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

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.

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

Blood ◽

10.1182/blood.v108.11.2544.2544 ◽

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.

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

Blood ◽

10.1182/blood-2002-12-3893 ◽

2003 ◽

Vol 101 (12) ◽

pp. 4680-4686 ◽

Cited By ~ 144

Author(s):

Kent W. Christopherson ◽

Scott Cooper ◽

Hal E. Broxmeyer

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mouse Bone Marrow ◽

Peptidase Activity ◽

Hematopoietic Stem ◽

Migratory Response ◽

Stromal Cell Derived Factor ◽

Marrow Cells

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

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Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽

10.1182/blood.v104.11.1293.1293 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1293-1293

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

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Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽

10.1182/blood.v104.11.2674.2674 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2674-2674

Author(s):

Seiji Fukuda ◽

Hal E. Broxmeyer ◽

Louis M. Pelus

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Leukemia Cells ◽

Flt3 Ligand ◽

Hematopoietic Progenitor ◽

Short Term ◽

Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

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Slug Plays an Essential Role in the Radioprotection of Hematopoietic Progenitors In Vivo by Antagonizing p53-Mediated Apoptotic Pathways.

Blood ◽

10.1182/blood.v104.11.31.31 ◽

2004 ◽

Vol 104 (11) ◽

pp. 31-31

Author(s):

Wen-Shu Wu ◽

Dong Xu ◽

Stefan Heinrichs ◽

A. Thomas Look

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Leukemia Cells ◽

Dependent Manner ◽

Wild Type ◽

Γ Irradiation ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Myeloid Progenitors

Abstract An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.

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Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

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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 ◽

10.1182/blood.v122.21.2476.2476 ◽

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.

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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 ◽

10.1182/blood-2003-07-2277 ◽

2004 ◽

Vol 104 (4) ◽

pp. 986-992 ◽

Cited By ~ 17

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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IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors

Journal of Experimental Medicine ◽

10.1084/jem.20200164 ◽

2020 ◽

Vol 217 (9) ◽

Cited By ~ 3

Author(s):

James W. Swann ◽

Lada A. Koneva ◽

Daniel Regan-Komito ◽

Stephen N. Sansom ◽

Fiona Powrie ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Chronic Inflammation ◽

Inflammatory Disease ◽

Erythropoietin Receptor ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment.

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Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽

10.1182/blood.v93.1.80 ◽

1999 ◽

Vol 93 (1) ◽

pp. 80-86 ◽

Cited By ~ 17

Author(s):

Shai Erlich ◽

Silvia R.P. Miranda ◽

Jan W.M. Visser ◽

Arie Dagan ◽

Shimon Gatt ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Progenitor Cells ◽

Fetal Liver ◽

Acid Sphingomyelinase ◽

Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

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