Figure 1: Overview of the morphological varieties and classified colonies of 4 hiPSC clones.

(A) Clustering of colony morphologies in the colony database according to morphological parameters. Horizontal branches show the hierarchical clustering results divided into 20 clusters at a threshold of 0.39451. Major clusters (A–E) are indicated with coloured branches. Red branches indicate colonies categorised into cluster-A, the irregular colony morphology cluster. Vertical branches show the correlation of measured morphological parameters categorized into 3 types (frequency, shape and volume). Based on the heat map colour gradients (blue: low; yellow: high), cluster-A can be described as the combination of a relatively mature morphology (high in volume parameters), with a boundary that is not round (low in shape parameters), and a very irregular morphology (low in frequency). Morphological interpretations of each cluster are described in Supplementary Table S5. (B) Representative images of the colonies in cluster-A/cluster-B. Left upper image, a and e, cluster-A colonies exhibit disrupted peripheral colony edges. The tightly packed colonies were partially disrupted by fibroblastic cellular morphology. The sky-blue overlay mask indicates the colony area recognised by our image analysis. Right upper image, c and g, colonies classified as cluster-B are typical ES-like growing colonies. Fixed colonies were immunohistochemically stained with an anti-OCT-3/4 b and d antibody and an anti-VIMENTIN antibody (f and h). Given that 8 × 8 tiling images were merged to form an image in our analysis, partially bright and biased fluorescent areas are occasionally observed in the images, derived from the edges of individual images. Scale bar = 500 μm.