cyclophosphamide. The multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in these mice were in a slowly cycling or noncycling state. Intravenous administration of 20,000 units of CSF-1 to these mice stimulated the hematopoietic progenitors into a rapidly cycling state in the marrow and spleen within three hours. Significant increases in absolute numbers of marrow and spleen CFU-GM and spleen BFU-E and CFU-GEMM were also detected. No endotoxin was detected in the CSF-1 preparation by Limulus lysate assay, and treatment of CSF-1 at 100 degrees C for 20 to 30 minutes completely inactivated the in vitro and in vivo stimulating effects. The effects of CSF-1 were not mimicked by the in vivo administration of 0.1 to 10 ng Escherichia coli lipopolysaccharide. These results suggest that the effects of CSF-1 in vivo were not due to contaminating endotoxin or to a nonspecific protein effect. CSF-1 did not enhance colony formation by BFU-E or stimulate colony formation by CFU-GEMM in vitro, thus suggesting that at least some of the effects of CSF-1 noted in vivo are probably indirect and mediated by accessory cells.

scholarly journals Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2229-2236 ◽  
Author(s):  
MJ Robertson ◽  
RJ Soiffer ◽  
AS Freedman ◽  
SL Rabinowe ◽  
KC Anderson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Red Blood Cell Transfusion ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Acute Myeloid

Abstract The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte- macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA- compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

scholarly journals Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3710-3719 ◽  
Author(s):  
C Mantel ◽  
Z Luo ◽  
J Canfield ◽  
S Braun ◽  
C Deng ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Proliferative Response ◽  
Myeloid Cell ◽  
Cyclin Dependent Kinase ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip- 1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip- 1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.

HoxA10 Regulates Myeloid Progenitor Proliferation by Activating Transcription of the Gene Encoding Transforming Growth Factor Beta 2.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1572-1572
Author(s):  
Chirag Shah ◽  
Hao Wang ◽  
Elizabeth A. Eklund
Keyword(s):  
Progenitor Cells ◽  
Transforming Growth Factor Beta ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Growth Factor Beta ◽  
Differentiation Block

Abstract Abstract 1572 HoxA10 is a homeodomain transcription factor which functions as a myeloid leukemia promoter. Correlative clinical studies found that increased expression of a group of HoxA proteins, including HoxA10, in acute myeloid leukemia (AML) was associated with poor prognosis. In murine models, overexpression of HoxA10 in the bone marrow was associated with development of a myeloproliferative disease which progressed to AML with time. These results suggested that HoxA10-overexpression dysregulated cell proliferation and/or survival, and predisposed to acquisition of additional mutations which led to differentiation block and AML. Additional investigations, we and others demonstrated that HoxA10 overexpression in murine hematopoietic stem cells (HSC) expanded the granulocyte/monocyte progenitor (GMP) population in vitro and in vivo. Despite this information about the impact of HoxA10 overexpression on myeloid leukemogenesis, the mechanisms by which HoxA10 exerts this effect are largely unknown. To investigate such mechanisms, we have been identifying HoxA10 target genes. In previous studies, we identified a number of HoxA10 target genes that encode phagocyte effector proteins. HoxA10 represses transcription of these gene in myeloid progenitors, and decreased HoxA10 repression activity contributes to phenotypic differentiation as myelopoiesis proceeds. This provided a potential mechanism for HoxA10 involvement in differentiation block, but not progenitor survival or expansion. We used a chromatin immuno-precipitation based approach to identify additional HoxA10 target genes involved in these activities. Previously, we reported that HoxA10 activated the DUSP4 gene in myeloid progenitor cells. This gene encodes Mitogen Activated Protein Kinase Phosphatase 2 (Mkp2) which inhibits Jnk-induced apoptosis in myeloid progenitor cells. This provided a mechanism for increased cell survival in HoxA10-overexpressing cells. In the current studies, we identified TGFB2 as a HoxA10 target gene. This gene encodes Transforming Growth Factor Beta 2 (TgfB2) a member of the TgfB super family of cytokines. Similar to TgfB1 and 3, TgfB2 interacts with TgfB-receptors I and II. However, unlike these more classical family members, TgfB2 induces proliferation of hematopoietic stem and progenitor cells. We found that HoxA10 activated the TGFB2 promoter via tandem cis elements in the proximal promoter. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing GMP and leukemia cells in vitro. Increased proliferation in HoxA10-overexpressing cells involved activation of the MAP kinase pathway in a TgfB2 dependent manner. These studies identify autocrine production of pro-proliferative cytokines as a novel mechanism for the function of Hox proteins. These findings have implications for ex vivo expansion of HSC and myeloid progenitors for tissue engineering. These result also have implications for therapeutic approaches to poor prognosis AML characterized by increased Hox expression. Disclosures: No relevant conflicts of interest to declare.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

Enforced expression of cyclin D2 enhances the proliferative potential of myeloid progenitors, accelerates in vivo myeloid reconstitution, and promotes rescue of mice from lethal myeloablation

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 986-992 ◽  
Author(s):  
Yutaka Sasaki ◽  
Christina T. Jensen ◽  
Stefan Karlsson ◽  
Sten Eirik W. Jacobsen
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Cyclin D2 ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Myeloid Progenitors

AbstractSevere and prolonged cytopenias represent a considerable problem in clinical stem cell transplantations. Cytokine-induced ex vivo expansion of hematopoietic stem and progenitor cells has been intensively explored as a means of accelerating hematopoietic recovery following transplantation but have so far had limited success. Herein, overexpression of D-type cyclins, promoting G0/G1 to S transition, was investigated as an alternative approach to accelerate myeloid reconstitution following stem cell transplantation. With the use of retroviral-mediated gene transfer, cyclin D2 was overexpressed in murine bone marrow progenitor cells, which at limited doses showed enhanced ability to rescue lethally ablated recipients. Competitive repopulation studies demonstrated that overexpression of cyclin D2 accelerated myeloid reconstitution following transplantation, and, in agreement with this, cyclin D2–transduced myeloid progenitors showed an enhanced proliferative response to cytokines in vitro. Furthermore, cyclin D2–overexpressing myeloid progenitors and their progeny were sustained for longer periods in culture, resulting in enhanced and prolonged granulocyte production in vitro. Thus, overexpression of cyclin D2 confers myeloid progenitors with an enhanced proliferative and granulocyte potential, facilitating rapid myeloid engraftment and rescue of lethally ablated recipients.

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