Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control

Abstract

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of 'hPSC colony morphology', permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

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.

Figure 2. Gene expression profiles of single hiPSC colonies classified as cluster-A, cluster-B, cluster-D, cluster-I and cluster-J 201B7 and 201B7-1A hPSC colonies (32 colonies), classified as cluster-A, cluster-B, cluster-D, cluster-I and cluster-J, were individually picked up from the culture vessel.

RNA extracted from these colonies was used to perform global gene microarray analysis. Gene expression profiles are normalised values, as described in the Methods section. (A) Comparisons between 201B7 and the aberrant subclone 201B7-1A classified as cluster-A, cluster-B, cluster-D, cluster-I and cluster-J employing representative stem cell markers. (B) Hierarchical clustering of the colonies based on 149 probes of stem cell-related markers proposed by the International Stem Cell Initiative. (C) Hierarchical clustering of the colonies based on 1,454 probes expressed at significantly higher levels in colonies classified in cluster-A vs. those in cluster-B, cluster-D, cluster-I and cluster-J. (D) PCA of colonies based on 29,445 global probes. Blue-filled diamonds: cluster-A colonies in 201B7; red-filled diamonds: cluster-A colonies in 201B7-1A; blue open circles: 201B7 colonies in other clusters; and red open circles: 201B7-1A colonies in other clusters. The numbers on the filled diamonds indicate colony ID numbers in 201B7. The red dotted area indicates biological similarities between colonies, reflecting the expression profile of 29,445 global probes.