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.

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.

Cripto Regulates Hematopoietic Stem Cells As a Hypoxic Niche Related Factor Through the Cell Surface Receptor GRP78

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2332-2332
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Matilda Rehn ◽  
Emma Rörby ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Cell Surface ◽  
Triosephosphate Isomerase ◽  
Cell Surface Receptor ◽  
Related Factor ◽  
Glycolytic Metabolism ◽  
Hematopoietic Stem ◽  
Blocking Antibody ◽  
Endosteal Niche ◽  
Hypoxic Environment ◽  
Surface Receptor

Abstract Abstract 2332 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of embryonic stem (ES) and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells (HSCs) has been unknown. We have reported that Cripto increases colony formation and maintains reconstitution ability of HSCs after growth in vitro and that this signaling is mediated through association of Cripto with cell surface receptor GRP78 which is expressed on a subset of HSCs (Miharada et al, Blood abstract, 2010). Here we show that Cripto/GRP78 signaling is important to sustain HSCs in the hypoxic niche. In order to clarify what pathway(s) are activated by Cripto/GRP78 signaling, we performed proteomics analysis using the HSC-like Lhx2 cell line. Two dimentional electrophoresis (2D-DIGE) of proteins was used to analyze Cripto stimulated- and unstimulated-Lhx2 cells. The findings show that several glycolytic metabolism-related proteins were up-regulated and/or phosphorylated by Cripto treatment, e.g. Pyruvate kinase, Phosphoglycerate kinase 1, and Triosephosphate isomerase. Glycolysis is active under hypoxia and it is known that HSC located in the endosteal area of bones are in a hypoxic environment. We therefore separately analyzed central HSCs (cHSCs) and endosteal HSCs (eHSCs) and their hypoxic/metabolic properties. The findings show that eHSCs exhibit a larger proportion of GRP78+HSCs than cHSC and are more dormant (18.9±4.3% of eHSCs and 5.2±3.2% of cHSCs). GRP78+HSCs exhibit a higher content of Pimonidazole high positive cells indicating more hypoxic cells (37.4±13.2% in GRP78+ and 10.2±6.4% in GRP78−, p=0.001), and they also have a higher proportion of MitoTracker (MT) low cells which indirectly represents higher glycolytic activity (0.29±0.02 in GRP78+ and 0.42±0.07 in GRP78−, as relative MFI, p=0.011). It is noteworthy that after 3 days culture with Cripto, GRP78+HSCs showed significantly lower increase in MT intensity relative to the control condition (7,993±3,223 with Cripto and 14,952±2,857 without Cripto, as MFI, p=0.018). To see how Cripto/GRP78 signaling is important in vivo, we analyzed endosteal niche cells. An endosteal cell analysis revealed that part of ALCAM+ and Sca-I+ cells which have been shown to have supportive ability for HSCs, expressed cell membrane associated Cripto on their cell surface. Moreover, injection of anti-GRP78 blocking antibody led to displacement of GRP78+HSCs from the endosteal area to the central marrow (16.9±5.2% with control IgG and 9.6±3.8% with blocking antibody, in eHSCs, p=0.023). Hypoxia-inducible factor 1α (HIF-1α) null mice, have been shown to exhibit failure in the maintenance of functional HSCs even though the immunophenotypic percentage of HSCs is normal. Since the Cripto promoter region has HIF-1 complex binding sites, we analyzed whether HIF-1α null mice exhibit alterations in the Cripto/GRP78 pathway. Analysis of HIF-1α null mice demonstrated a reduced number of GRP78+HSCs within the eHSC compartment (13.8±1.6% in Mx-Cre control and 5.3±2.5% in HIF-1α null mice, p=0.001) and decreased number of cell surface Cripto+ endosteal niche cells. Taken together, our study strongly suggests that the Cripto/GRP78 pathway is an important signal, as an intermediary of HIF-1 regulation, to maintain HSCs in the hypoxic environment of the endosteal niche, by inducing glycolytic metabolism in dormant HSCs. Disclosures: No relevant conflicts of interest to declare.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1169-1169
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Yorky Tsin Sik Wong ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional screen identifies regulators of murine hematopoietic stem cell repopulation

2016 ◽  
Vol 213 (3) ◽  
pp. 433-449 ◽  
Author(s):  
Per Holmfeldt ◽  
Miguel Ganuza ◽  
Himangi Marathe ◽  
Bing He ◽  
Trent Hall ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Surface Receptor ◽  
The Novel ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Extracellular Matrix Components ◽  
Murine Hematopoietic Stem Cell ◽  
Surface Receptor ◽  
First Time

Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted &gt;1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp521. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3−/− HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host.

scholarly journals Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4378-4378
Author(s):  
Yasuyuki Saito ◽  
Jana M. Ellegast ◽  
Rouven Müller ◽  
Richard A. Flavell ◽  
Markus G. Manz
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Organs ◽  
Humanized Mice ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Lymphoid System ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014). To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice. Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals. Disclosures No relevant conflicts of interest to declare.

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.

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.

Protein Phosphatase 2A Plays an Essential Role in Migration of CD34+ Cells to SDF-1/CXCL12.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1131-1131
Author(s):  
Sunanda Basu ◽  
Nicole T. Ray ◽  
Simon J. Atkinson ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Leading Edge ◽  
Hematopoietic Stem ◽  
Directional Movement ◽  
Cd34 Cells ◽  
Phosphatase 2A

Abstract Although cord blood contains significant numbers of hematopoietic stem and progenitor cells (HSPC), its applicability has remained largely in children (1Broxmeyer and Smith, 2004). Homing of hematopoietic stem progenitor cells (HSPC) to bone marrow is a critical determinant for success of transplantation. Enhancement of homing of HSPC from cord blood has the potential to increase the applicability of cord blood transplantation in adults. Therefore, it is important to understand the molecules that underlie directional movement of HSPC. Stromal derived factor-1 (SDF-1)/CXCL12 is the most potent chemoattractant for both mouse and human HSPC and has been shown to play an important role in the homing and retention of HSPC in bone marrow. Earlier studies have shown that PI-3 kinase plays a critical role in chemotaxis by attracting various pleckstin homology (PH) containing domain proteins at the leading edge and spatial localization of PI3Kinase and PTEN is critical for maintaining the leading edge. Controlled activation of Akt/PKB, one of the various PH containg domains, has been described to be required for efficient chemotaxis. In this study we have demonstrated that protein phosphatase 2A (PP2A), a serine-threonine phosphatase, plays an important role in chemotaxis of cord blood CD34+ cells towards SDF-1, primarily by modulating Akt activity. Inhibition of PP2A by okadaic acid (OA) or siRNA impairs chemotaxis; this involved impairment in the ability of CD34+ cells to polarize and reduced speed of movement. This was associated with robust and prolonged Akt phosphorylation. Indeed, over expression of constitutive active Akt in CD34+ cells impaired SDF-1 directed chemotaxis. Co-immunoprecipitation experiments demonstrated increased association of Akt with PP2A following SDF-1 stimulation and this increased association of Akt and PP2A-catalytic subunit was observed at the plasma membrane of SDF-1 stimulated CD34+ cells by confocal microscopy. The importance of PP2A in maintaining a critical level and duration of activated Akt was also supported by our finding that inhibition of PI-3kinase by low dose of LY294002, partially recovered chemotactic activity in CD34+ cells pretreated with OA. Interestingly, glycogen synthase kinase-3 (GSK-3) associated with Akt following SDF-1 stimulation, although it was found to be constitutively associated with PP2A-C. Inhibition of GSK-3 using GSK-3 IX inhibitor impaired chemotaxis, raising the possibility that GSK-3 is a downstream target for Akt in SDF-1 directed chemotaxis. The physiological relevance of our in vitro findings is established from the observation that OA pretreatment impaired engraftment potential of CD34+ cells in NOD-SCID mice. Our findings contribute to the growing understanding of molecules that affect directional movement and may have the potential implications for homing and engraftment of HSPC.

Role of the Urokinase Receptor (uPAR) in the Cross-Talk of Hematopoietic Stem Cells with the Bone Marrow Microenvironment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 324-324 ◽  
Author(s):  
Nunzia Montuori ◽  
Patrizia Ricci ◽  
Bianca Serio ◽  
Valeria Visconte ◽  
Claudio La Penna ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Mirror Image ◽  
Cell Surface Receptor ◽  
Hematopoietic Stem ◽  
Stromal Microenvironment ◽  
Surface Receptor ◽  
Cell Adhesion And Migration

Abstract The urokinase-type plasminogen activator receptor (uPAR) is a cell-surface receptor involved in cell adhesion and migration. uPAR binds urokinase (uPA) and vitronectin (VN) and interacts with integrins and chemotaxis receptors. Soluble forms of uPAR (suPAR) have been detected in human plasma and urine. A cleaved form of suPAR (c-suPAR), lacking the N-terminal domain and exposing the sequence SRSRY (aa 88–92), stimulates cell migration by activating fMLP receptors. We recently demonstrated uPAR involvement in G-CSF-induced CD34+ hematopoietic stem cell (HSC) mobilization. We also demonstrated that c-suPAR could induce mobilization of hematopoietic stem/progenitor cells in mice. Since HSC mobilization and homing to bone marrow (BM) are mirror image processes which utilize the same mediators and similar signaling pathways, we investigated whether uPAR and its ligands could play a role in regulating CD34+ HSC interactions with the BM stroma, thus also contributing to HSC homing and engraftment to the BM. We found expression of uPA and VN in cultures of human BM stroma cells. Interestingly, stroma cells also produced suPAR and high amounts of c-suPAR, exposing the chemotactic SRSRY sequence. The role of the different soluble forms of uPAR produced by stroma cells in regulating HSC interactions with the BM microenvironment was analyzed by long term cultures (LTC) of BM and G-CSF mobilized CD34+ HSCs, in the presence of suPAR or the uPAR-derived uPAR84–95 peptide, corresponding to the active site of c-suPAR. Both suPAR and the uPAR84–95 peptide increased the number of adherent and released clonogenic progenitors from LTC of BM and G-CSF mobilized HSCs. To elucidate the mechanism of suPAR and c-suPAR effects on CD34+ HSC interactions with the stromal microenvironment, in vitro adhesion and proliferation assays were performed on CD34+ KG1 cells. suPAR treatment determined a significant increase in CD34+ KG1 cell adhesion whereas c-suPAR increased cell proliferation. Taken together, our results indicate that BM stroma produces soluble forms of uPAR that regulate CD34+ HSC interactions with BM microenvironment, their local proliferation and trafficking from and to BM.

Optimization Of Vascular Niches To Increase Hematopoietic Engraftment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4456-4456 ◽  
Author(s):  
Saloomeh Mokhtari ◽  
Evan Colletti ◽  
Christopher D Porada ◽  
Graca Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Lentiviral Vector ◽  
In Utero ◽  
Type I ◽  
Cellular Interactions ◽  
Inherited Disorders ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Hsc Transplantation

In utero hematopoietic stem cell transplantation (IUHSCT) is a promising approach for correcting selected congenital hematologic and immunologic disorders. However, if higher levels of donor hematopoietic stem/progenitor (HSC) cell engraftment could be achieved, a wider range of inherited disorders could be targeted. We have previously shown that adult bone marrow (BM) derived-CD34+ cells adhere less efficiently to fetal stromal cells than to their adult counterpart. Furthermore, it has been shown that perivascular cells are able to support, through cellular interactions, the long-term engrafting HSC. Here, we hypothesized that by transplanting bone marrow (BM)-derived endothelial progenitor cells (EPC) prior to HSC transplantation, it would be possible to establish HSC donor-optimized vascular niches within the recipient’s BM, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Adult sheep BM HSC were immunoselected with an antibody against sheep CD34, while EPC were isolated by adherence to collagen type I. Characterization of these cells demonstrated that they were spindle-shaped, and they expressed fetal liver kinase (flk-1/KDR), vonWillebrand factor (vWF), and melanoma cell adhesion molecule (MCAM/CD146). In addition, these cells formed capillary-like structures in Matrigel-based media. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered, intraperitoneally, 1.4X105 CD34+ cells transduced with an eGFP-encoding lentiviral vector (HSCeGFP) in combination with 7.1X105 EPC transduced with an mKate-encoding lentiviral vector (EPCmKate) (n=4), from the same donor, either concurrently, or 3 days prior to HSCeGFP transplantation. At 60 days post-transplant, we performed flow cytometry on peripheral blood (PB) and BM to assess the levels of donor cell engraftment. We also performed confocal microscopic analysis of bone sections to identify the localization and interaction between transplanted cells. Our results demonstrate that animals receiving EPCmKate 3 days prior to HSC transplantation displayed 13-fold higher levels of eGFP(+) hematopoietic cells in their BM (6.5±0.5%), when compared with animals receiving EPC and HSC simultaneously (0.39±0.29%). Confocal microscopy analysis showed that, regardless of the time-point of transplant, donor cells that engrafted in the diaphysis localized to the perivascular area, and a correlation was found between the levels of CD146(+)mKate cells and HSCeGFP engraftment. By contrast, in the metaphysis, only eGFP(+) cells were detected, and these cells co-expressed osteopontin, a marker of osteoblasts. These results show that in IUHSCT, delivery of EPC,CD146(+), cells prior to CD34+HSC results in modification of the vascular niches by donor-derived cells, leading to significantly higher levels of HSC engraftment. Furthermore, a considerable percentage of CD34+eGFP(+) cells did not contribute to the hematopoietic pool, but rather, contributed to the developing bone, suggesting that a more effective selective process for HSC might be necessary for improving engraftment in IUHSCT. Disclosures: No relevant conflicts of interest to declare.

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