Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells

Abstract

Embryonic stem (ES) cells have a broad potential application in regenerative medicine and can be differentiated into cells of all three germ layers. Adhesion of ES cells to extracellular matrix (ECM) proteins is essential for the differentiation pathway; Cell-ECM adhesion is mediated by integrins that have the ability to activate many intracellular signaling pathways. Therefore, we hypothesize that the expression and function of integrin receptors is a critical step in ES differentiation. Using functional cell adhesion assays, our study demonstrates that α5β1 is a major functional integrin receptor expressed on the cell surface of undifferentiated mouse ES-D3 cells, which showed significantly higher binding to fibronectin as compared to collagens. This adhesion was specific mediated by integrin α5β1 as evident from the inhibition with a disintegrin selective for this particular integrin. Differentiation of ES-D3 cells on fibronectin or on a collagen type1/fibronectin matrix, caused further selective up-regulation of the α5β1 integrin. Differentiation of the cells, as evaluated by immunofluorescence, FACS analysis and quantitative RT-PCR, was accompanied by the upregulation of mesenchymal (Flk1, isolectin B4, α-SMA, vimentin) and endodermal markers (FoxA2, SOX 17, cytokeratin) in parallel to increased expression of α5β1 integrin. Taken together, the data indicate that fibronectin-mediated, upregulation of α5β1 integrin and adhesion of ES-D3 cells to specific ECM molecules are linked to early stages of mouse embryonic stem cells commitment to meso-endodermal differentiation.

Figure 1

LIF-deprivation increased adhesion of ES-D3 cells grown on fibronectin to α5 disintegrins and anti-α5 antibody. (A) 1.5 × 10⁶ cells were differentiated on flasks coated with 0.1% gelatin (control), 30 µg/ml collagen IV or 10 µg/ml fibronectin for three days (Method I), harvested with 0.5 mM EDTA, loaded with CMFDA dye and submitted for adhesion assay on plates coated with 2 µg/ml per well of VLO4 (white bar), Bitisgabonin-1 (gray bar), EC3 (light gray bar) and VP12 (black bar). The data represents mean ± SEM of three experiments, each performed in triplicates. *p < 0.05 compared to cells grown on gelatin. (B) 1.5 × 10⁶ cells grown on flasks coated with 10 µg/ml fibronectin were cultured for three days in the presence (undifferentiated) or absence (differentiation, Method I) of LIF. The cells were harvested tested for adhesion to plates coated with the different monoclonal anti-α and anti-β antibodies (10 µg/ml per well). White bars, undifferentiated; gray bars, differentiated. The data represent mean ± SEM of six experiments, each performed in triplicates. *p < 0.05 compared to α1; **p < 0.05 compared to α5; ⁺p < 0.05 compared to β3.

Figure 2

Growth of ES-D3 cells on fibronectin increased adhesion to anti-α5 antibody (A) and fibronectin (B). (A) 1.5 × 10⁶ cells were cultured in flasks coated with, respectively, 0.1% gelatin, 30 µg/ml collagen I and IV, VCAM or 10 µg/ml fibronectin. Either left undifferentiated (white bars) or induced to differentiate by LIF deprivation (Method I, grey bars), the cells were harvested after three days with 0.5 mM EDTA, loaded with CMFDA and assayed for adhesion to anti-α5 antibody. The data represent mean ± SEM of three independent experiments, each performed in triplicates. *p < 0.05 compared to cells grown on gelatin; **p < 0.05 compared to undifferentiated cells grown on fibronectin. (B) The cells previously grown on either 0.1% gelatin (white bars) or 10 µg/ml fibronectin (gray bars) for three days in the absence of LIF (differentiation Method I) were harvested with 0.5 mM EDTA, loaded with CMFDA and assayed for adhesion to 30 µg/ml collagen I, 30 µg/ml collagen IV or 10 µg/ml fibronectin. The data represents mean ± SEM compared to undifferentiated control of three independent experiments, each performed in triplicates. *p < 0.05 compared to cells grown on gelatin and tested on fibronectin.

Figure 3

Light micrographs of undifferentiated and differentiated ES-D3 cells. The cells were cultured on 0.1% gelatin (A and B) or 10 µg/ml fibronectin (C and D) for 6 h (A and C) or three days (B and D) in the presence (A and B undifferentiated) or absence (C and D differentiated) of LIF (Method I). The cell morphology was evaluated by phase contrast and typical fields are presented. Bar = 20 µm. Inserts: fluorescence staining of single cells; blue -DAPI, nuclear staining; red, rhodamine-phalloidin staining of the actin cytoskeleton.

Figure 4

Time course of ES-D3 cells spreading on gelatin or fibronectin. The cells were cultured in the presence or absence of LIF (Method I) on 0.1% gelatin (white bars) or 10 µg/ml fibronectin (gray bars) for 24 h. Every 6 h the cells were photographed. % cell spreading was estimated as the ratio of spread elongated cells out of the total number of cells in the field ×100.

Figure 5

Expression of pluripotency and differentiation markers in ES-D3 cells. The cells were cultured for eight days in the presence (undifferentiated) or absence (differentiated, Method II) of LIF and stained with specific antibodies as indicated in Materials and Methods, or incubated with 10 µg/ml FITC-isolectin B4 (Lectin) as endothelial marker. Inserts: nuclear staining (upper part) and marker staining (lower part) of undifferentiated cells. The cells were analyzed by fluorescent microscopy; typical photographs are presented. Nucleus: the nuclear staining was performed with DAPI (blue). Markers: labeled with primary antibodies followed by appropriate secondary antibodies labeled with Alexa 488 (green) and Alexa 594 (red); Merge: superposition of nuclear and specific marker staining; bars = 50 µm.

Figure 6

Expression of Flk-1 in ES-D3 cells analyzed by FACS and immunoprecipitation. The cells were grown on 10 µg/ml fibronectin for three days in presence or absence of LIF (Method I). (A) Cell extracts from undifferentiated and differentiated cells were immunoprecipitated by anti-Flk-1 monoclonal antibody and separated by 7.5% SDS-PAGE followed by western blotting with anti-Flk-1 polyclonal antibody. VEGFR-2, the position of mature glycosylated receptor. For experimental details, see Materials and Methods. (B) Flk-1expression assessed by flow cytometry in the differentiated cells. The cells were fixed, permeabilized, stained with anti Flk-1 antibody followed by second antibody (right trace) or stained only with second antibody (left trace, control).

Figure 7

Time course of expression of pluripotency and meso-endodermal markers in ES-D3 cells. (A) mRNA expression levels of pluripotency marker gene Oct3/4 and meso-endodermal marker genes FoxA2 and Sox17 were evaluated in ES-D3 cells differentiated for up to eight days (Method II). Oct3/4 (triangles), FoxA2 (squares), Sox17 (diamonds). Insert: Decreased Oct3/4 mRNA level over time. Data is presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 compared to day 0 control. (B) Transcription factor protein expression in ES-D3 cells differentiated for 8 days (Method II), as determined by immunostaining using anti-Sox17 and anti-FoxA2 antibodies (1:100), nuclear staining by DAPI and morphology evaluated by phase contrast; bars = 50 µm.