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Blood, 1 October 2007, Vol. 110, No. 7, pp. 2399-2407. Prepublished online as a Blood First Edition Paper on June 22, 2007; DOI 10.1182/blood-2006-10-051276. This Article Abstract Full Text (PDF) Supplemental Figure All Versions of this Article: blood-2006-10-051276v1 110/7/2399    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Qian, H. Articles by Ekblom, M. Search for Related Content PubMed PubMed Citation Articles by Qian, H. Articles by Ekblom, M. Related Collections Hematopoiesis and Stem Cells Cell Adhesion and Motility Stem Cells in Hematology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMATOPOIESIS Distinct roles of integrins 6 and 4 in homing of fetal liver hematopoietic stem and progenitor cells Hong Qian1, Elisabeth Georges-Labouesse2, Alexander Nyström3, Anna Domogatskaya4, Karl Tryggvason4, Sten Eirik W. Jacobsen1,5, and Marja Ekblom1,5 1 Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden; 2 Institute of Genetics and Molecular and Cellular Biology (GBMC), Centre National de la Recherche Scientific (CNRS)/Institut National de la Santé et de la Recherche Medicale (INSERM)/University Louis Pasteur (ULP), Illkirch, France; 3 Department of Cell and Matrix Biology, Lund University, Lund, Sweden; 4 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; 5 Department of Hematology, Lund University Hospital, Lund, Sweden    Abstract Top Abstract Introduction Materials and methods Results Discussion Authorship References   Homing of hematopoietic stem cells (HSCs) into the bone marrow (BM) is a prerequisite for establishment of hematopoiesis during development and following transplantation. However, the molecular interactions that control homing of HSCs, in particular, of fetal HSCs, are not well understood. Herein, we studied the role of the 6 and 4 integrin receptors for homing and engraftment of fetal liver (FL) HSCs and hematopoietic progenitor cells (HPCs) to adult BM by using integrin 6 gene–deleted mice and function-blocking antibodies. Both integrins were ubiquitously expressed in FL Lin–Sca-1+Kit+ (LSK) cells. Deletion of integrin 6 receptor or inhibition by a function-blocking antibody inhibited FL LSK cell adhesion to its extracellular ligands, laminins-411 and -511 in vitro, and significantly reduced homing of HPCs to BM. In contrast, the anti-integrin 6 antibody did not inhibit BM homing of HSCs. In agreement with this, integrin 6 gene–deleted FL HSCs did not display any homing or engraftment defect compared with wild-type littermates. In contrast, inhibition of integrin 4 receptor by a function-blocking antibody virtually abrogated homing of both FL HSCs and HPCs to BM, indicating distinct functions for integrin 6 and 4 receptors during homing of fetal HSCs and HPCs.    Introduction Top Abstract Introduction Materials and methods Results Discussion Authorship References   During transplantation, intravenously injected hematopoietic stem and progenitor cells (HSPCs) selectively transmigrate through the sinusoidal walls into the bone marrow (BM) niches to engraft and reconstitute hematopoiesis. This process, called homing, is a rapid multistep process involving multiple adhesion receptor and ligand pairs, leading first to reversible adhesion of circulating HSPCs to endothelial cells of BM sinusoids and subsequently transmigration through the sinusoidal walls into the specific hematopoietic niches.1 The adhesive interactions mediated by distinct receptors on hematopoietic cells regulate the localization, migration, and other HSPC functions within their native microenvironments, and are likely to define their homing potential in a transplantation setting.2 The rate of reconstitution following transplantation varies depending on the source of hematopoietic stem cells (HSCs), suggesting developmentally regulated and cell type–specific differences in expression and function of homing receptors. Cord blood grafts are associated with a particularly delayed hematopoietic reconstitution.3 In contrast, experimental studies have shown a more rapid and robust reconstitution of both human and mouse fetal liver (FL) HSCs than adult BM HSCs.4,5 Increasing HSC homing and engraftment efficiency might offer a means to enhance reconstitution, which may be critically important if donor cell numbers are limiting, as frequently observed in cord blood units.3 Therefore, knowledge of the specific molecular mechanisms involved in homing and engraftment of different types of HSPCs is of clinical significance for the development of clinical transplantation methods. Mouse models for mammalian hematopoiesis are widely used to study developmentally regulated HSC functions, including mobilization, homing, and engraftment.6 During mammalian ontogeny, HSCs develop sequentially in distinct hematopoietic tissues, beginning in the yolk sac and subsequently in the aorta-gonad-mesonephros region in the embryo proper. At embryonic day 10 (E10) in mice, definitive hematopoiesis arises in the liver, which is the main site of hematopoiesis throughout subsequent fetal development.7 In later gestation, hematopoiesis expands into spleen (E15) and thereafter into BM (E17). This shift in hematopoiesis is accomplished by migration of liver HSPCs into spleen and BM, and consequently, is associated with mobilization of large numbers of HSPCs into the bloodstream.6 Despite the central role of migration and homing in stem-cell biology and in stem-cell therapy, the molecular pathways underlying FL HSC homing into BM remain to be elucidated. Members of the integrin family of adhesion receptors are major mediators of cell migration during development and in the adult organism, including the hematopoietic tissue.8 All integrins are heterodimeric transmembrane molecules consisting of and ß subunits. They mediate binding to extracellular matrix (ECM) and cell surface ligands by their extracellular domains and provide a link between cytoskeletal proteins and the cytoplasmic domain, thereby acting as bidirectional signaling molecules.9 In vivo studies using gene-deleted mice and function-blocking antibodies have established the fundamental role for ß1 integrins during HSC migration both into FL and fetal and adult BM.10–12 So far, 12 distinct ß1 integrins, containing different chains (1-11, v), have been characterized.8 Of these, the function of integrin 4 receptor during embryonic hematopoiesis has been studied by using gene-ablated mice and function-blocking antibodies.13–16 Although these data indicate an important role for the integrin 4 chain during fetal hematopoiesis, its role in homing of FL HSPCs has not been defined. The integrin 6 chain assembles with integrin ß1 or ß4 chains to form major receptors for ECM laminins.17,18 Integrin 6 is ubiquitously expressed in both human and adult mouse primitive hematopoietic cells.19–21 In adult BM, the major ligands of 6 integrins, laminin isoforms laminin-411 and laminin-511, previously called laminin-8 and laminin-10, respectively,22 are present in subendothelial basement membranes of sinusoids.23–25 We have previously shown that the integrin 6 receptor promotes migration of human hematopoietic progenitor cells (HPCs) in vitro21 and functions as a homing receptor for adult mouse BM HSPCs in vivo.19 Herein, we have studied the role of the integrin 6 receptor for homing, engraftment, and mobilization of FL HSPCs in vivo by using function-blocking antibodies and gene-deleted mice.26 Our results show that the 6 integrin receptor is functional during homing of FL HPCs, but not during homing and engraftment of multilineage repopulating HSCs in vivo. In contrast, we show the critical role of integrin 4 receptor for homing of both FL HPCs and multilineage repopulating HSCs to adult BM, indicating distinct functions for integrin 6 and 4 receptors during fetal hematopoiesis.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion Authorship References   Animals and genotyping Integrin 6+/– (C57BL/6 CD45.2) and wild-type congenic (CD45.1, CD45.2) mice were maintained under specific pathogen-free conditions. All adult mice used in the study were 8 to 14 weeks old. All animal protocols were approved by the local ethics committee at Lund University. The integrin 6+/– mouse strain26 has been backcrossed to a C57BL/6 background for more than 10 generations. Integrin 6–/– or wild-type CD45.2 embryos were obtained by interbreeding integrin 6+/– mice. Genotyping was performed by polymerase chain reaction (PCR). The wild-type integrin 6allele was detected by using a forward primer (5'-GTGATAACTCCAGCTTGTGTGTCAAG-3') and a reverse primer (5'-CCTCTGCAGCGGGAGTGCTTC-3'),27 producing a 500-bp DNA fragment. The forward primer 5'-ATCGTCATGCTCCTGTTTCTCC-3' and the reverse primer 5'-TCAGAGCAGCCGATTGTC-3', which is located in the neomycin cassette, produced a 1000-bp band in the mutant integrin 6-allele. The PCR parameters were as follows: 1.5 minutes denaturation at 94°C; 40 cycles of amplification at 94°C for 1 minute; 61°C for 0.5 minutes; and 72°C for 1 minute, followed by 72°C for 10 minutes. Hematopoietic growth factors Human granulocyte colony-stimulating factor (G-CSF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF) were provided by Amgen (Thousand Oaks, CA). Human fms-like tyrosine kinase-3 ligand was a kind gift of Immunex (Seattle, WA). Mouse interleukin-3 was from PeproTech (Rocky Hill, NJ). Proteins and function-blocking antibodies Recombinant laminin-411 and laminin-511 were produced as described.28,29 Human placental laminin-511/521 (laminin-10/11) was from Chemicon (Chandlers Ford, United Kingdom). The purified preservative-free antibody GoH3 against integrin 6 (CD49f) was from Immunotech (Marseille, France), and PS/2 against integrin 4 (CD49d) was from Chemicon. HA2/5 against integrin ß1 (CD29) and the rat IgG2a and hamster IgM antibodies were from PharMingen (San Diego, CA). Isolation and analysis of FL HSPCs Single cell suspensions from E14.5 FLs were prepared with 25-gauge needles and 70-µm filters. Cells not expressing lineage markers (Lin– cells) were selected by using antibodies against B220 (RA3–6B2), CD5 (53-7.3), CD8a (53-6.7), Gr-1 (RB6–8C5), and Ter-119 (Ly76) (PharMingen) and sheep anti–rat IgG (Fc)–conjugated immunomagnetic beads (Dynal, Oslo, Norway), and thereafter stained with goat antirat-tricolor (Caltag, Burlingame, CA), rat antimouse Sca-1-FITC (E13-161.7), c-Kit-APC (2B8), Flt3-PE (AZF10.1), or isotype-matched control antibodies (PharMingen). 7-amino actinomycin (Sigma-Aldrich, St Louis, MO) was used to exclude dead cells. Lin–Sca-1+Kit+ (LSK) cells were sorted on a FACSVantage or FACSDiva (Becton Dickinson, San Jose, CA). Reanalysis reproducibly showed a purity of more than 95%. Integrin expression on FL LSK and Lin–Sca-1–c-Kit+ cells was analyzed by using PE-conjugated antibodies against integrin 6 (GoH3), 5 (5H10-27), or 4 (R1-2) (PharMingen). Analyses were performed using by FACSCalibur using FlowJo (TreeStar, San Carlos, CA) or CELLQuest (Becton Dickinson) programs. Cell adhesion assay Flat-bottom 96-well plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated with ECM proteins in PBS (Gibco, Carlsbad, CA) at 10 to 30 µg/mL. The cell adhesion and antibody inhibition assays were performed as described.19 Assays for HPC homing E14.5 FL cells were incubated with anti-integrin or control antibodies (2 µg/106 cells) in IMDM, 5% fetal calf serum (FCS) on ice for 30 to 40 minutes. The cells (5 x 106/mouse) were injected into tail vein of adult recipient mice, which were irradiated 4 to 8 hours before transplantation with a single 900-cGy dose using a cesium Cs 137 source (Instrument AB Scanditronix, Uppsala, Sweden). Three hours after injection, blood, BM, and spleen were harvested for granulocyte-macrophage colony-forming units (CFU-GM) assay, as described.19 The myeloid colonies (CFU-GMs) containing more than 50 cells were scored using an inverted Olympus IX 70 microscope (Tokyo, Japan). The mean value of each duplicate was calculated. The number of recovered CFU-GMs was corrected to represent the whole BM, estimating that one femur represents 5.9% of the total BM.30 The number of injected CFU-GMs was assayed in a fraction of the donor FL cells. Host-derived residual CFU-GMs (0-2 in BM and spleen, 0 in blood) were assessed in irradiated mice but not receiving a transplant (1-2/experiment) and were subtracted from CFU-GMs recovered after transplantation. The percentage of recovered CFU-GMs of injected cells was calculated. To analyze homing of integrin 6–/– FL HPCs, FLs were dissected in separate dishes containing PBS, 5% FCS. The cells from each liver were transplanted to 1 to 2 mice (3.5 to 5 x 106 cells/mouse), and CFU-GM homing was analyzed. Antibody perturbation of FL HSC homing Wild-type CD45.2 E14.5 FL cells (donor) were incubated with anti-integrin or control antibodies (2µg/106 cells) for 30 to 40 minutes on ice.19 The anti-integrin 6 antibody was used in a concentration previously shown to inhibit homing of adult HSCs to BM.19 The cells (10 x 106 cells/mouse) were injected intravenously into lethally irradiated CD45.1 mice. Because of a possible transient effect of function-blocking antibodies, homing was assayed at 3 hours. The BM cells were harvested and washed with IMDM, 5% FCS to remove nonbound antibodies. The cells from one femur of each primary recipient were injected together with 2 x 105 BM cells from adult CD45.1 mice (competitor) into lethally irradiated CD45.1 secondary recipient mice. Blood was harvested at 3, 6, and 16 weeks after transplantation and analyzed for reconstitution of donor HSCs (CD45.2) and competitor/recipient cells (CD45.1). The reconstitutions at 3 and 6 weeks are achieved by both short-term and long-term HSCs, and the reconstitution at 16 weeks is derived from LT-HSCs only.31–33 Lineage reconstitution was examined using fluorochrome-conjugated antibodies against CD4 and CD8 (T cells), B220 (B cells), and Mac-1 (CD11b) (myeloid cells) along with antibodies against CD45.1 (A20) and CD45.2 (104) (PharMingen) as described.19 Since myeloid cells, including myeloid progenitors, in contrast to lymphocytes, are short lived and must be continuously generated from the HSCs, sustained myeloid reconstitution is the best indicator of HSC reconstitution.34,35 Competitive repopulation assay of integrin 6–/– FL and fetal bone marrow (FBM) HSCs Integrin 6+/+ or 6–/– E14.5 FL cells (1 x 106/mouse) were injected intravenously together with 1.5 x 106 wild-type adult BM competitors (CD45.1) into lethally irradiated CD45.1 recipients. Seventeen weeks after transplantation, a half femur equivalent BM cells were transplanted into lethally irradiated secondary recipient mice. Reconstitution was analyzed at 3 to 40 weeks after primary transplantation and 12 weeks after secondary transplantation. E18.5 FBM cells from integrin 6–/– fetuses or WT littermates were obtained by homogenizing femurs and tibias through a 70-µm filter. Competitive repopulating units (CRUs) were measured by transplanting limiting doses of cells (1/15 and 1/7.5 leg equivalents corresponding to 2.0-2.2 x 104 and 4.0-4.4 x 104 cells, respectively) into lethally irradiated recipients (7 mice/dose) together with 2 x 105 BM competitor cells. Reconstitution was analyzed at 8 and 16 weeks after transplantation. Reconstituted mice were defined as having a minimum of 0.1% CD45.2+ total cells and 0.02% CD45.2+ cells of each of the myeloid, B, and T lineages with an analysis of a minimum 50 000 blood cells.36 The specificity of the fluorescence-activated cell sorting (FACS) staining was controlled by analyzing the blood of CD45.1 mice that did not undergo transplantation, and in all the assays the background CD45.2 staining was less than 0.01% and CD45.2+Mac-1+ staining was less than 0.002% of total nucleated blood cells. CRUs were calculated based on the frequencies of positive mice at each dose,37 using Limit Dilution Analysis software (StemCell Technologies, Vancouver, BC). Mobilization assays The mice reconstituted with integrin 6–/– or 6+/+ FL cells (CD45.2) together with CD45.1 competitor BM cells 5 to 8 months after transplantation were injected with G-CSF (100 µg/kg subcutaneously, twice daily) for 3 days.38 The following day, blood and femora were collected. Lin–Sca-1+ cells expressing CD45.1 or CD45.2 markers in blood and BM were analyzed as described above. Reverse-transcription (RT)–PCR analysis of integrin 6 splice variants Total RNA from adult BM and E14.5 FL LSK and Lin–Sca-1–c-Kit+ cells was isolated using Trizol (Life Technologies, Bethesda, MD). First-strand synthesis was performed by using oligo dT primer and Superscript II RT (Invitrogen, Frederick, MD). The primers for the integrin 6A and 6B variants were as follows: forward, 5'-GTGAACGTGAGGTGTGTGAAC-3' and reverse, 5'-CGCATGGTATCGGGGAATGC-3'39; and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward, 5'-TTGTCAGCAATGCATCCTGC-3' and reverse, 5'-CCGTTCAGCTCTGGGATGAC-3'.40 The cycling conditions were 94°C for 2 minutes, followed by 35 cycles of 94°C for 15 seconds, 50°C for 30 seconds, and 72°C for 2 minutes. Statistical analysis Statistical significance was determined using the unpaired t test for parametric or Mann-Whitney t test for nonparametric comparisons.    Results Top Abstract Introduction Materials and methods Results Discussion Authorship References   Expression integrin 4, 6, and 5 on FL HSCs and HPCs We analyzed the expression of integrin 4 and 6 chains on LSK cells, which are highly enriched for HSCs and contain all short-term and long-term reconstituting activity in mouse FL.5,41 All LSK cells from E14.5 FL expressed integrin 4 and 6 receptors (Figure 1). View larger version (50K): [in this window] [in a new window]   Figure 1. Integrin expression in mouse fetal liver HSCs. E14.5 FL cells were stained with lineage antibodies (Lin) and gated for Lin– and viable cells. Lin– viable cells were gated for Sca-1–c-Kit+ clonogenic progenitor cells and the more primitive Sca-1+c-Kit+ (LSK) cells. The left panels show gates for Sca-1+c-Kit+ and Sca-1–c-Kit+ on Lin– cells. A ubiquitous expression of integrin 6, 4, and 5 chains was seen in LSK cells (right panels) and Lin–Sca-1–c-Kit+ progenitors (middle panels). Staining with the anti-integrin antibodies is shown as open histograms and staining with isotype control antibodies, as gray histograms. Shown is 1 representative analysis of 2.   Furthermore, 100% and 95% of committed myeloid progenitors, which have the phenotype Lin–Sca-1–c-Kit+,6 expressed the integrin 4 and 6 chains, respectively. Expression of integrin 5 was similar on FL cells. In agreement with this, a ubiquitous expression of these integrins has been reported in less well-defined fetal HSPCs.16,42,43 Integrin 6ß1 mediates FL LSK adhesion to laminins In in vitro adhesion assay (Figure 2), 10% to 15% of the isolated FL LSK cells adhered to recombinant laminin-411 and -511 isoforms and 25% adhered to placental laminin-511/521. Pretreatment of the cells with a protein kinase C activator, TPA, did not increase cell adhesion to the laminins (not shown). In addition to integrin 6ß1, other laminin receptors, including integrin 2ß1, -dystroglycan, p67 receptor, and heparan sulfate proteoglycans are also expressed on HSPCs.20,44–46 We therefore analyzed the contribution of integrin 6ß1 receptor for FL LSK adhesion to laminin-411 and -511 by using integrin 6–/– FL LSK cells and function-blocking antibodies against 6 and ß1 chains. Deletion of 6 integrin receptor effectively reduced LSK cell adhesion to all laminin isoforms in vitro (Figure 2A). In agreement with this, adhesion of wild-type LSK cells to laminins was inhibited by both antibodies (Figure 2B-D), indicating that integrin 6ß1 is a major lamininreceptor in FL LSK cells. However, some cells still adhered to laminins after inhibition of integrin 6 function, which might indicate expression of other laminin receptors in a fraction of FL LSK cells. View larger version (25K): [in this window] [in a new window]   Figure 2. Adhesion of mouse fetal liver LSK cells to laminins and inhibition of cell adhesion by antibodies against integrin 6 and ß1 chains. (A) The adhesion of integrin 6–/– FL LSK cells (6–/–) and wild-type littermates (6+/+) to laminin-411 (LM-411) (30 µg/mL), laminin-511 (LM-511) (10 µg/mL), and laminin-511/521 (LM-511/521) (10 µg/mL). PBS indicates adhesion to wells coated with PBS instead of proteins; FN, adhesion to wells coated with fibronectin. Data represent mean (± SEM) from 2 experiments performed in triplicate. Asterisks indicate differences compared with wild-type cells. (B-D) Adhesion of LSK cells to wells coated with LM-411 (B), LM-511 (C), and LM-511/521 (D) in the presence of antibodies GoH3 against integrin 6 chain (6), Ha2/5 against integrin ß1 chain (ß1), or control rat or hamster monoclonal antibodies (rIgG and hIgM, respectively). PBS indicates cell adhesion to wells coated with PBS instead of proteins. The results are shown as percentage adherent cells of mean adhesion obtained after treatment with rIgG control antibody. Data are mean (± SEM) from 2 to 3 experiments performed in triplicate.   Normal development of FL HSPCs in integrin 6–/– mice FL hematopoiesis was studied in E14.5 integrin 6 gene–deleted embryos and wild-type littermates (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). The numbers of LSK cells, the more primitive Flt3– fractions,47 and CFU-GMs were similar in gene-deleted and in wild-type embryos, indicating normal development of FL HSPCs in the absence of 6 integrin. Homing of integrin 6–/– FL HPCs to adult BM is impaired To analyze the role of integrin 6 receptor for FL HPC homing, FL cells from integrin 6–/– embryos or wild-type littermates were injected into lethally irradiated mice. About 12% of the injected wild-type FL CFU-GMs were recovered in BM (Figure 3A). Deletion of integrin 6 receptor resulted in a partially but significantly reduced homing of FL HPCs to BM (67% of wild type). In contrast, CFU-GM homing to spleen was not affected by the absence of integrin 6 receptor. The numbers of 6–/– and 6+/+ CFU-GMs recovered in blood were similar. Since only a fraction of the transplanted CFU-GMs were recovered in spleen and in blood, it is likely that the injected FL CFU-GMs transiently home to other organs, such as lung and liver, as previously described.48 The colony growth of integrin 6–/– FL cells was similar to that of wild-type cells (Figure 3B). View larger version (36K): [in this window] [in a new window]   Figure 3. The role of integrin 6 and 4 receptors for homing of fetal liver progenitors to BM and spleen. FL cells were injected into lethally irradiated recipient mice. Three hours after injection, cells from blood, BM, and spleen were collected for CFU-GM assay. Data are pooled from 3 independent experiments. (A) Percentages of injected FL HPCs (GFU-GM) from integrin 6–/– FL (6–/–) embryos and wild-type littermates (6+/+) recovered in blood, BM, and spleen. (B) Frequency of CFU-GMs in integrin 6–/– and wild-type (6+/+) FLs. (C) Percentages of FL CFU-GMs recovered in blood, BM, and spleen after treatment with a function-blocking antibody against integrin 6 receptor (6) or isotype control antibody (rIgG). (D) Frequency of CFU-GMs in FL cells cultured after treatment with the antibody against integrin 6 receptor (6) or isotype control antibody (rIgG), or without pretreatment with antibodies (no ab). (E) Percentages of FL CFU-GMs recovered in blood, BM, and spleen after treatment with a function-blocking antibody against integrin 4 receptor (4) or isotype control antibody (rIgG). (F) Frequency of CFU-GMs in FL cells cultured after treatment with the antibody against integrin 4 receptor (4) or isotype control antibody (rIgG), or without pretreatment with antibodies (no ab). The results in panels C and E are pooled from 2 separate experiments. *P < .05, **P < .01, ***P < .001, compared with results obtained from wild-type FL cells (A,B) or cells treated with the isotype control antibodies (C-F). Each dot represents mean of 2 duplicate measurements from one recipient mouse. The horizontal bars show mean values.   Integrin 6 antibody inhibits FL HPC homing to BM and spleen Since the integrin 6 gene deletion resulted in only partial reduction of HPC homing, it is possible that the loss of function may have been compensated by other cell adhesion molecules. To circumvent this possibility of compensation, we also used a function-blocking antibody to evaluate the effect of a more immediate loss of integrin 6 function for progenitor cell homing. The anti-integrin 6 antibody GoH3 used has previously been shown to inhibit transendothelial migration of neutrophils in vivo49,50 and homing of adult mouse HPCs and HSCs to BM vivo.19 FL cells were preincubated with the anti-integrin or control antibodies and injected intravenously into lethally irradiated recipient mice. Three hours after transplantation, 14.9% and 4.0% of the CFU-GMs pretreated with the control antibody were recovered in BM and spleen, respectively (Figure 3C). In full agreement with the reduced homing efficiency of integrin 6 gene–deleted HPCs, the function-blocking antibody against integrin 6 receptor significantly inhibited homing of CFU-GMs into BM (60% of control). Notably, treatment with the anti-integrin 6 antibody also resulted in reduced homing of HPCs to spleen and significant retention of HPCs in blood, demonstrating that HPC migration to extramedullary sites is more effectively inhibited by the antibody than gene deletion. The inhibition of FL CFU-GM homing to BM by the anti-integrin 6 antibody was similar to that previously observed with adult BM cells.19 However, homing of FL but not adult BM CFU-GM to spleen was reduced by the anti-integrin 6 antibody treatment, suggesting developmentally regulated differences in receptor engagement in homing of embryonic and adult BM HPCs to spleen. Importantly, antibody treatment did not influence colony formation of FL HPCs (Figure 3D). Integrin 4 antibody abrogates FL HPC homing to BM The partial inhibition of FL HPC homing to BM after treatment with anti-integrin 6 antibody suggests functional collaboration of additional adhesion molecules. Previous16,42,43 and our present results indicate that integrin 4 is ubiquitously expressed on FL HSPCs. Thus, we analyzed the role of integrin 4 receptor for homing of FL HPCs into adult BM, using a function-blocking antibody PS/2, previously shown to be functionally active in vitro and in vivo.19,51,52 Treatment of FL cells with the antibody almost completely inhibited CFU-GM homing into BM (< 10% of control). The anti-integrin 4 antibody also inhibited HPC homing to spleen, in contrast to the reported increase in uptake of adult HPCs to spleen after blocking of integrin 4 receptor or its counterreceptor VCAM-1.51 Correspondingly, significantly more CFU-GMs were retained in blood after treatment with the anti-integrin 4 antibody, compared with treatment with the control antibody (Figure 3E). Pretreatment of cells with anti-integrin 4 antibody did not affect colony growth (Figure 3F). Integrin 6-deficient FL HSCs engraft normally in adult BM To study the contribution of integrin 6 receptor to HSC homing and engraftment in BM, FL cells from integrin 6–/– embryos or wild-type littermates were transplanted together with adult BM competitor cells into lethally irradiated recipients. Engraftment of short-term and long-term integrin 6–/– FL HSCs was similar to that of wild-type cells. Reconstitution of total and myeloid cells (Figure 4A), as well as lymphoid cells (not shown), from both genotypes was identical at 3 to 40 weeks after transplantation. The homing efficiency of most primitive HSCs was studied by transplanting BM cells from the primary recipients into lethally irradiated secondary recipients. At 12 weeks after serial transplantation, reconstitution was still similar in both groups (Figure 4B), suggesting that FL cells lacking integrin 6 home and engraft efficiently in adult BM. View larger version (16K): [in this window] [in a new window]   Figure 4. Reconstitution and mobilization of integrin 6–/– fetal liver HSCs. (A) FL cells (106) from integrin 6–/– (6–/–) embryos or wild-type littermates (6+/+) expressing CD45.2 isotype together with 1.5 x 106 CD45.1 competitor adult BM cells were injected into lethally irradiated CD45.1 mice. Blood was harvested at 3, 6, 16, 30, and 40 weeks after transplantation. Shown is reconstitution of CD45.2+ cells and CD45.2 myeloid cells (Mac-1) of all nucleated cells (mean ± SD; n = 13-14/group at 3, 6, and 16 weeks and n = 3-4/group at 30 and 40 weeks). (B) After 17 weeks, BM cells were transplanted into secondary recipients and reconstitution was analyzed 12 weeks after transplantation. Data are pooled from 2 independent experiments. The horizontal bars show mean values for each measurement. (C) Schematic representation of HSPC mobilization. Mice reconstituted with integrin 6–/– (6–/–) or wild-type (6+/+) FL cells (CD45.2), together with CD45.2 competitor cells, 5 and 8 months after transplantation, were injected subcutaneously with G-CSF for 3 days. On the fourth day, the femora and blood were collected and the cells in blood and BM were analyzed by flow cytometry. (D) Shown is the proportion of CD45.2+Lin–Sca-1+ cells of all Lin–Sca-1+ cells in BM and blood. The data are pooled since there was no difference of mobilization response in the 2 experiments.   Normal mobilization of integrin 6–/– HSPCs by G-CSF To test whether integrin 6 receptor is involved in HSPC mobilization, HSPCs were mobilized by G-CSF in mice stably reconstituted with integrin 6–/– or wild-type FL cells together with competitor cells at 5 and 8 months after transplantation. The proportions of FL-derived Lin–Sca-1+ cells in blood and BM were analyzed by flow cytometry (Figure 4C). The proportion of integrin 6–/– Lin–Sca-1+ cells in blood and BM was similar to that derived from wild-type littermates (Figure 4D), showing that absence of 6 integrin does not influence HSPC mobilization from BM. Integrin 4, but not 6, receptor is required for homing of FL HSCs to BM Since FL HSCs have a greater proliferative capacity than adult HSCs,4 a possible homing defect in integrin 6 gene–deleted HSCs might be masked by the high proliferation rate during engraftment. In addition, the efficient engraftment of integrin 6–/– FL HSCs might be due to functional compensation by other molecules. To specifically address the impact of 6 integrin receptor on HSC homing, we studied the effect of inhibition of integrin function by antibody on short-term homing of HSCs to BM as described.30 The HSCs were assayed by the competitive repopulation assay (Figure 5A). Homing of FL HSCs, in contrast to adult HSCs,19 was not affected by inhibition of integrin 6 receptor function (Figure 5B). In contrast, treatment with the anti-integrin 4 antibody nearly abrogated homing of both short-term and long-term HSCs, indicating a dominant role for integrin 4-receptor in recruiting FL HSCs into BM. View larger version (34K): [in this window] [in a new window]   Figure 5. Perturbation of fetal liver HSC homing by antibodies against integrin 6 and 4. (A) Schematic representation of the experimental setup: FL cells from CD45.2 mice were incubated with rat IgG2a antibody (rIgG) or antibodies against integrin 4 (4) or integrin 6 (6) and injected into lethally irradiated CD45.1 mice. BM cells were harvested 3 hours after injection and cells from one femur each were injected together with CD45.2 competitor BM cells into lethally irradiated CD45.1 secondary recipient mice. Blood was harvested at 3, 6, and 16 weeks after transplantation and reconstitution was analyzed by flow cytometry. (B) Peripheral blood reconstitution of CD45.2 cells and CD45.2 myeloid cells at 3, 6, and 16 weeks. ***P < .001 compared with reconstitution from cells incubated with rat IgG. Data are pooled from 2 independent experiments. The horizontal bars show mean values. (C) Representative FACS dot plots showing blood reconstitution 16 weeks after secondary transplantation. The left panels show reconstitution of CD45.2 FL cells. The right panels show blood reconstitution of myeloid (Mac-1) cells derived from CD45.2 cells. The numbers indicate the mean percentage of cells in the corresponding fraction (n = 11-14).   Normal HSC colonization to FBM in integrin 6–/– mice The role of integrin 6 receptor during physiological migration of HSCs from FL into FBM was studied in E18.5 integrin 6 gene–deleted embryos and wild-type littermates. Fetal BM cellularity (Figure 6A) was not affected by integrin 6 gene deletion. Furthermore, the CRU numbers in 6–/– FBM were similar to that in WT littermates (Figure 6B), suggesting normal HSC homing and engraftment in the absence of integrin 6. View larger version (13K): [in this window] [in a new window]   Figure 6. Normal FBM hematopoiesis in absence of integrin 6. E18.5 FBM from integrin 6 gene–deleted mice (6–/–) and wild-type littermates (6+/+) was analyzed for total cell number (A) total cell number (horizontal bars show mean values), and (B) competitive repopulating units (CRUs). CRUs were assayed by transplanting 2 to 4.4 x 104 FBM cells together with 2 x 105 adult BM cells into lethally irradiated adult mice. Three of 13 and 3 of 12 mice that received integrin 6–/– FBM cells, and 3 of 14 and 2 of 12 mice that received wild-type FBM cells, were reconstituted with FBM cells at 8 weeks and 16 weeks, respectively. The CRU numbers/2 legs (femur and tibia) are shown as mean (± 95% confidence interval).   Expression of integrin 6 mRNA splice variants in BM and FL HSPCs The integrin 6 polypeptide chain is expressed in 2 functionally different variants, 6A and 6B, with different cytoplasmic domains generated by alternative mRNA splicing.53 Of these, the larger 6A polypeptide is more active in promoting migration than the smaller isoform.54 We studied the expression of the 2 integrin 6 mRNA splice variants on FL and adult BM LSK cells and the more mature Lin–Sca-1–c-Kit+ cells. Both the 510-bp and 378-bp cDNA fragments corresponding to 6A and 6B mRNAs, with a higher expression of the larger splice variant, were detected in LSK and Lin–Sca-1–c-Kit+ fractions from both FL and adult BM (Figure 7), indicating that the differences in integrin 6 receptor function in FL and adult BM HSCs are not due to differential expression of the functionally different splice variants. View larger version (39K): [in this window] [in a new window]   Figure 7. Expression of integrin 6A and 6B mRNA splice variants in adult mouse BM and FL HSPCs. Adult mouse BM and E14.5 FL LSK and Lin–Sca-1–c-Kit+ cells were gated for sorting (left panels). The vertical and horizontal bars were set on the basis of isotype-matched negative control profiles (99.3% of cells negative). Reanalysis of each cell population showed more than 99% purity. RT-PCR analysis for integrin 6A and 6B mRNA splice variants of FL and BM LSK and Lin–Sca-1–c-Kit+ cells is shown in the right panel. MW indicates positions of 500-bp and 400-bp molecular weight markers on the gel. The bands corresponding to 510-bp and 378-bp integrin 6A and 6B splice variants are seen in all 4 cell populations. PCR for GAPDH shows the relative amounts of mRNA in the corresponding samples. Shown is 1 representative of 2 experiments.      Discussion Top Abstract Introduction Materials and methods Results Discussion Authorship References   Clinical and experimental studies have revealed several phenotypic and functional differences in fetal and adult HSCs.6 However, the molecular differences underlying differential homing and engraftment capacities of primitive and definitive HSC populations are still largely unknown. In the present study, we have specifically addressed the role of integrin 6 and 4 receptors for homing and engraftment of FL HSC and HPC populations. In addition, we have studied the consequences of integrin 6 gene deletion for FL hematopoiesis and reconstitution of FL HSCs in BM. Embryonic deletion of integrin 6 chain leads to severe skin blistering, cerebral malformations, and neonatal death. However, specific aspects of hematopoiesis in integrin 6–deficient mice have not been previously studied.26 We here show that 10% to 15% of wild-type FL HPCs, injected into recipient mice after treatment with control antibody or without antibody treatment, were recovered in BM within 3 hours. This fraction of homed FL HPCs is similar as previously observed in adult BM cells.19,30,51 In contrast to our results, one study43 reported a severely reduced homing capacity of FL HPCs compared with adult BM HPCs, with 1% to 1.5% of transplanted FL and 6% of adult BM HPCs recovered in BM 3 hours after intravenous injection. The reason for the difference and for the overall lower frequencies of homed HPCs in this study might be a more extensive manipulation of cells before the colony assay. Furthermore, in another study, approximately 10% of transplanted HSCs from both mouse FL and adult BM were recovered in BM of lethally irradiated recipients 24 hours after injection,55 suggesting constant homing efficiency of HSCs and HPCs during ontogeny. We show here that homozygous deletion of integrin 6 receptor resulted in reduced homing of FL CFU-GMs into BM. In agreement with this, the function-blocking antibody against integrin 6 chain significantly inhibited homing of FL CFU-GMs into BM, with a slightly more pronounced effect than obtained by the gene deletion. However, HPC homing in both experimental assays was only partially inhibited, and approximately 60% of HPCs were still recovered in the BM, compared with wild-type FL cells or cells treated with control antibody. A similar degree of inhibition by a function-blocking anti-integrin 6 antibody was previously shown for adult BM HPCs,19 indicating a role for integrin 6 receptor for HPC homing throughout ontogeny. In contrast to FL HPCs, homing of long-term or short-term repopulating HSCs was not inhibited by the function-blocking antibody against 6 integrin. In line with this, homing and engraftment of integrin 6 gene–deleted FL HSCs, assayed 3 to 40 weeks after transplantation, did not differ from that of wild-type HSCs. Reconstitution analysis 12 weeks after serial transplantation did not show any engraftment defect of 6–/– FL HSCs. Furthermore, the HSC frequencies were similar in BM of integrin 6 gene–deleted fetuses and in wild-type littermates, indicating that fetal HSC development is normal in the absence of 6 integrin. Taken together, the present and previous results19 show ubiquitous expression but distinct developmentally regulated functions of the integrin 6 receptor in fetal and adult HSPCs. The observed differences in integrin 6 receptor function in HSCs suggest differences in integrin activational status, influencing the binding capacity to particular ligands.56 Activation of integrin receptors can be modulated both by ligand-dependent extracellular signals and intracellular signaling influenced by a variety of cytokines and chemokines.57,58 Such modulation might be an important regulatory mechanism influencing transendothelial or stromal migration and homing of HSPCs.58–62 Our results show a relatively low frequency of LSK cells adherent to laminins, which might indicate an inactive conformation of the 6 integrin receptor in most cells. Protein kinase C pathway activator TPA, which mimics the effect of physiological agonists,63 did not increase adhesion, indicating a protein kinase C–independent mechanism. Other possible mechanisms for integrin activation, determined by the specific cellular environments, including posttranslational modifications, interactions with growth factor receptors,58 or association with other membrane molecules such as tetraspanins,44 should be addressed in further studies. A role of integrin 6 receptor for mobilization of HSPCs was studied by using mice reconstituted with integrin 6–/– or wild-type FL HSCs, together with adult BM competitor cells. In line with the reported high proliferation and engraftment capacity of FL cells,5,64 the majority of Lin–Sca-1+ cells in BM 5 to 8 months after transplantation was derived from FL cells. Mobilization of Lin–Sca-1+ cells was not influenced by deletion of integrin 6 receptor, in agreement with our previous findings, which did not show any mobilizing effect of intravenously injected anti-integrin 6 antibodies in adult mouse HSPCs.19 Recently, a p67 nonintegrin laminin receptor was reported to be functionally active in homing and G-CSF–induced mobilization of human HPCs.45,65 In addition, several other laminin receptors including integrin 2ß1, -dystroglycan, Lutheran glycoprotein, and heparan sulfate proteoglycans,20,44–46,66,67 have been reported in adult hematopoietic cells. The possible expression and role of these receptors during fetal HSPC mobilization and homing should be addressed in further studies. Our present findings show a ubiquitous expression of integrin 4 receptor in FL LSK cells and more mature Lin–Sca-1–c-Kit+ progenitors. We therefore analyzed its contribution for FL HSPC homing. Remarkably, homing of FL HPCs to BM was completely inhibited by a function-blocking antibody against integrin 4 chain. Furthermore, homing of short-term and long-term repopulating HSCs was abrogated by inhibition of integrin 4 function. Previous studies where integrin 4–/– embryos and chimeric mice were used have established an important role for 4 integrin for maintenance and development of multilineage progenitors in fetal liver, BM, and spleen, as well as for postnatal multilineage hematopoietic development.13,14,68 In vivo homing of fetal 4–/– HSPCs was not specifically addressed in these studies, although a functional defect in transmigration through adherent stromal cell layer in cultures, restricted to erythroid and B-lineage cells, was noted in vitro. In a recent study,69 reconstitution, albeit in a relatively low level, from E12.5 integrin 4–/– FL cells was reported after transplantation. However, the significance of this is unclear since a homing defect might have been compensated by increased proliferative potential of integrin gene–deleted HPCs, as recently reported in adult mice.70,71 In contrast, studies on inducible integrin 4 gene deletion during adult hematopoiesis have shown decreased self-renewal capacity of HSCs and selective impairment in homing of 4-deficient HPCs to BM.70,72 Our recent findings19 have shown that inhibition of integrin 4 receptor by a function-blocking antibody dramatically reduces homing of both short-term and long-term HSCs from adult BM. Taken together, the present and previous findings suggest a role for integrin 4 during homing of both fetal and adult HSPCs into myeloablated recipients after transplantation, whereas its role during shift of hematopoiesis from liver to BM during fetal development is currently unknown. In conclusion, our results from in vivo transplantation assays show that 6-integrin receptor functions as a homing receptor for FL HPCs during their transmigration into adult BM, whereas homing and engraftment of FL HSCs is independent of 6-integrin receptor function. Furthermore, our findings indicate an essential role for integrin 4-receptor during homing of both FL HPCs and multilineage reconstituting HSCs into BM. Altogether, these and previous findings show intrinsic differences in function of cell adhesion receptors in HSCs and HPCs from developing hematopoietic tissues compared with those from adult sources,6,19 which may be one of the mechanisms underlying the differences in reconstitution in the transplantation setting.    Authorship Top Abstract Introduction Materials and methods Results Discussion Authorship References   Contribution: K.T. and A.D. provided laminin-411 and laminin-511; E.G.-L. provided integrin 6–/– mice; H.Q. and M.E. designed and performed research, analyzed data, and wrote the paper; A.N. performed research and analyzed data; and S.E.W.J. designed research and analyzed data. Conflict-of-interest disclosure: K.T. and A.D. have declared a financial interest in a company (BioStratum) whose products (recombinant laminin-411 and -511) were studied in the present work. All other authors declare no competing financial interests. Correspondence: Marja Ekblom, Hematopoietic Stem Cell Laboratory, BMC B12, Lund University, 221 84 Lund, Sweden; e-mail:marja.ekblom{at}med.lu.se.    Acknowledgments   This work was supported by grants from ALF (Government Public Health Grant), Kungliga fysiografiska sällskapet in Lund, the Swedish Cancer Society, University Hospital of Lund Foundation, and Georg Danielsson's Foundation. We thank Yutaka Sasaki, Jens Nygren, Lilian Wittman, Zhi Ma, and Anna Fossum for technical help and scientific discussions.    Footnotes   Submitted October 10, 2006; accepted June 15, 2007. Prepublished online as Blood First Edition Paper, June 22, 2007 DOI: 10.1182/blood-2006-10-051276 The online version of this article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.    References Top Abstract Introduction Materials and methods Results Discussion Authorship References   Lapidot T, Dar A, Kollet O. How do stem cells find their way home? Blood 2005; 106:1901–1910.[Abstract/Free Full Text] Nilsson SK, Simmons PJ, Bertoncello I. Hemopoietic stem cell engraftment. Exp Hematol 2006; 34:123–129.[CrossRef][Medline] [Order article via Infotrieve] Zheng Y, Watanabe N, Nagamura-Inoue T, et al. Ex vivo manipulation of umbilical cord blood-derived hematopoietic stem/progenitor cells with recombinant human stem cell factor can up-regulate levels of homing-essential molecules to increase their transmigratory potential. Exp Hematol 2003; 31:1237–1246.[CrossRef][Medline] [Order article via Infotrieve] Holyoake TL, Nicolini FE, Eaves CJ. Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp Hematol 1999; 27:1418–1427.[CrossRef][Medline] [Order article via Infotrieve] Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood 1996; 87:3500–3507.[Abstract/Free Full Text] Kondo M, Wagers AJ, Manz MG, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol 2003; 21:759–806.[CrossRef][Medline] [Order article via Infotrieve] Medvinsky AL and Dzierzak EA. Development of the definitive hematopoietic hierarchy in the mouse. Dev Comp Immunol 1998; 22:289–301.[CrossRef][Medline] [Order article via Infotrieve] Danen EH and Sonnenberg A. Integrins in regulation of tissue development and function. J Pathol 2003; 200:471–480.[CrossRef][Medline] [Order article via Infotrieve] Giancotti FG and Ruoslahti E. Integrin signaling. Science 1999; 285:1028–1032.[Abstract/Free Full Text] Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fassler R. Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature 1996; 380:171–175.[CrossRef][Medline] [Order article via Infotrieve] Potocnik AJ, Brakebusch C, Fassler R. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 2000; 12:653–663.[CrossRef][Medline] [Order article via Infotrieve] Williams DA, Rios M, Stephens C, Patel VP. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991; 352:438–441.[CrossRef][Medline] [Order article via Infotrieve] Arroyo AG, Yang JT, Rayburn H, Hynes RO. Alpha4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 1999; 11:555–566.[CrossRef][Medline] [Order article via Infotrieve] Arroyo AG, Yang JT, Rayburn H, Hynes RO. Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell 1996; 85:997–1008.[CrossRef][Medline] [Order article via Infotrieve] Gribi R, Hook L, Ure J, Medvinsky A. The differentiation programme of embryonic definitive hematopoietic stem cells is largely {alpha}4 integrin independent. Blood 2006; 108:501–509.[Abstract/Free Full Text] Hamamura K, Matsuda H, Takeuchi Y, Habu S, Yagita H, Okumura K. A critical role of VLA-4 in erythropoiesis in vivo. Blood 1996; 87:2513–2517.[Abstract/Free Full Text] Tunggal P, Smyth N, Paulsson M, Ott MC. Laminins: structure and genetic regulation. Microsc Res Tech 2000; 51:214–227.[CrossRef][Medline] [Order article via Infotrieve] Colognato H and Yurchenco PD. Form and function: the laminin family of heterotrimers. Dev Dyn 2000; 218:213–234.[CrossRef][Medline] [Order article via Infotrieve] Qian H, Tryggvason K, Jacobsen SE, Ekblom M. Contribution of alpha6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha4 integrins. Blood 2006; 107:3503–3510.[Abstract/Free Full Text] Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin expression are associated with altered homing properties of Lin(-/lo) Thy1. 1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Exp Hematol 2002; 30:176–185.[CrossRef][Medline] [Order article via Infotrieve] Gu YC, Kortesmaa J, Tryggvason K, et al. Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells. Blood 2003; 101:877–885.[Abstract/Free Full Text] Aumailley M, Bruckner-Tuderman L, Carter WG, et al. A simplified laminin nomenclature. Matrix Biol 2005; 24:326–332.[CrossRef][Medline] [Order article via Infotrieve] Siler U, Rousselle P, Muller CA, Klein G. Laminin gamma2 chain as a stromal cell marker of the human bone marrow microenvironment. Br J Haematol 2002; 119:212–220.[CrossRef][Medline] [Order article via Infotrieve] Gu Y, Sorokin L, Durbeej M, Hjalt T, Jonsson JI, Ekblom M. Characterization of bone marrow laminins and identification of alpha5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-Mix cells. Blood 1999; 93:2533–2542.[Abstract/Free Full Text] Siler U, Seiffert M, Puch S, et al. Characterization and functional analysis of laminin isoforms in human bone marrow. Blood 2000; 96:4194–4203.[Abstract/Free Full Text] Georges-Labouesse E, Messaddeq N, Yehia G, Cadalbert L, Dierich A, Le Meur M. Absence of integrin alpha 6 leads to epidermolysis bullosa and neonatal death in mice. Nat Genet 1996; 13:370–373.[CrossRef][Medline] [Order article via Infotrieve] Miller BJ, Georges-Labouesse E, Primakoff P, Myles DG. Normal fertilization occurs with eggs lacking the integrin alpha6beta1 and is CD9-dependent. J Cell Biol 2000; 149:1289–1296.[Abstract/Free Full Text] Kortesmaa J, Yurchenco P, Tryggvason K. Recombinant laminin-8 (alpha(4)beta(1)gamma(1)): production, purification, and interactions with integrins. J Biol Chem 2000; 275:14853–14859.[Abstract/Free Full Text] Doi M, Thyboll J, Kortesmaa J, et al. Recombinant human laminin-10 (alpha5beta1gamma1): production, purification, and migration-promoting activity on vascular endothelial cells. J Biol Chem 2002; 277:12741–12748.[Abstract/Free Full Text] Katayama Y, Hidalgo A, Furie BC, Vestweber D, Furie B, Frenette PS. PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin. Blood 2003; 102:2060–2067.[Abstract/Free Full Text] Uchida N, Aguila HL, Fleming WH, Jerabek L, Weissman IL. Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1. 1lo Lin-Sca-1+ hematopoietic stem cells. Blood 1994; 83:3758–3779.[Abstract/Free Full Text] Traver D, Miyamoto T, Christensen J, Iwasaki-Arai J, Akashi K, Weissman IL. Fetal liver myelopoiesis occurs through distinct, prospectively isolatable progenitor subsets. Blood 2001; 98:627–635.[Abstract/Free Full Text] Forsberg EC, Serwold T, Kogan S, Weissman IL, Passegue E. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors. Cell 2006; 126:415–426.[CrossRef][Medline] [Order article via Infotrieve] Oostendorp RA, Ghaffari S, Eaves CJ. Kinetics of in vivo homing and recruitment into cycle of hematopoietic cells are organ-specific but CD44-independent. Bone Marrow Transplant 2000; 26:559–566.[CrossRef][Medline] [Order article via Infotrieve] Helgason CD, Antonchuk J, Bodner C, Humphries RK. Homeostasis and regeneration of the hematopoietic stem cell pool are altered in SHIP-deficient mice. Blood 2003; 102:3541–3547.[Abstract/Free Full Text] Buza-Vidas N, Antonchuk J, Qian H, et al. Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK. Gene Dev 2006; 20:2018–2023.[Abstract/Free Full Text] Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci U S A 1990; 87:8736–8740.[Abstract/Free Full Text] Craddock CF, Nakamoto B, Andrews RG, Priestley GV, Papayannopoulou T. Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice. Blood 1997; 90:4779–4788.[Abstract/Free Full Text] Kabayama K, Ito N, Honke K, Igarashi Y, Inokuchi J. Suppression of integrin expression and tumorigenicity by sulfation of lactosylceramide in 3LL Lewis lung carcinoma cells. J Biol Chem 2001; 276:26777–26783.[Abstract/Free Full Text] Hager M, Gawlik K, Nystrom A, Sasaki T, Durbeej M. Laminin {alpha}1 chain corrects male infertility caused by absence of laminin {alpha}2 chain. Am J Pathol 2005; 167:823–833.[Abstract/Free Full Text] Rebel VI, Miller CL, Thornbury GR, Dragowska WH, Eaves CJ, Lansdorp PM. A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp Hematol 1996; 24:638–648.[Medline] [Order article via Infotrieve] Mazo IB, Quackenbush EJ, Lowe JB, von Andrian UH. Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow. Blood 2002; 99:4182–4191.[Abstract/Free Full Text] Szilvassy SJ, Meyerrose TE, Ragland PL, Grimes B. Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver. Blood 2001; 98:2108–2115.[Abstract/Free Full Text] Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM, Weissman IL. Differential expression of novel potential regulators in hematopoietic stem cells. PLoS Genet 2005; 1:0281–0294. Bonig H, Chang KH, Nakamoto B, Papayannopoulou T. The p67 laminin-receptor identifies human erythroid progenitor and precursor cells and is functionally important for their bone marrow lodgment. Blood 2006; 108:1230–1233.[Abstract/Free Full Text] Selleri C, Montuori N, Ricci P, et al. Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization. Blood 2005; 105:2198–2205.[Abstract/Free Full Text] Adolfsson J, Mansson R, Buza-Vidas N, et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 2005; 121:295–306.[CrossRef][Medline] [Order article via Infotrieve] Dooner M, Cerny J, Colvin G, et al. Homing and conversion of murine hematopoietic stem cells to lung. Blood Cells Mol Dis 2004; 32:47–51.[CrossRef][Medline] [Order article via Infotrieve] Dangerfield J, Larbi KY, Huang MT, Dewar A, Nourshargh S. PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane. J Exp Med 2002; 196:1201–1211.[Abstract/Free Full Text] Dangerfield JP, Wang S, Nourshargh S. Blockade of {alpha}6 integrin inhibits IL-1{beta}- but not TNF-{alpha}-induced neutrophil transmigration in vivo. J Leukoc Biol 2004; 77:159–165.[CrossRef][Medline] [Order article via Infotrieve] Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf NS. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 1995; 92:9647–9651.[Abstract/Free Full Text] Miyake K, Weissman IL, Greenberger JS, Kincade PW. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med 1991; 173:599–607.[Abstract/Free Full Text] Tamura RN, Cooper HM, Collo G, Quaranta V. Cell type-specific integrin variants with alternative alpha chain cytoplasmic domains. Proc Natl Acad Sci U S A 1991; 88:10183–10187.[Abstract/Free Full Text] de Melker AA and Sonnenberg A. Integrins: alternative splicing as a mechanism to regulate ligand binding and integrin signaling events. Bioessays 1999; 21:499–509.[CrossRef][Medline] [Order article via Infotrieve] Szilvassy SJ, Ragland PL, Miller CL, Eaves CJ. The marrow homing efficiency of murine hematopoietic stem cells remains constant during ontogeny. Exp Hematol 2003; 31:331–338.[CrossRef][Medline] [Order article via Infotrieve] Chen MS, Almeida EA, Huovila AP, et al. Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM. J Cell Biol 1999; 144:549–561.[Abstract/Free Full Text] Ginsberg MH, Partridge A, Shattil SJ. Integrin regulation. Curr Opin Cell Biol 2005; 17:509–516.[CrossRef][Medline] [Order article via Infotrieve] ffrench-Constant C and Colognato H. Integrins: versatile integrators of extracellular signals. Trends Cell Biol 2004; 14:678–686.[CrossRef][Medline] [Order article via Infotrieve] Levesque JP and Simmons PJ. Cytoskeleton and integrin-mediated adhesion signaling in human CD34+ hemopoietic progenitor cells. Exp Hematol 1999; 27:579–586.[CrossRef][Medline] [Order article via Infotrieve] Levesque JP, Haylock DN, Simmons PJ. Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors. Blood 1996; 88:1168–1176.[Abstract/Free Full Text] Peled A, Kollet O, Ponomaryov T, et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000; 95:3289–3296.[Abstract/Free Full Text] Schofield KP, Rushton G, Humphries MJ, Dexter TM, Gallagher JT. Influence of interleukin-3 and other growth factors on alpha4beta1 integrin-mediated adhesion and migration of human hematopoietic progenitor cells. Blood 1997; 90:1858–1866.[Abstract/Free Full Text] Bazzoni G and Hemler ME. Are changes in integrin affinity and conformation overemphasized? Trends Biochem Sci 1998; 23:30–34.[CrossRef][Medline] [Order article via Infotrieve] Pawliuk R, Eaves C, Humphries RK. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 1996; 88:2852–2858.[Abstract/Free Full Text] Selleri C, Ragno P, Ricci P, et al. The metastasis-associated 67 kDa laminin receptor is involved in G-CSF-induced hematopoietic stem cell mobilization. Blood 2006; 108:2476–2484.[Abstract/Free Full Text] Chan JY, Lee-Prudhoe JE, Jorgensen B, et al. Relationship between novel isoforms, functionally important domains, and subcellular distribution of CD164/endolyn. J Biol Chem 2001; 276:2139–2152.[Abstract/Free Full Text] Parsons SF, Lee G, Spring FA, et al. Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity. Blood 2001; 97:312–320.[Abstract/Free Full Text] Arroyo AG, Taverna D, Whittaker CA, et al. In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins. J Immunol 2000; 165:4667–4675.[Abstract/Free Full Text] Gribi R, Hook L, Ure J, Medvinsky A. The differentiation programme of embryonic definitive hematopoietic stem cells is largely {alpha}4 integrin independent. Blood 2006; 108:501–509.[Abstract/Free Full Text] Scott LM, Priestley GV, Papayannopoulou T. Deletion of alpha4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing. Mol Cell Biol 2003; 23:9349–9360.[Abstract/Free Full Text] Bungartz G, Stiller S, Bauer M, et al. Adult murine hematopoiesis can proceed without {beta}1 and {beta}7 integrins. Blood 2006; 108:1857–1864.[Abstract/Free Full Text] Priestley GV, Scott LM, Ulyanova T, Papayannopoulou T. Lack of {alpha}4 integrin expression in stem cells restricts competitive function and self-renewal activity. 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