Cell position within human pluripotent stem cell colonies determines apical specialization via an actin cytoskeleton-based mechanism

Abstract

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.

Keywords: actin dynamics; apical specialization; cell polarity; human pluripotent stem cells; stem cell colony; topology; transcriptome analysis.

Graphical abstract

Figure 1

Apical characteristics of edge and center cells in a micropatterned hPSC colony (A) A procedure for micro-contact printing. A polydimethylsiloxane stamp with micropatterns was covered with Matrigel and coated overnight at 4°C. Then, the stamp was placed on the coverslip for 10 min. Immediately thereafter, stamps were removed and cells were seeded on coverslips. Scale bar, 2,000 μm. (B) Localization of apical specialization proteins in hPSC colony. Confocal images were taken in 1-μm steps along the z axis. “Apical” images were generated by stacking images from the top of the edge of the cell nucleus to the top of apical proteins of center cells (approximately 13 μm) and “Basal” images from the very bottom to the end of the apical (approximately 16 μm) using z stack maximum projection. Depth coding indicates the apical side in red and the bottom side in blue. Images of side view (XZ) are displayed. Scale bar, 20 μm. (C) Ultrastructure of the surface of micropatterned H9 cell colony was examined by SEM. Scale bars, 100, 50, and 5 μm (from left). The protrusions on the surface of center cells are highlighted with arrows.

Figure 2

Live labeling of hPSCs cultured on micropattern with fluorescence dyes (A) hPSCs were seeded onto the micropattern and live cells were labeled with Hoechst 33342. After bright-field and fluorescent images were taken in live cells, cells were fixed and labeled with Syto16. Hoechst 33342 images from live cells and Syto16 images from fixed cells were merged. Arrowheads indicate mitotic cells. Scale bar, 200 μm. (B) Dye-labeling intensity at each region in the colony was divided by the highest intensity in the same colony for normalization, and the value was converted to the percent peak. Data represent the mean ± SEM (n = 10 colonies, three independent experiments). (C) Fluorescence live imaging of PSCs expressing EGFP-ZO1 upon Hoechst labeling. Edge area varies depending on the region. Triangles indicate edge area is within one cell layer (white) or two cell layers (red) of micropattern boundary. Scale bars, 200 μm (white) and 50 μm (yellow). (D) Various dye labeling in an hPSC colony. Live cells were simultaneously stained with Hoechst 33342 and DiI, and images were merged. Live cells were labeled with Syto16, Mitotracker, or TMRM. Scale bar, 200 μm. (E) Quantification of center and edge cell dye intensity in (D). Dye intensity of edge and center cells were measured and percent peak intensity was calculated. The length of the edge was defined as 12.3 ± 0.73 μm (mean ± SEM). Each dot represents percent peak intensity of an individual colony randomly collected in three independent experiments (n = 26–30). ^∗∗∗∗p < 0.0001 from t test. (F) The effect of colony size on dye labeling in an hPSC colony. The size of the hPSC colony was regulated by micropattern diameter. Scale bar, 50 μm. (G) Quantification of dye-labeling intensity in (F) (n = 9 colonies, three independent experiments). Data represent the mean ± SEM. AU, arbitrary units. (H) Dye diffusion kinetics at the edge and center of the colony. Fluorescent images of the Hoechst 33342-labeled colony were taken at 5, 10, 15, 30, and 60 min after dye addition. Temporal order of images is presented in a clockwise direction. Scale bar, 200 μm. (I) Quantification of dye intensity and ratio of edge to center in (H). Left: y axis with line graphs indicates the intensity (AU, arbitrary units). Each dot represents average intensity of edge or center cells. Right: y axis with the bar graphs indicates intensity ratio of edge to center (n = 10 colonies, three independent experiments). Data represent mean ± SEM.

Figure 3

Separation of edge and center cells by FACS and RNA-seq analysis (A) To isolate edge and center cells for mRNA sequencing, micropatterned hPSC colonies were labeled with TMRM for 15 min at 37°C and dissociated as single cells. Low- and high-intensity populations were separated by FACS analysis (①). To clarify the high-intensity peak, dissociated single cells were also labeled with TMRM for 15 min at 37°C and analyzed (②). The high-intensity populations from micropatterned colonies (marked with red arrowhead) was overlapped with the single-cell TMRM-labeled population (marked with the black arrowhead). The quantification graph shows each cell population as a percentage (n = 5). Data represent the mean ± SEM. (B) Volcano plot shows fold change versus p value. Significant DEGs were selected when the fold change (FC) value gap was higher than 1.2 and p < 0.01. Genes higher expressed in the edge are colored red, whereas lower expressed genes are colored green. (C) Heatmap of 48 genes that are differentially expressed between the edge and center cell groups (edge sample, n = 4; center sample, n = 5). Six genes were highly expressed in the center (green annotation bar) and 42 genes at the edge (red annotation bar). The grayscale annotation bar represents statistical significance between edge and center. (D) KEGG enrichment analyses are depicted as a dot plot. DEGs were annotated and the top 30 pathways were selected. (E) Verification of RNA-seq data. Edge and center cells cultured on micropatterned polyethylenenaphthalate membrane were cut using a laser under LCM. The white line indicates a cutting margin (top). Scale bar, 150 μm. Ten to 15 colonies were cut and collected from each experiment, and four independent experiments were performed. qPCR data from collected edge and center cells (bottom). FC (center/edge) of indicated genes was calculated, and the ΔCT values of center and edge cells for each gene were used for statistical analysis. Data represent mean ± SEM. Each dot represents fold change of an individual batch of experiment (n = 3 or 4). ^∗p < 0.05 from t test. (F) Validation of RNA-seq data by immunostaining of ANNEXIN A1 and SERPIN E1. Both proteins were highly expressed in edge cells and polarized in distribution. Data represent mean ± SEM (right side). Each dot represents percent peak intensity of an individual colony randomly collected in three independent experiments (n = 26–30). ^∗∗∗∗p < 0.0001 from t test. Scale bars, 20 μm (yellow) and 5 μm (magenta).

Figure 4

Differential kinematics and mechanical dynamics in an hPSC colony (A) Cell trajectory with a path length indicated by a purple gradient (left) and the mean path lengths (black circles) and mid-quartile (gray) obtained along the radial lines from the center to the edge of the cell colony for 24 h (right). (B) A color-coded map of cell speed with migration direction (black arrows) (left) and the mean speed (black circles) and mid-quartile (green) obtained along the radial lines from the center to the edge of the cell colony (right) at 60 min. (C) A color-coded map of radial coordinated cellular traction force with traction direction (white arrows) (left) and the mean traction (black circles) and mid-quartile (blue) obtained along the radial lines from the center to the edge of the cell colony (right) at 60 min. (D) A color-coded map of tension (left) and the mean tension (black circles) and mid-quartile (red) obtained along the radial lines from the center to the edge of the cell colony (right) at 60 min. All the data for the analysis of distribution of cellular migration was obtained from the number of data points with a size of 64 × 64 points (n = 732) acquired from center to edge.

Figure 5

Effect of actin disruption on dye diffusion and apical polarization and characterization of differentiated micropatterned colonies (A) Micropatterned hPSC colonies were treated with ROCK inhibitor Y-27362 for 24 h. Dye diffusion and apical specialization markers were examined. The top row indicates control groups and the bottom row indicates the ROCK inhibitor treatment group. Y-27632 treatment perturbed polarized distribution of apical markers in the center cells. Scale bar, 50 μm. (B) The polarized distribution of ANNEXIN A1 and SERPIN E1 in the center cells was disrupted with Y-27632 treatment. Quantification of fluorescence intensity along the z axis (height) in the center and edge cells. In three independent experiments, 26–30 colonies were randomly collected and quantified. Fluorescence intensity at each point was converted into percent total intensity and data represent mean ± SEM. Scale bars, 50 μm (yellow) and 5 μm (magenta). (C) hPSCs grown on a 500-μm micropattern were treated with Wnt 3A (100 ng/mL) and Activin A (100 ng/mL) for 24 h. Live control and differentiated colonies were labeled with Hoechst and fixed colonies were immunostained with anit-SOX17 and anti-ZO1 antibodies. High-magnification images of boxed areas are shown in the right column. Scale bars, 200 μm (white) and 50 μm (yellow). (D) Two cell populations upon differentiation were isolated by FACS based on Hoechst labeling intensity (①). To clarify the high-intensity population, dissociated single cells were labeled and analyzed (②). Definitive endoderm marker SOX17 and MIXL1 were examined with qPCR analysis using sorted cells. Fold changes (high/low) of indicated genes were calculated. Data represent mean ± SEM in three independent experiments. Each dot represents fold change obtained from an individual batch of experiment (n = 3).

Figure 6

Diagram of position-dependent differences in hPSC colonies and relevant contribution to biological functions Differential mechanical force and actin dynamics in edge and center cells induce differential gene expression profiles in hPSC colonies, leading to position-dependent differences in biological functions including endocytosis, dye diffusion, migration, and actin fence formation.