Transgene expression study of CXCR4 active mutants. Potential prospects in up-modulation of homing and engraftment efficiency of hematopoietic stem/progenitor cells.
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ABSTRACT:
SUBMITTER: Sharma M
PROVIDER: S-EPMC4594461 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:SDF-1/CXCR4 axis plays a principle role in the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs), a process that defines cells ability to reach and seed recipient bone marrow niche following their intravenous infusion. However, the proper functioning of CXCR4 downstream signaling depends upon consistent optimal expression of both SDF-1 ligand and its receptor CXCR4, which in turn is variable and regulated by several factors. The constitutive active mutants of CXCR4 (N119A and N119S) being able to induce autonomous downstream signaling, overcome the limitation of ligand-receptor interaction for induction of CXCR4 signaling. Therefore, we intended to explore their potential in Chemotaxis; a key cellular process which crucially regulates cells homing to bone marrow. In present study, Tet-on inducible gene expression vector system was used for doxycycline inducible regulated transgene expression of CXCR4 active mutants in hematopoietic stem progenitor cell line K-562. Both of these mutants revealed significantly enhanced Chemotaxis to SDF-1 gradient as compared to wild type. Furthermore, gene expression profiling of these genetically engineered cells as assessed by microarray analysis revealed the up-regulation of group of genes that are known to play a crucial role in CXCR4 mediated cells homing and engraftment. Hence, this study suggest the potential prospects of CXCR4 active mutants in research and development aimed to improve the efficiency of cells in the mechanism of homing and engraftment process.
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Project description:Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.
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Project description:Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of immunologic disorders. For effective transplant, HSCs must traffic from the peripheral blood to supportive bone marrow niches. We previously showed that HSC trafficking can be enhanced by ex vivo treatment of hematopoietic grafts with 16-16 dimethyl prostaglandin E2 (dmPGE2). While exploring regulatory molecules involved in dmPGE2 enhancement, we found that transiently increasing the transcription factor hypoxia-inducible factor 1-? (HIF1?) is required for dmPGE2-enhanced CXCR4 upregulation and enhanced migration and homing of stem and progenitor cells and that pharmacologic manipulation of HIF1? is also capable of enhancing homing and engraftment. We also now identify the specific hypoxia response element required for CXCR4 upregulation. These data define a precise mechanism through which ex vivo pulse treatment with dmPGE2 enhances the function of hematopoietic stem and progenitor cells; these data also define a role for hypoxia and HIF1? in enhancement of hematopoietic transplantation.
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Project description:Adhesion is a key component of hematopoietic stem cell regulation mediating homing and retention to the niche in the bone marrow. Here, using an RNA interference screen, we identify cytohesin 1 (CYTH1) as a critical mediator of adhesive properties in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs). Knockdown of CYTH1 disrupted adhesion of HSPCs to primary human mesenchymal stroma cells. Attachment to fibronectin and ICAM1, 2 integrin ligands, was severely impaired, and CYTH1-deficient cells showed a reduced integrin β1 activation response, suggesting that CYTH1 mediates integrin-dependent functions. Transplantation of CYTH1-knockdown cells to immunodeficient mice resulted in significantly lower long-term engraftment levels, associated with a reduced capacity of the transplanted cells to home to the bone marrow. Intravital microscopy showed that CYTH1 deficiency profoundly affects HSPC mobility and localization within the marrow space and thereby impairs proper lodgment into the niche. Thus, CYTH1 is a novel major regulator of adhesion and engraftment in human HSPCs through mechanisms that, at least in part, involve the activation of integrins.
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Project description:Migration and bone marrow (BM) homing of hematopoietic stem progenitor cells (HSPCs) is regulated by several signaling pathways, and here we provide evidence for the involvement in this process of hematopoietic-specific phospholipase C-β2 (PLC-β2). This enzyme is involved in release of intracellular calcium and activation of protein kinase C (PKC). Recently we reported that PLC-β2 promotes mobilization of HSPCs from BM into peripheral blood (PB), and this effect is mediated by the involvement of PLC-β2 in the release of proteolytic enzymes from granulocytes and its role in disintegration of membrane lipid rafts. Here we report that, besides the role of PLC-β2 in the release of HSPCs from BM niches, PLC-β2 regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including SDF-1, S1P, C1P, and ATP. Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release in PLC-β2-KO mice and a high baseline level of heme oxygenase 1 (HO-1), an enzyme that negatively regulates cell migration.
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Project description:Cell-based therapy is a possible avenue for the treatment of Duchenne muscular dystrophy (DMD), an X-linked skeletal muscle-wasting disease. We have demonstrated that cultured myogenic progenitors derived from the adult skeletal muscle side population can engraft into dystrophic fibers of non-irradiated, non-chemically injured mouse models of DMD (mdx(5cv)) after intravenous and intraarterial transplantation, with engraftment rates approaching 10%. In an effort to elucidate the cell-surface markers that promote progenitor cell extravasation and engraftment after systemic transplantation, we found that expression of the chemokine receptor CXCR4, whose ligand SDF-1 is overexpressed in dystrophic muscle, enhances the extravasation of these cultured progenitor cells into skeletal muscle after intraarterial transplantation. At 1 day post-transplantation, mice that received CXCR4-positive enhanced green fluorescent protein (eGFP)-positive cultured cells derived from the skeletal muscle side population displayed significantly higher amounts of eGFP-positive mononuclear cells in quadriceps and tibialis anterior than mice that received CXCR4-negative eGFP-positive cells derived from the same cultured population. At 30 days posttransplantation, significantly higher engraftment rates of donor cells were observed in mice that received CXCR4-positive cells compared with mice transplanted with CXCR4-negative fractions. Our data suggest that CXCR4 expression by muscle progenitor cells increases their extravasation into skeletal muscle shortly after transplantation. Furthermore, this enhanced extravasation likely promotes higher donor cell engraftment rates over time.
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Project description:Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine stromal cell-derived factor-1 (SDF-1) and its G-protein-coupled receptor C-X-C motif chemokine receptor 4 (CXCR4), which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1-directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13, and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cells not only inhibited SDF-1-directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13, and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis, and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.
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Project description:The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in ?-1,4-galactosyltransferase-1 (?4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in ?4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24?h after transplantation of ?4GalT-1-deficient BM cells, suggesting that ?4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.
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Project description:Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2?(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.
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Project description:Mobilized blood has supplanted bone marrow (BM) as the primary source of hematopoietic stem cells for autologous and allogeneic stem cell transplantation. Pharmacologically enforced egress of hematopoietic stem cells from BM, or mobilization, has been achieved by directly or indirectly targeting the CXCL12/CXCR4 axis. Shortcomings of the standard mobilizing agent, granulocyte colony-stimulating factor (G-CSF), administered alone or in combination with the only approved CXCR4 antagonist, Plerixafor, continue to fuel the quest for new mobilizing agents. Using Protein Epitope Mimetics technology, a novel peptidic CXCR4 antagonist, POL5551, was developed. In vitro data presented herein indicate high affinity to and specificity for CXCR4. POL5551 exhibited rapid mobilization kinetics and unprecedented efficiency in C57BL/6 mice, exceeding that of Plerixafor and at higher doses also of G-CSF. POL5551-mobilized stem cells demonstrated adequate transplantation properties. In contrast to G-CSF, POL5551 did not induce major morphological changes in the BM of mice. Moreover, we provide evidence of direct POL5551 binding to hematopoietic stem and progenitor cells (HSPCs) in vivo, strengthening the hypothesis that CXCR4 antagonists mediate mobilization by direct targeting of HSPCs. In summary, POL5551 is a potent mobilizing agent for HSPCs in mice with promising therapeutic potential if these data can be corroborated in humans.
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