SVZ neurospheres were prepared from 1- to 3-day-old C57BL/6 WT or GFP mice in serum-free medium (SFM) supplemented with 10 ng/ml epidermal growth factor (EGF) and 5 ng/ml FGF-2 (Invitrogen) (Agasse et al., 2008). BEC were obtained from adult (6–8 weeks) mice whole brain fragments (excluding the brain stem and the cerebellum) digested with 1 mg/ml of collagenase/dispase (Roche) and resuspended in EC medium containing 10% of fetal bovine serum (FBS) (Wu et al., 2003). BEC were selected using 4 μg/ml puromycin for 2 days (Perrière et al., 2005). Cells were plated on 1% gelatin A (Sigma-Aldrich)-coated petri-dishes, grown until confluence (10 days), trypsinized and collected. BEC looked healthier and maintained better as subconfluent cultures, compared to confluent cultures. This was especially evident at higher passages. At increased density of BEC, the cells were more quiescent, and eventually lifted off the substrate. Thus, BEC were grown to confluency only for expansion purposes. In cocultures, we used BEC at no more than 60% confluency.

For cocultures, BEC were plated on gelatin-coated glass coverslips in 24-well plates (20,000 cells/well), in EC medium for 24 h, treated with or without (Control) the protein synthesis inhibitor cycloheximide (CHX; 1 μg/ml; Sigma-Aldrich) for 1 h and carefully washed 3 times in sterile PBS to completely remove traces of FBS and/or CHX. SVZ spheres were seeded on top of BEC in SFM devoid of growth factors. The contribution of BEC soluble factors was evaluated in SVZ neurospheres plated on CHX-treated BEC in SFM plus BEC SFM-conditioned medium (CM) (1:1). After 24 h, cells were fixed in 4% paraformaldehyde. For cell proliferation studies, 10 μM 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich) was added to the medium for the last 4 h of coculture session.

For Western blot (see Western blot section), SVZ cells were obtained from the dissociation of primary neurospheres and plated as single cells on ECM proteins to allow a homogeneous activation of stemness and the Notch pathway rather than a selective activation affecting only cells present at the bottom of the neurospheres contacting the substrate. For the Cell pair assay (see Cell pair assay section), SVZ cells were obtained from the dissociation of SVZ fragments and plated as single cells on ECM proteins, to allow the testing of SVZ cells that had not been previously exposed to growth factors.

SVZ neurospheres obtained from floxed β1 mice (β1^flox/flox) (1- to 4-day) were grown in DMEM/F12 supplemented with B27 in the presence of EGF (10 ng/ml) and FGF-2 (5 ng/ml) for 3 days, then dissociated and infected with an adenoviral vector expressing Cre recombinase (Eton Bioscience Inc.) using 50 virus particles per cell (Leone et al., 2005). Cells were replated in the same medium. The culture medium was changed after 3 days to medium without adenovirus. Recombination was confirmed 10 days after infection by the expression of β-galactosidase in the primary infected neurospheres, as excision of the β1 gene activates a lacZ reporter gene.

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, Roche) method was used to stain apoptotic nuclei (Rosa et al., 2010). BrdU immunostaining was performed as described previously (Rosa et al., 2010). Cells were incubated overnight with primary antibodies as listed in Supplementary Table 1, and for 1 h with the appropriate Alexa 594 and 488 secondary antibodies (1:200 in PBS, Invitrogen).

SVZ neurospheres and BEC were bound to slides using cytocentrifugation, and labeled for α6 integrin and laminin as aforementioned. SVZ labeling for β1 integrin was performed in living cells incubated at 37°C for 3 h in SFM containing anti-β1 antibody before fixation. BEC in cocultures were stained for cluster of differentiation 31 (CD31). Nuclei were stained with 2 μg/ml Hoechst 33342 (Invitrogen) and preparations were mounted in Dako mounting medium (Dako).

Detection of α6β1 integrin and laminin was performed in SVZ primary neurospheres and BEC collected in lysis buffer as previously described (Rosa et al., 2010). Total brain proteins were used as a positive control for α6 and β1 integrins. Activation of the Notch pathway was evaluated in SVZ cells obtained from the dissociation of primary neurospheres (Neurocult chemical dissociation kit, STEMCELL Technologies) and plated, as single cells, at a density of 900 000 cells per well of a 6-well plate coated with poly-D-lysine (20 μg/ml) alone or coated with either laminin-1 (25 μg/ml, reference: L2020), fibronectin (5 μg/ml) or vitronectin (5 μg/ml) (all from Sigma-Aldrich). Cells were grown for 72 h in SFM and harvested in lysis buffer. Western blot was performed as previously detailed (Rosa et al., 2010) using primary and secondary antibodies as listed in Supplementary Tables 2, 3, followed by visualization using ECF™ reagent on a Storm 860 Gel and Blot Imaging System (GE Healthcare) or using fluorescence-conjugated secondary antibodies on an Odyssey® Infrared Imaging System (Licor Biosciences). Band intensities were measured using ImageJ (NIH Image).

Dissociated cells from SVZ explants were plated onto 10 mm diameter glass coverslips coated with poly-D-lysine (20 μg/ml) alone or with laminin-1, fibronectin or vitronectin as aforementioned, at a density of 2500 cells per coverslip. Cells were grown in SFM containing 5 ng/ml EGF and 2.5 ng/ml FGF-2 (low EGF/FGF-2) for 24 h. Involvement of the mTOR signaling pathway was tested by incubating cells for the 24 h of the assay in the presence of 20 nM rapamycin (Tocris Bioscience). Cells were immunostained for Sox2 and stained with Hoechst 33342.

Fluorescent images were acquired using a LSM 510 Meta confocal microscope or an Axioskop 2 Plus fluorescent microscope (Carl Zeiss Inc.). In cocultures, 10 photos (40x magnification) of each coverslip were taken using a LSM 510 Meta confocal microscope (Carl Zeiss). Countings were performed in the vicinity of neurospheres where SVZ cells migrate out of the neurosphere to form a pseudomonolayer of cells. Some of these SVZ cells associate closely with BEC. Comparisons regarding cell proliferation, cell death, stemness, and neuronal differentiation were made between the population of cells that contacts BEC vs. the one that does not contact BEC. Due to the high density of cells, no countings are performed within neurospheres. The percentages of BrdU+/TUNEL+/Sox2+/Mash1+ SVZ cells were therefore calculated within the population of SVZ cells contacting CD31+ BEC (i.e., Hoechst-labeled SVZ cell nuclei that are located <10 μm from BEC) as well as in areas where no BEC are present, for comparison. Numbers of DCX+ ramifications contacting or crossing BEC as well as DCX+ cell bodies adjacent to BEC were counted. Numbers of Sox2+/Sox2+, Sox2+/Sox2− and Sox2−/Sox2− cell pairs were expressed as a percentage of total cell divisions. Unless otherwise specified, experiments were replicated at least in 3 independent cultures. Within each experiment, 3 coverslips for each condition were analyzed. In Western blots, the ratio of intensity between the bands and their respective loading controls (β-actin or GAPDH) were performed. The Western blots presented are representative of blots performed with at least 3 different cultures. Data are expressed as means ± s.e.m. The unpaired Student t-test, and the one-way ANOVA followed by the Dunnett post-test for comparison with the control condition or the Bonferroni for multiple comparisons were used; P ≤ 0.05 was considered statistically significant.

BEC primary cultures were grown in direct contact with SVZ cells for 24 h. The maximum duration of the coculture experiment was determined according to the capacity of BEC to survive in SFM and SFM conditioned by SVZ cells (evaluated by Methylthiazol Tetrazolium Assay and TUNEL staining; Supplementary Figure 1). Figure 1 provides images of the interaction between BEC and SVZ cells, demonstrating GFP neurospheres adhering to BEC and extending a pseudomonolayer of cells (Figure 1A, at t = 0 h, and Figure 1B, at t = 24 h). After 24 h of coculture, SVZ cells including Nestin positive (+) immature cells and GFAP+ astrocytes contact CD31+ BEC (Figures 1C,D).

Heterocellular contacts between EC and stem/progenitor cells or cancer stem cells involve binding of laminin to α6β1 integrin (Shen et al., 2008; Lathia et al., 2010). As depicted in Figure 2, free-floating SVZ spheres express α6 and β1 integrin subunits (Figures 2A,B,D,E) and BEC secrete laminin (Figures 2C,F). In order to impair laminin-integrin interactions and test functional implications of these interactions, a pulse of 1 h with 1 μg/ml of the protein synthesis inhibitor cycloheximide (CHX) was applied to BEC followed by a chase of 24 h in SFM to mimic coculture conditions. This treatment was expected to decrease the turnover of ECM and membrane-bound proteins. Laminin protein levels, evaluated by Western blot (Figure 2G) and expressed as a percentage of GAPDH expression (Figure 2H), showed a marked decrease in CHX-treated BEC as compared to untreated cocultures (Untreated BEC vs. CHX-treated BEC: P < 0.05). The CHX treatment did not induce apoptosis in BEC as evaluated by TUNEL staining and compared to BEC cultured in EC medium (Control) (Control 24 h: 1.50 ± 0.33%, 3182 cells counted; CHX-treated: 2.54 ± 0.36%, 2271 cells counted).

The effect of SVZ-BEC contact on SVZ cell proliferation was evaluated in cocultures incubated with 10 μM BrdU (Figure 3A). Within the population of cells contacting CD31+ BEC (1533 Hoechst-labeled SVZ cells in contact with BEC were counted), 17.08 ± 1.22% were BrdU+. This number was normalized to 100% (Figures 3B,E). In contrast, in pseudomonolayers of SVZ cells that did not contact BEC, fewer cells were proliferating (P < 0.001, 4377 Hoechst-labeled SVZ cells not contacting BEC were counted, Figure 3E) indicating that the contact with BEC sustains SVZ cell proliferation. To impair normal SVZ-BEC contacts and evaluate the impact on proliferation of SVZ cells, BEC were cultured for 1 h with 1 μg/ml CHX prior to coculture (Figure 3A). The number of proliferating SVZ cells associated with CHX-treated BEC drastically decreased (P < 0.001, Figures 3C,E). As demonstrated above, CHX treatment leads to a decline of laminin protein expression to ~70% of control levels. However, since CHX does not specifically inhibit the synthesis of membrane-bound and ECM proteins as laminin, the observed decrease in proliferation could be due to a reduction in BEC-secreted soluble factors with a proliferative effect on SVZ cells. Nonetheless, in cocultures with CHX-treated BEC where BEC-derived soluble factors were restored by incubation in BEC conditioned media (Figure 3D), the pro-proliferative effects were not recovered (P < 0.001, Figure 3E). We have previously shown that the BEC diffusible factor angiopoietin 1 (Ang-1) promotes proliferation in SVZ cells (Rosa et al., 2010). Also, accumulation of diffusible molecules in the ECM may influence progenitor cell dynamics (Kerever et al., 2007). BEC-secreted Ang-1 may therefore accumulate in the ECM and stimulate proliferation. To test this, cocultures were performed in the presence of 5 μg/ml of an anti-Tie2 receptor neutralizing antibody. No difference in the numbers of proliferating SVZ cells contacting BEC was observed (Figure 3E) suggesting that proliferation was mediated by cell contact rather than by Ang-1.

Regarding survival, there were no differences in the numbers of apoptotic TUNEL+ cells between the population of SVZ cells contacting BEC (15.74 ± 1.72% apoptotic nuclei, 1023 cells counted) and the population of SVZ cells not contacting BEC (15.67 ± 2.51% apoptotic nuclei, 4330 cells counted). Moreover, BEC pre-treatment with CHX did not affect the number of apoptotic SVZ cells in contact with BEC (19.34 ± 2.48% apoptotic nuclei, 735 cells counted). These results indicate that SVZ-BEC interactions promote SVZ cell proliferation without interfering with cell survival.

To specifically target cell contacts through α6β1 integrin, cocultures were performed in the presence of an anti-α6 neutralizing antibody (5 μg/ml). We verified by TUNEL staining that the neutralizing antibody did not affect cell death in the population of SVZ cells contacting BEC (13.25 ± 1.86% apoptotic nuclei, 679 cells counted) nor in the pseudomonolayer (14.90 ± 1.52%, 2572 cells counted), as compared to cocultures performed in the absence of the antibody. Regarding proliferation, in the presence of the antibody, the number of BrdU+ cells contacting BEC decreased (P < 0.001, Figure 3F) compared with control cocultures. To further confirm this, SVZ neurospheres were obtained from neonatal mice containing floxed β1 alleles (Campos et al., 2004). When exposed to an adenovirus carrying Cre recombinase, SVZ neurospheres with the floxed β1 genetic background lost their capacity to express β1 integrin (β1^flox/flox treated with Cre referred to as “β1^−/−”) (Leone et al., 2005). Control neurospheres expressed β1 integrin (β1^flox/flox not treated with Cre, referred to as “No Cre”). No Cre and β1^−/− neurospheres were cocultured for 24 h with BEC. The numbers of No Cre BrdU+ cells contacting BEC were similar to that obtained with WT SVZ cells (20.25 ± 5.86%, 893 cells counted). This value was normalized to 100%. In line with the results using anti-α6 neutralizing reagent, the percentage of β1^−/− BrdU+ cells in contact with BEC was decreased (P < 0.001, Figure 3G). We verified that these effects were not due to a decrease in the proliferative capacities of β1^−/− cultures compared to No Cre (data not shown). These data indicate that α6β1 integrin-mediated signaling is responsible, at least in part, for the proliferation of SVZ cells.

To assess the involvement of direct contact between BEC and SVZ cells in stemness, cocultures were stained for Sox2, a stem/progenitor cell marker. Within the population of SVZ cells contacting BEC, 42.45 ± 2.19% were Sox2+ (3013 Hoechst-labeled SVZ cells in contact with BEC were counted). This number was normalized to 100% (Figures 4A,D). In contrast, the percent of Sox2+ cells in the SVZ population that were not in contact with BEC was significantly smaller (P < 0.001, 12652 Hoechst-labeled SVZ cells not contacting BEC were counted, Figure 4D), which indicates that contact with BEC plays a significant role in the maintenance of the SVZ stem cell state. Accordingly, the percentage of Sox2+ cells decreased within cells contacting CHX-treated BEC (P < 0.001, Figures 4B,D). Interestingly, incubation of cocultures with CHX-treated BEC with BEC-conditioned media partially restored the expression of Sox2 in SVZ cells contacting CHX-treated BEC (P < 0.05 as compared to cocultures with CHX-treated BEC, Figures 4C,D) demonstrating that BEC-derived soluble factors also contribute to stemness maintenance in SVZ cells. However, Ang-1 was not involved in this process as coculture with an anti-Tie2 receptor antibody did not modify the number of Sox2+ SVZ cells contacting BEC (96.38 ± 6.21%, Figure 4D). Cocultures were then performed in the presence of the anti-α6 neutralizing antibody. The number of Sox2+ SVZ cells contacting BEC decreased as compared to non-treated cocultures (P < 0.001, Figure 4E). Furthermore, the number of No Cre Sox2+ cells contacting BEC was 35.71 ± 6.21% (588 cells counted) and was similar to that obtained with WT SVZ cultures This number was normalized to 100%. The percentage of β1^−/− Sox2+ cells in contact with BEC was decreased (P < 0.05, Figure 4F). These effects were not due to a decrease in Sox2 expression in the β1^−/− cultures compared to No Cre cells as verified by Western blot (data not shown). Together, these data show that the close interaction between SVZ and BEC can modulate stemness in SVZ cells, and that this function is regulated through α6β1 integrin in SVZ cells.

Neuronal differentiation was evaluated based on staining for doublecortin (DCX) in untreated (Control) and CHX-treated BEC cocultures (Figures 5A–C). There were no differences in the number of DCX+ cell bodies or neurites contacting BEC in Control (1221 Hoechst-labeled SVZ cells contacting BEC were counted) and in CHX-treated BEC cocultures (1461 Hoechst-labeled SVZ cells contacting BEC were counted, Figure 5C). Similar results were obtained when analyzing SVZ progenitors labeled with Mash1, a neuronal transcription factor, further demonstrating that EC contacts did not affect neuronal commitment and differentiation (1938 Hoechst-labeled SVZ cells contacting BEC, 1563 Hoechst-labeled SVZ cells contacting CHX-treated BEC, and 4461 Hoechst-labeled SVZ cells that did not contact BEC, were counted, Figure 5D).

To further understand the role of the ECM protein laminin and α6β1 integrin in SVZ cells' stemness, SVZ cells were cultured for 72 h in SFM devoid of growth factors in culture dishes coated with poly-D-lysine alone or with either laminin-1 or other BEC-derived ECM molecules such as fibronectin or vitronectin. Levels of Sox2 were determined in cultures plated onto poly-D-lysine alone and set to 100%. Laminin-1, but not fibronectin nor vitronectin, induced an increase in Sox2 levels (poly-D-lysine vs. laminin-1: P < 0.05, Figure 6A). Levels of Sox2 protein in free-floating neurospheres cultures in the presence of EGF and FGF-2 were evaluated as positive controls as these conditions promote stemness in SVZ cells (Figure 6A). These results indicate that laminin-1 specifically sustains stemness in SVZ cells.

Studies have shown that the Notch signaling pathway regulates stem cell maintenance (Androutsellis-Theotokis et al., 2006; Aguirre et al., 2010). Therefore, levels of the Notch intracellular domain (NICD), the cleaved and activated form of Notch1 receptor, and of Hes1, a downstream effector of Notch, were quantified in SVZ cells cultured for 72 h on BEC-derived substrates as a measure of Notch activation. The results showed that laminin-1 triggers greater Notch activation as compared to the other ECM substrates tested (levels of NICD, poly-D-lysine vs. laminin-1: P < 0.05, Figure 6B; levels of Hes1: poly-D-lysine vs. laminin-1: P < 0.01, Figure 6C).

The capacity of self-renewal is a central feature of the stem cell state and is defined by the possibility of stem cells to divide and generate two daughter cells that are identical to the “mother” stem cell or a stem cell and a progenitor cell. However, there is loss of self-renewal capacity when a stem cell terminally divides into two progenitor cells. The capacity of laminin-1, compared to other ECM molecules, to favor self-renewing divisions was determined on SVZ single cells plated for 24 h and stained for Sox2. Cell pairs either Sox2+/Sox2+, Sox2+/Sox2− or Sox2−/Sox2− were identified (Figure 7A). Numbers of cell pairs were determined in each condition and compared to numbers obtained on poly-D-lysine set to 100% (corresponding to a total of 400 cell pairs counted). Only cells grown on laminin-1 increased self-renewing divisions (Sox2+/Sox2+) as compared to poly-D-lysine (poly-D-lysine vs. laminin-1: P < 0.001). Consistent with the idea that laminin-1 predominantly induces self-renewal, laminin-1 significantly decreased the number of differentiating divisions (Sox2−/Sox2−) (poly-D-lysine vs. laminin-1: P < 0.001; Figure 7B).

It has been shown that Notch receptor activation leads to the expression of Hes genes through cytoplasmatic intermediate mediators including the serine-threonine kinase mTOR (Androutsellis-Theotokis et al., 2006). To disclose whether mTOR mediated laminin-induced self-renewing divisions on SVZ precursors, cell pair assays were performed in the presence of 20 nM of the mTOR inhibitor rapamycin. While laminin-1 increased the numbers of Sox2+/Sox2+ pairs (poly-D-lysine vs. laminin-1: P < 0.001) at the expense of Sox2−/Sox2− (P < 0.001), these effects were inhibited in the presence of rapamycin (P < 0.001; Figure 7C) (Numbers of cell pairs counted were compared to numbers obtained on poly-D-lysine set to 100%, corresponding to a total number of 763 cell pairs counted). Taken together, these results suggest that laminin sustains stemness via activation of the Notch and mTOR signaling pathways.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.