Phenotypic and Functional Evidence for the Expression of CXCR4 Receptor During Megakaryocytopoiesis by Christel Rivière, Frédéric Subra, Karine Cohen-Solal, Véronique Cordette-Lagarde, Remi Letestu, Christian Auclair, William Vainchenker, and Fawzia Louache Blood Volume 93(5):1511-1523 March 1, 1999 ©1999 by American Society of Hematology

Detection of CXCR4 transcripts by RNase protection assay Detection of CXCR4 transcripts by RNase protection assay. mRNA was extracted from peripheral blood mononuclear cells, PHA-activated mononuclear cells, CD41+ cells, and platelets. tRNA served as a negative control. Detection of CXCR4 transcripts by RNase protection assay. mRNA was extracted from peripheral blood mononuclear cells, PHA-activated mononuclear cells, CD41+ cells, and platelets. tRNA served as a negative control. Samples of mRNA were hybridized with a specific CXCR4 riboprobe and a specific actin riboprobe, then digested with RNase T1. Protected fragments were analyzed on denaturing acrylamide gels. Sizes of the fragments were determined using labeled Msp I–digested pBR322. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

CXCR4 expression on megakaryocytes and platelets. CXCR4 expression on megakaryocytes and platelets. (A, B) Representative data of flow cytometric analysis of CD41a and CXCR4 on megakaryocytes grown in culture are shown. Cord blood CD34+ cells were cultured for 6 days in the presence of a combination of SCF and PEG-rhuMGDF. Cells were stained using an anti-CD41a MoAb in combination with either an isotype control antibody (A) or the anti-CXCR4 MoAb (B). (C) CXCR4 expression on freshly isolated megakaryocytes after percoll enrichment. (D) Platelets, depleted in leukocytes, were labeled using a similar procedure. Labeling with control MoAb is shown by the thin line. The thick line shows the staining with the anti-CXCR4 MoAb. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Calcium flux response of megakaryocytes to SDF-1. Calcium flux response of megakaryocytes to SDF-1. After CD41a labeling, cells were loaded with Fluo-3 and exposed to SDF-1 (300 ng/mL). Changes in fluorescence were monitored over time by flow cytometry after SDF-1 addition. The results are derived from one representative experiment of a total of three separate experiments. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Actin polymerization in megakaryocytes after SDF-1 addition. Actin polymerization in megakaryocytes after SDF-1 addition. Cultured cells were incubated at 37°C for 30 seconds and 2 minutes in the presence of either SDF-1 (300 ng/mL) or thrombin (Stago, 1 U/mL). Intracellular F actin (phalloidin) or G actin (oregon green conjugated DNase 1) was determined by flow cytometry after gating the CD41a+ cells. Results of one representative experiment are shown. Two other experiments gave similar results. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Effects of SDF-1 on the expression of CD62 by megakaryocytes. Effects of SDF-1 on the expression of CD62 by megakaryocytes. (A,B,C) Examples of double-color staining with anti-CD62 in combination with anti-CD41a (A) or with control antibodies (B) and (C). Primary cultured cells were either left unstimulated (thick line) or stimulated for 10 minutes with either thrombin (1 U/mL, thin line) or SDF-1 (300 ng/mL, broken line). CD62 staining in combination with CD41a staining was then compared by FACS analysis as described in Materials and Methods. The analysis was performed in CD41alow (D) and CD41ahigh (E) gates as defined in (A) and in platelets (F). These data are representative of four experiments. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Chemotaxis assay and cell surface expression of CD41a. Chemotaxis assay and cell surface expression of CD41a. (A) Chemotaxis responses of the CD41a+ cell population. CD34+ cells were seeded in serum-free liquid culture containing SCF and PEG-rhuMGDF for up to 6 days. The cells were subjected to chemotaxis through 5-μm pores to various concentrations of SDF-1 and stained with an anti-CD41a MoAb (black bars). Checkerboard analysis was performed by adding SDF-1 (500 ng) both in the bottom and in the top well (white bar). This migration is inhibited by preincubating the cells with pertussis toxin (PTX; hatched bar). Data are expressed as chemotaxis index and represent the mean for one representative experiment done in triplicate. (B) Effect of a blocking MoAb against CXCR4 (12G5). The cells were preincubated either with increasing concentrations of 12G5 MoAb (white squares) or an isotype-control MoAb (black squares) before the migration assay. (C) Chemotaxis responses of megakaryocytes. After migation in response to SDF-1 (500 ng/mL), the cells were stained with an anti-CD41a MoAb. The percentage of CD41+cells was determined in starting and migrated cells. The results shown are the mean and SD of five experiments. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Morphological characteristics of cells migrating in response to SDF-1. Morphological characteristics of cells migrating in response to SDF-1. After migration, cultured cells were analyzed for their morphological characteristics. Dot plot analysis (FSC versus SSC) of starting population (A), cells migrated in response to SDF-1 (B), and cells migrated in absence of SDF-1 (C) are shown. May-Grunwald-Giemsa staining of the starting cell fraction (D) and the cell fraction that migrated in response to SDF-1 (E) are shown. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Flow cytometric analysis of cell surface expression of CD41a and CD42a on starting and migrated cell populations. Flow cytometric analysis of cell surface expression of CD41a and CD42a on starting and migrated cell populations. After migration, cultured cells were labeled with an R-PE–conjugated anti-CD41a MoAb and a FITC-conjugated anti-CD42a MoAb. (A, B, C) An example of double-color fluorescence beween CD41a and CD42a (A) and the respective controls (B, C). The cells within the lymphoid blast window (R1, Fig 7) were analyzed for the expression of CD42a (D) and CD41a (E). The thick line shows the staining of cells that migrated in response to an optimal concentration of SDF-1 (500 ng/mL). Broken lines represent the staining of the starting cell population, and dotted lines represent the staining with control antibodies. In two experiments, CD42a (F) and CD41a (G) staining was compared between the cells that migrated in response to SDF-1 (thick line) and to control media (thin line). (H) Migration of CD41alow/CD42alow cells. The mean and SD percentages of CD42alow/CD41alow were determined by gating on the entire population of CD41a+cells. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Chemotaxis responses of CFU-MK in response to various concentrations of SDF-1. Chemotaxis responses of CFU-MK in response to various concentrations of SDF-1. CD34+ cells were seeded in serum-free liquid culture containing SCF and PEG-rhuMGDF for up to 6 days. After 6-day culture, the cells were subjected to chemotaxis through 5-μm pores and plated in CKU-MK assay. Results are shown of one representative experiment performed in triplicate. Two other experiments gave similar results.*P < .05, **P < .01 when compared with control. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Effects of SDF-1 on CFU-MK growth. Effects of SDF-1 on CFU-MK growth. After CD34+ selection, 1 × 103cells were assayed in fibin-clot cultures in presence of increasing concentrations of SDF-1. Data are expressed as the mean and SD of two experiments performed in triplicate. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology

Effects of SDF-1 on platelet production. Effects of SDF-1 on platelet production. CD34+ cells were seeded in serum-free liquid culture containing SCF and PEG-rhuMGDF for up to 6 days. After two washes, SDF-1 was added at optimal concentration (500 ng/mL) in a second phase of the culture in the presence of increasing concentrations of PEG-rhuMGDF without any other factor (A) or in presence of an optimal concentration of SCF (25 ng/mL) (B). Conversely, platelet production was tested in the presence of an optimal concentration of PEG-rhuMGDF(10 ng/mL) and increasing concentrations of SCF (C). Six days after SDF-1 addition, platelet production was assessed by flow cytometry after anti-CD41a labeling. Cells from each culture condition without SDF-1 (white bar) and in presence of SDF-1 (black bar) were distributed in the same volume (400 μL), and for each sample the acquisition rate was 1 μL/s for 100 seconds. Data represent the mean for two experiments done in triplicate. Christel Rivière et al. Blood 1999;93:1511-1523 ©1999 by American Society of Hematology