Fig 1.
Background schematic and CE workflow overview.
A) Studying cells in isolation disregards the effects of community interactions, which are known to direct cell fate decisions (left). Powerful micropatterning in vitro assays increase control over the cellular microenvironment and facilitate the study of context dependent cell fate acquisitions (middle). Our analysis software enhances these assays by allowing researchers to analyze cell behavior within its population context instead of as independent isolated events (right). B) CE fits into existing image analysis pipelines after initial measurements have been extracted from the images. C) Overview of the CE workflow, each step is described in detail in the methods section.
Fig 2.
Classification of cells into colonies.
A) mPSCs restricted to grow on micropatterned ECM spots (left) can be classified into colonies via the DBSCAN algorithm (middle). Merged and partial colonies can be excluded by applying filters to the DBSCAN clustering results (right). Each data point represents a cell and colonies are encircled with green lines. Scale bar = 500 μm. B) Number of cells per colony in wells from a 96-well plate after running DBSCAN. C) Number of cells per colony in wells after excluding merged and partial colonies from the DBSCAN results. D) Number of colonies per well after running DBSCAN with and without filters, and from a visual inspection of the images. E) Colony identification for colonies of different sizes in the same well (left), colonies of non-circular geometries (middle), and colonies in wells without pre-patterned ECM spots (right). Scale bar = 500 μm.
Fig 3.
Quantification of radial expression trends.
A) Comparison of SOX2 expression between cells growing inside and outside of colonies in microcontact-printed wells. B) Cells from multiple colonies at similar locations within their respective colony are aggregated together in bins according to a hexagonal grid system. The heatmap is colored by the desired measure of variation or central tendency. C) Multiple colonies of hPSCs are aggregated to reveal general tendencies in the spatial protein expression pattern of SOX2 (left). Each bin shows the mean expression of cells from multiple colonies. Trends are visualized as line plots, where joint data points represent the mean intensity of cells at each distance bin throughout the colony (right). Error bars represent 95% confidence intervals between cells from the same location in different colonies. D) Hexagonally binned cells in triangular hPSC colonies (left). Cells grouped in concentric bins according to their distance from the colony centroid (middle) or to the closest colony edge (right). The color is used for illustration purposes to distinguish bins from each other. E) Visualization of the Sox2 expression levels in the hexagonal bins (left) and the concentric bins (middle). The number of cells per bin for the different concentric binning strategies (right).