Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures

Abstract

Introduction: In vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood.

Methods: Here, using in vitro assay with engineered MDCK cells expressing H2B-mCherry (nucleus) and gp135/Podocalyxin-GFP (apical marker), we show in multi-dimensions that such coordination for epithelial morphogenesis can be determined by cell-soluble ECM interaction in the fluidic phase.

Results: The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time-lapse two-photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135-GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi-lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment.

Conclusion: Our results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell-soluble ECM interaction and self-assembly.

Keywords: cell-ECM interaction; epithelial polarity; epithelial self-assembly; epithelial topology; fluidic phase; tubulogenesis.

FIGURE 1

Soluble ECM components are required to form epithelial polarization on Matrigel. (A) Experimental set‐up. MDCK cells were cultured on the top of basement membrane (BM) gels with or without soluble ECM components in the medium. Cells express H2B‐mCherry and gp135‐GFP to indicate their nucleus and polarity, respectively. Note the distribution of nucleus with respect to gp135 in polarized lumens (on the right). (B) Left in each row: Represented phase‐contrast (bright field) and 3‐D projected fluorescent images (gp135: green, H2B: red, and their overlay) for cells seeded on BM gels and cultured for 12 days (seen in Methods for 3‐D projected imaging and processing). The medium contained (top row) no soluble ECM, (middle row) 2% BM or (bottom row) 20 µg/mL type I collagen (COL). Nuc: cell nucleus. Right in each row: Fluorescence intensities of H2B‐mCherry and gp135‐GFP along the indicated white dotted lines (from left to right). The overlay of the red and green curves indicates the positions of cell nucleus (red) with respect to the apical marker (green). a. u.: arbitrary unit. (C and D) 3‐D view of the polarized lobular and tubular structures. The images from confocal scanning microscopy (with 20x objective) were reconstructed into 3‐D structures. The 3‐D views showed the relative position of cell nucleus (red) and apical marker gp135 (green) in the closed lobular (C) and elongated tubular (D) structures

FIGURE 2

Distinct dynamics of cell coordination in response to soluble BM/COL. MDCK cells express H2B‐mCherry and GFP‐gp135 or β‐actin‐GFP to indicate cell nucleus and apicobasal polarity, respectively. Cells were cultured on BM gel with or without soluble ECM in the medium and positioned on the microscope by change with fresh medium every day. Time‐lapse fluorescence images were taken with 22 min interval time per cycle in the following days. (A and B) Represented time series of 3‐D projected epifluorescence images (gp135: green, H2B: red) for cells in medium (A) without ECM (n > 10), (B) with 2% basement membrane (BM) components (n > 10) (also seen in Movies 1&2, interval = 22 min). (C) Represented optical sectioning from scanning microscopy β‐actin: green, H2B: red) to demonstrate lumen formation with polarized actin distribution after culturing cells in medium containing 2% BM for 14 days. (D) Represented time series of 3‐D projected epifluorescence images for cells in medium with 20 µg/mL COL (also seen in Movie S3) (n = 14). The purple arrows indicate the polarization initiating from the local regions of the tubule. (E) The collective apicobasal polarization along the tubular axis (n = 6). The time‐sequence images (interval: 22 min*5 = 110 min) showed the spatial distribution of gp135 relative to cell nuclei (H2B) during the polarization. (F) Size quantification of the diameters (along the long axis) of unpolarized clusters without ECM (n = 21) and polarized lobular lumens with BM (n = 50), and the length of polarized tubes with COL (n = 14) around Day 5. (G) The timing quantification when lumens (n = 38) or tubules (n = 14) got polarized under culture with 2% BM or 20 µg/mL COL in the medium. N/A refers to ‘not applicable for polarization’ without ECM in the medium. The data quantification (mean ± SD) was performed by using ImageJ and Origin. *** and **** represent significant difference with P < 10^‐2 and 10^‐6 in comparison with the group ‘with COL’ from Student's t test analysis

FIGURE 3

Cell polarization in the fluidic phase with and without direct cell‐ECM contact. (A, B) Lateral assembly of laminin around the luminal structure. MDCK cells were cultured for 1‐7 days on BM gels with 2% BM in the medium, followed by laminin immuno‐staining. (A) Represented fluorescent images of gp135‐GFP (green), H2B‐mCherry (red), laminin staining (cyan) and their overlay. (B) Fluorescence quantification of laminin staining on the cultured MDCK clusters (A). The graph data with individual dots (mean ± SD) show the average fluorescence intensity over the entire clusters with typical staining signals. a.u.: arbitrary unit. (C) Represented images of 3‐D reconstructed, top and bottom views of cell nucleus (red) and laminin (cyan) on the spherical lobules after 12‐day culture (also seen in Movie S4). (D) Represented fluorescent images of gp135‐GFP (green), H2B‐mCherry (red), COL staining (cyan) and their overlay of the tubules (n = 15). MDCK cells were cultured for 12 days on BM gels with COL (20 µg/mL) in the medium, followed by COL immuno‐staining. Note the formation of linear structure of COL (pink arrow). (E) Represented images of 3‐D reconstructed, top and bottom views of cell nucleus (red) and COL (cyan) on the tubule after 12‐day culture. For (C & E), images were taken by confocal scanning microscope and created by ImageJ 3‐D reconstructions. Note the lateral condensation of laminin (COL) around the lobules (tubule) (outlined by nucleus) and the absence of laminin (COL) assembly on the top and bottom

FIGURE 4

The expansion of dividing cells into polarized lobule. The MCDK cells expressing gp135‐GFP and H2B‐mCherry were seeded on BM gel with 2% BM in medium, and two‐photon confocal imaging started one day later. The wavelengths of excitation light were 890 nm for GFP and ~ 1100 nm for mCherry; the step size in z‐direction was 1.0 µm. Time‐lapse imaging was taken with the interval time of 3 or 4 h. Medium was changed every day along with focus/position corrections. Acquired confocal images were processed by two channels (GFP and mCherry) overlay and 3‐D reconstructions. Each image here shows the 3‐D view of 4 or 5 time‐points at the labelled time (in hours, and the starting time was set as zero), and two images for each time point were displayed from different angles (generally from the top and the bottom views). (A) The polarity distribution at early stage in the cluster with a few cells. (B) The polarity distribution during growth of the cluster into fine 3‐D lobular structure. More detailed 3‐D views are shown in Movie S5. A note: the images of (A, B) were acquired at the same position in one experiment, but may not be from the exact same sample during the re‐focusing process as cells were motile at the early stage

FIGURE 5

Cells form tubular and lobular structures above non‐adhesive substrates. (A) The design of suspension culture on agarose gel. Details are described in Methods. (B) Representative bright‐field and 3‐D projected epifluorescence images (Green: gp135, Red: H2B) for MDCK cells cultured in medium containing no ECM materials (Left), 2% BM (Middle), or 20 μg/mL COL components (Right) above 1% agarose gel for 12 days. Initial cell density: ~10⁴ cells/mL. To enhance visualization, out‐of‐focus signals were removed and only fluorescence signals from cells near the focal plane (indicated by blue arrows) were shown. The red arrows indicated the scaffolds self‐assembled from soluble BM or COL. (C) Fluorescence intensities of H2B‐mCherry and gp135‐GFP along the white dotted lines (from left to right) in the indicated H2B/gp135 overlay. The cell nucleus (red) located outside the apical marker (green) indicates polarity formation. (D) The 3‐D projected images of spherical lobules from suspension culture with 2% BM (~12 days). The laminin staining (blue) indicated the assembled scaffolds from soluble BM under suspension. The images were taken by confocal microscopy (20x objective). 3‐D projection of the images was obtained by collecting pixels with the maximal intensity through the entire z‐stack into a single plane, and the 3‐D view was constructed by the software ImageJ. (E) Formation of multi‐lumen polarity on the tubules under suspension culture. MDCK cells were cultured above agarose gel with 20 µg/mL COL in the medium for 12 days. The arrows indicate some of these small lumens on the tubules