Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

Abstract

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.

Figure 1

Fibronectin is produced and assembled by undifferentiated mES cells. (A) Domain structure of fibronectin (adapted from [21]). (B) mES cells grown on gelatin in mES cell medium in the presence of LIF were fixed, stained with R457 anti-fibronectin antiserum followed by rhodamine-goat anti-rabbit IgG, and analyzed by phase contrast (left) and fluorescence (right) microscopy. Scale bars = 50 μm.

Figure 2

Increased substrate fibronectin induces mES cell spreading. (A–D) mES cells were plated in the presence of LIF on a gelatin-coated surface (A) or on surfaces coated with fibronectin [FN] at 0.1 (B), 1 (C), or 10 (D) μg/ml. After 6 hours, cells were fixed, permeabilized, and stained with anti-vinculin monoclonal antibody followed by rhodamine-goat anti-mouse IgG. Scale bars = 50 μm. Insets show magnified images of cells. (E) Cell areas were quantified as described in Materials and Methods for mES cells grown on the indicated ECM coatings in the presence of LIF for 4 hours. Bars represent average cell areas relative to average area of cells on gelatin and are from a representative experiment. Error bars represent standard error in the mean for each condition, relative to that for gelatin. Statistical analyses were performed relative to Gelatin. ** p < 0.01.

Figure 3

Fibronectin affects self-renewal markers and FAK phosphorylation. (A, B) Nanog-GFP mES cells were grown on the indicated surfaces for six days, passaging every other day. After six days, expression of GFP (A) or SSEA-1 (B) was quantified by flow cytometry. SSEA-1 was detected with anti-SSEA-1 antibody and fluorescent secondary antibody. Black profile is from non-fluorescent mES cells (A) or unstained mES cells (B). (C) RNA was extracted from cells grown as in (A–B), and quantitative RT-PCR was performed using primers for Nanog and Oct4. Values for fibronectin samples were normalized to gelatin [Gel] (set at 1). Graphs represent the average of triplicate samples from two independent experiments, and error bars indicate +/− one standard deviation from the mean. * p < 0.05. (D, E) mES cells grown on indicated substrates were lysed after 6 days, equal amounts of protein were separated by SDS-PAGE, proteins were detected by immunoblotting with anti-pFAK-Y397, anti-FAK, or anti-pStat3-Y705 antibodies. pStat3 blots were stripped and re-probed with anti-Stat3 antibodies. Blots are representative of at least two experiments.

Figure 4

siRNA knockdown of fibronectin causes loss of cell adhesion mES cells were treated with fibronectin (FN) siRNAs (A) or control siRNAs (B) followed by plating on gelatin in the presence of LIF in Knockout medium. (C) As in A, but with addition of 1 μg FN/ml to culture medium. (D) mES cells were treated with fibronectin siRNAs and plated in the presence of LIF on tissue culture plastic coated with 50 μg/ml III9–10 protein. Phase contrast images were taken 24 hours after plating. (E) Total colonies were counted in multiple microscopic fields (n > 4), wells were washed with PBS, and attached colonies were counted in multiple fields (n > 4). Numbers of colonies per field were averaged. Graphs represent average number of colonies/field for two independent experiments. Error bars represent one standard deviation from average value. Scale bars = 50 μm.

Figure 5

Fibronectin binds to the gelatin substrate. (A) Phase contrast images of mES cells plated on gelatin-coated (i) or uncoated (ii) tissue culture plastic. The graph shows total and attached mES cell colonies on gelatin-coated (Gelatin) or uncoated (Plastic) surfaces 24 hours after plating. Averages for two independent experiments were calculated as in Figure 4 legend. (B) mES cells grown for 24 hours were removed from the indicated surfaces by treatment with EDTA. An ELISA was performed to quantify surface-bound fibronectin using R457 anti-fibronectin antiserum. (B, inset) mES cells on either uncoated or gelatin-coated tissue culture wells were labeled with ³⁵S-methionine for 24 hours. ³⁵S-labeled fibronectin was isolated from the media, separated by SDS-PAGE, and analyzed using a phosphorimager. FN indicates location of fibronectin band. (C) Phase contrast images of mES cells plated on gelatin, either in the presence of CAPS buffer (i) or in the presence of 0.7 μM (50 μg/ml) 70 kD fibronectin fragment (ii). Graph shows total and attached cell colonies without [Gelatin] and with 70 kD [+70 kD], averaged from two independent experiments.

Figure 6

Fibronectin knockdown results in loss of self-renewal. (A) Nanog-GFP mES cells were treated with siRNAs against fibronectin and then grown under the indicated conditions in the presence of LIF in Knockout medium. GFP expression was monitored by flow cytometry on day 6. Day 0 profile is of siRNA-treated cells before replating. Black profile is of non-fluorescent mES cells. (B) Nanog-GFP mES cells were treated with siRNAs against FN, and then grown either in the presence (+FN) or absence (-FN) of exogenous fibronectin. RNA was extracted from cells on day 6, and quantitative RT-PCR was performed using primers for Nanog and Oct4. Values for each sample are normalized to siRNA-treated cells plated + FN. Graphs represent the average of triplicate samples in two independent experiments, and error bars indicate +/− one standard deviation from the mean. ** p < 0.01. (C, D) Cells grown as in (B) with (+) or without (−) fibronectin added were lysed on day 6. Equal amounts of protein were separated by SDS-PAGE, and proteins were detected by immunoblotting with anti-pFAK-Y397 or anti-FAK (C) or with anti-pStat3-Y705 (D) antibodies. pStat blots were stripped and reprobed with anti-Stat3 antibodies (D). Blots are representative of at least two experiments.