Upregulation of MT1-MMP by Molecules Present in Leukaphereis Products Primes CD34+ Cell Homing.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5273-5273
Author(s):  
Neeta Shirvaikar ◽  
Jencet Montano ◽  
A.Robert Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Mmp Inhibitor ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood ◽  
Stimulation Of

Abstract Hematopoietic stem/progenitor cells (HSPC) derived from mobilized peripheral blood engraft faster than those from bone marrow (BM) or cord blood (CB). We recently postulated that leukapheresis product supernatants (collected from G-CSF-mobilized patients) and their components (fibrinogen, fibronectin, thrombin, hyaluronic acid (HA) and C3 cleavage fragments) have a priming effect on HSPC homing by enhancing chemotactic responses to SDF-1 through increased incorporation of CXCR4 into membrane lipid rafts and stimulation of matrix metalloproteinase (proMMP-2 and proMMP-9) secretion in these cells (Blood2005; 105:40). As MT1-MMP has been shown to activate latent forms of MMP-2 and (indirectly) MMP-9 and is known to localize proteolytic activity at the leading edge of migrating cells, we decided to investigate MT1-MMPs’ role in the priming of HSPC homing. We observed that stimulation of BM and CB CD34+ cells with priming agents (HA, fibrinogen or thrombin) not only increased secretion of proMMP-2 and proMMP-9 (zymography) but also highly upregulated levels of MT1-MMP transcript (RT-PCR) and protein (Western blotting) in these cells. Moreover, trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), enhanced by priming agents, was inhibited by the potent MT1-MMP inhibitor epigallocatechin-3-gallate. Furthermore, priming agents (HA, fibrinogen and thrombin) increased levels of active MMP-2 in co-cultures of stromal cells (endothelial cells and BM fibroblasts) with CD34+ cells. To elucidate the mechanism of MT1-MMP upregulation by priming agents, we evaluated whether they affected MT1-MMP incorporation in ganglioside M1-enriched membrane lipid rafts in the same way as for CXCR4. We found that stimulation of hematopoietic and THP-1 cells with HA, fibrinogen and thrombin increased incorporation of MT1-MMP into membrane lipid rafts and these observations were confirmed using confocal microscopy. Hence it appears that MT1-MMP, like CXCR4, localizes in membrane lipid rafts, and this is enhanced by priming agents, leading to better homing of HSPC.

A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3374-3374
Author(s):  
Neeta Shirvaikar ◽  
Ali Jalili ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Protein Expression ◽  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
And Migration ◽  
Stimulation Of

Abstract Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.

Fibrinogen Enhances Homing-Related Responses of CD34+ by Incorporating Membrane Type1-Matrix Metalloproteinase into Membrane Lipid Rafts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3188-3188
Author(s):  
Neeta Shirvaikar ◽  
A. Robert Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Lipid Rafts ◽  
Stromal Cells ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Chemokine Gradient ◽  
Cord Blood Cells ◽  
First Time ◽  
Mobilized Peripheral Blood

Abstract Clinical studies have shown that transplanted mobilized peripheral blood (mPB) hematopoietic stem/progenitor cells (HSPC) home faster to the bone marrow (BM) than harvested BM or cord blood cells. We previously showed that fibrinogen has a priming effect on HSPC homing by enhancing the chemotactic responses of CD34+ cells towards a low SDF-1 gradient by incorporating CXCR4 into membrane lipid rafts (Blood2005; 105:40). In this study we further investigated the mechanism of priming by determining the effect of fibrinogen on matrix metalloproteinases (MMPs) expressed by CD34+ cells, particularly MT1-MMP which is localized on the leading edge of migrating cells and not only activates latent MMPs but by itself degrades several extracellular matrix (ECM) components such as collagens, laminin, fibrin and fibronectin. We found that fibrinogen highly upregulated MT1-MMP protein expression (Western blot) in CD34+ cells as well as proMMP-2 and proMMP-9 secretion (zymography); primed trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP; and stimulated MMP-2 activation in co-cultures of stromal cells (BM fibroblasts and HUVEC) with CD34+ cells. Moreover, we demonstrate by confocal microscopy, for the first time, that in CD34+ cells MT1-MMP is localized in the GM1-fraction of lipid rafts where it co-localizes with CXCR4; this co-localization is enhanced when CD34+ cells are stimulated with fibrinogen. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibited MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards an SDF-1 gradient. Thus we conclude that fibrinogen enhances homing-related responses of CD34+ cells towards SDF-1 by increased incorporation and co-localization of CXCR4 and MT1-MMP in membrane lipid rafts. Further, we postulate that while the presence of CXCR4 in lipid rafts allows the cells to better sense the SDF-1 chemokine gradient, the upregulated MT1-MMP in the lipid rafts facilitates their migration through the ECM and possibly towards the BM niches.

MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3536-3536
Author(s):  
Neeta Shirvaikar ◽  
Leah A. Marquez-Curtis ◽  
Andrew Shaw ◽  
A. Robert Turner ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Flow Cytometry ◽  
Steady State ◽  
Western Blot ◽  
Lipid Rafts ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3536 Poster Board III-473 Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures: No relevant conflicts of interest to declare.

Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 40-48 ◽  
Author(s):  
Marcin Wysoczynski ◽  
Ryan Reca ◽  
Janina Ratajczak ◽  
Magda Kucia ◽  
Neeta Shirvaikar ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Lipid Rafts ◽  
Adhesion Molecule ◽  
Actin Polymerization ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
Calcium Flux ◽  
Urokinase Plasminogen Activator Receptor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte–colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)– and bone marrow (BM)–derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.

Impaired Engraftment of Hematopoietic Stem/Progenitor Cells (HSPC) in Immunodefcient Mice Supports an Important Role of Complement System for Optimal Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 338-338
Author(s):  
Reca Ryan ◽  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Progenitor Cells ◽  
Lipid Rafts ◽  
Membrane Lipid ◽  
Scid Mice ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cell Counts ◽  
Cd34 Cells

Abstract Recently we demonstrated that conditioning for transplantation (radio-chemotherapy) activates complement (C) in bone marrow (BM) and that the third complement component (C3) cleavage fragments (C3a and desArgC3a) increase responsiveness of hematopoietic stem/progenitor cells (HSPC) to stromal-derived factor (SDF)-1 gradient by enhancing the incorporation of CXCR4 into membrane lipid rafts – what enables its better interaction with small GTPases from the Rho/Rac family (Blood2003, 101, 3784, Blood2005, 105, 40–48). Based on these data we hypothesized that C could affect the homing/engraftment of HSPC. Thus we performed transplant experiments in several strains of immunodeficient animals. First, we noticed that lethally irradiated NOD/SCID mice engrafted worse with wt HSPC as compared to wt animals (~30% decrease in a presence of donor-derived clonogeneic CFU-GM in marrow cavities 24 hrs after transplantation). This impaired engraftment correlated with the lack of C activation in BM after conditioning for transplantation by lethal irradiation. The lack of C activation in NOD/SCID mice after conditioning for transplant could be explained by a lack of IgM antibodies that activate C by classical IgM-dependent pathway. Next, to learn more on the molecular mechanisms of C cascade activation during conditioning for transplantation and the role of the C3a-C3aR axis in engraftment of HSPC we studied engraftment i) of wild type (wt) murine HSPC in immunodeficient mice (C3−/− and C3aR−/−) and ii) murine HSPC derived from C3aR−/− or C3−/− deficient mice in wild type littermates. The engraftment of HSPC was evaluated by i) recovery of peripheral blood cell counts in transplanted animals, ii) number of CFU-S colonies and iii) number of clonogeneic progenitors in marrow cavities 16 day after transplantation. We noticed that both C3−/− and C3aR−/− mice had impaired engraftment with wt HSPC. At the same time HSPC from C3aR−/− mice but not C3−/− animals showed poor engraftment in wt recipients. This suggests that i) C3aR expressed on HSPC interacts with C3a generated during C-activation in BM environment and ii) that this interaction is important for optimal homing of HSPC. To support further this notion, human CD34+ cells were exposed to nontoxic doses of C3aR antagonist SB290157 and transplanted into NOD/SCID mice. Subsequently, 24 hrs after transplantation cells were isolated from the marrow cavities and stimulated to grow human CFU-GM colonies. By employing this assay we noticed reduced engraftment of human CD34+ cells (~30%, p<0.0001) as compared to mice transplanted with control CD34+ cells unexposed to SB290157. These data allow for the following conclusions: i) C is activated in BM during conditioning for transplantation by irradiation ii) C is activated after exposure of a natural neoepitope in damaged marrow which is recognized by natural IgM activating C via the classical pathway, iii) C3 cleavage product C3a binds to C3aR on transplanted HSPC and increases incorporation of CXCR4 into membrane lipid rafts enhancing responsiveness of HSPC to an SDF-1 gradient, and finally iv) a proper interplay between the C system and SDF-1-CXCR axis ensures optimal homing of HSPC.

Upregulation of MT1-MMP Expression by Hyaluronic Acid Enhances Homing-Related Responses of Hematopoietic CD34+ Cells to an SDF-1 Gradient.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2889-2889 ◽  
Author(s):  
Neeta Shirvaikar ◽  
Jencet Montano ◽  
Andrew R. Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Hyaluronic Acid ◽  
Leading Edge ◽  
Cell Surface Receptor ◽  
Migration And Invasion ◽  
Hematopoietic Stem ◽  
Tumor Cell Migration ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Surface Receptor ◽  
And Migration

Abstract Hyaluronic acid (HA), the bone marrow (BM) extracellular matrix microenvironment (ECM) component, not only supports cell adhesion but also promotes migration and homing of hematopoietic stem/progenitor cells (HSPC) by interacting with its cell surface receptor CD44. CD44 has been shown to co-localize with matrix metalloproteinases (MMPs), particularly membrane-type (MT)-1 MMP and MMP-9, at the leading edge of migrating tumor cells, and the cleavage of CD44 by MT1-MMP is critical for tumor cell migration and invasion. MT1-MMP has strong pericellular proteolytic activity and also activates latent forms of MMP-2 and MMP-9. In this study we examined the effect of HA on MT1-MMP expression and migration of BM, peripheral blood and cord blood CD34+ cells. We found that HA upregulates mRNA for MT1-MMP and MMP-9, increases MT1-MMP protein (as evaluated by Western blotting) and stimulates MMP-9 and MMP-2 activity (as determined by zymography) in CD34+ cells. In chemotaxis assays HA alone did not show any chemotactic activity but primed the chemotaxis of CD34+ cells to a low SDF-1 gradient (10 ng/mL) and their trans-Matrigel chemoinvasion to a low SDF-1 gradient. Similarly, SDF-1 besides stimulating MMP-2 and MMP-9 (as we previously described in Exp Hematol2000; 28:1274), also upregulated MT1-MMP in CD34+ cells. In addition, we found that the HA enhanced activation of latent MMP-2 in co-cultures of CD34+ cells with HUVEC. In conclusion, we demonstrate for the first time that (i) HA primes CD34+ cell chemotaxis and chemoinvasion to a low SDF-1 gradient; (ii) that both HA and SDF-1 stimulate MT1-MMP, MMP-2 and MMP-9 in CD34+ cells, and (iii) HA enhances activation of proMMP-2 in the ECM. Hence the interaction of HA and SDF-1 with MT1-MMP could play an important role in HSPC migration and homing and we postulate that pretreatment of HSPC with HA before transplantation could enhance their homing by inducing expression of MMPs that is optimal for stem cell engraftment.

Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1652-1652
Author(s):  
Patrick Ziegler ◽  
Steffen Boettcher ◽  
Hildegard Keppeler ◽  
Bettina Kirchner ◽  
Markus G. Manz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Human Mesenchymal Stem Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

scholarly journals A Novel View of the Role of Prostaglandin E2 (PGE2) in Facilitating Engraftment of HSPCs By Activating the NOX2-ROS-Nlrp3 Inflammasome Axis to Incorporate the CXCR4 Receptor into Membrane Lipid Rafts

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Magdalena Kucia ◽  
Kamila Bujko ◽  
Arjun Thapa ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Nlrp3 Inflammasome ◽  
Membrane Lipid ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Cxcr4 Receptor ◽  
Nlrp3 Inflammasomes

Background . It is known that prostaglandin E2 (PGE2) increases the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs). However, aside from its role in upregulation of CXCR4 receptor expression on the surface of these cells, the exact mechanism has not been proposed. We have demonstrated in the past that an important step enabling the migration of HSPCs is the incorporation of CXCR4 into membrane lipid rafts on the leading surface (leading edge, in two dimensions) of migrating cells, which facilitates its interaction with cell migration signaling pathways (Wysoczynski M et al. Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient. Blood. 2005;105(1):40-48). Recently, we reported that Nlrp3 inflammasome-deficient HSPCs show a defect in lipid raft formation that results in defective migration of these cells in response to an SDF-1 gradient and their defective homing and engraftment after transplantation (Adamiak, M et al. Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts. Stem Cell Rev and Rep (2020). https://doi.org/10.1007/s12015-020-10005-w). An important activator of Nlrp3 inflammasomes is reactive oxygen species (ROS). Importantly, the enzyme that generates ROS, known as NADPH oxidase 2 (NOX2), is also associated with cell membrane lipid rafts. Hypothesis. Given the known roles of PGE2, membrane lipid rafts, and the Nlrp3 inflammasome in migration, homing, and engraftment of HSPCs, we hypothesized that PGE2 signaling promotes Nlrp3 inflammasome activation in a Nox2-ROS-dependent manner that results in incorporation of CXCR4 into membrane lipid rafts, which better explains the role of PGE2 in these phenomena.Materials and Methods. To test this hypothesis, murine SKL and human CD34+ cells enriched for HSPCs were stimulated with PGE2 to evaluate activation of genes of the Nlrp3 inflammasome complex at the mRNA and protein levels. Next, HSPCs from Nox2-KO mice were tested for membrane lipid raft formation in functional chemotaxis assays in response to SDF-1 gradients under conditions promoting membrane lipid raft formation. Formation of membrane lipid rafts in Nox2-KO cells was also evaluated by confocal analysis in the presence or absence of PGE2. Finally, the effect of the PGE2-Nox2-Nlrp3 inflammasome axis on the formation of membrane lipid rafts was evaluated in the presence of the ROS scavenger N-acethyl-cysteine (NAC). Results. We provide for the first time evidence that PGE2 activates Nlrp3 inflammasomes in HSPCs in a Nox2-ROS-dependent manner. This Nlrp3 inflammasome activation increases at the leading surface of migrating HSPCs with incorporation of the CXCR4 receptor into membrane lipid rafts. Formation of membrane lipid rafts was absent in Nox2-KO and Nlrp3-KO mouse HSPCs and in normal wild type cells after their exposure to NAC. Moreover, we also observed that Nox2-KO and Nlrp3-KO mice had a lower basal level of CXCR4 expression. Conclusions. Our results for the first time explain the role of PGE2 in promoting homing and migration of HSPCs, which occurs in response to PGE2 by activation of the Nox2-ROS-Nlrp3 inflammasome axis and thereby promotes incorporation of the CXCR4 receptor into membrane lipid rafts. Moreover, basal expression of the CXCR4 receptor was at a low level on the surface of HSPCs from Nlrp3-KO mice. Thus, our results provide evidence for the importance of the Nox2-ROS-Nlrp3 inflammasome axis in PGE2-mediated homing and engraftment of HSPCs and the role of PGE2-mediated lipid raft formation for optimal responsiveness of CXCR4 to SDF-1 in the BM microenvironment. Disclosures No relevant conflicts of interest to declare.

STAT5-Induced Self-Renewal and Impaired Myelopoiesis of Human Hematopoietic Stem/Progenitor Cells Involves Downmodulation of C/EBPα.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 268-268
Author(s):  
Jan Jacob Schuringa ◽  
Bart-Jan Wierenga ◽  
Hein Schepers ◽  
Malcolm A.S. Moore ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Rt Pcr ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Affymetrix Microarray ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
Proliferative Advantage

Abstract Previously, we demonstrated that enforced activation of STAT5 in human cord blood (CB)-derived stem/progenitor cells results in enhanced long-term stem cell self-renewal and impaired myelopoiesis (J.J.Schuringa et al, J.Exp.Med. 2004;200:623). Now, C/EBPα was identified as a critical transcription factor that is downregulated by STAT5. Affymetrix microarray analysis on STAT5A(1*6)-transduced CD34+ cells identified C/EBPα as the most prominently downregulated gene (−3.3 fold), and these data were confirmed by RT-PCR and Western blotting. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34+ cells by using a retroviral approach. Re-expression of C/EBPα in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis as determined by morphological analyses, FACS analyses for myeloid markers such as CD11b, CD14 and CD15, and RT-PCR for myeloid-restricted genes such as g-csfr. While enforced activation of STAT5A resulted in accelerated erythropoiesis, this was blocked when C/EBPα was re-introduced into STAT5A(1*6) cells. Similarly, the proliferative advantage imposed on CD34+ cells by STAT5A(1*6) depended on the downmodulation of C/EBP as reintroduction of C/EBPα in these cells induced a quick cell cycle arrest and the onset of myeloid differentiation. At the stem/progenitor cell level, LTC-IC frequencies were elevated from 0.5% to 11% by STAT5A(1*6) as compared to controls, but these elevated LTC-IC frequencies were strongly reduced when C/EBPα was reintroduced in STAT5A(1*6) cells. Enumeration of progenitors in methylcellulose assays revealed similar results, the number of CFCs was reduced over 10-fold when C/EBPα was expressed in STAT5A(1*6) cells. Also, secondary CAFCs and long-term cultures could only be generated from STAT5A(1*6) expressing cells, but not from cells that co-expressed STAT5A(1*6) and C/EBPα. Taken together, these data indicate that STAT5-induced self-renewal and impaired myelopoiesis involves downmodulation of C/EBPα.

Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5195-5195
Author(s):  
Lulu Lu ◽  
Yongping Song ◽  
Baogen Ma ◽  
Xiongpeng Zhu ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Lymphocyte Subsets ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P &lt;0.05). The difference between cord blood and mobilized peripheral blood was not significant (P&gt;0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P&lt;0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P&lt;0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P &lt;0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P&lt;0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

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