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

View Article: PubMed Central - PubMed

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.

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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.
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f1: 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.

Mentions: Second, colony morphology in the database was analysed by performing hierarchical clustering using the open source clustering software Cluster 3.0 and sorting colonies into morphological categories depending on the patterns exhibited by multiple parameters (Supplemetary Fig. S2, Step 1–3). The following clusters comprising >5% of total colonies in the database were defined as ‘major clusters’: cluster-A, cluster-B, cluster-C, cluster-D and cluster-E (Fig. 1A and Supplementary Table S5). This analysis eliminated arbitrary interference by selecting specific thresholds, parameters or categories and automatically determining ‘commonly observed morphology’. The proportion of 201B7-1A colonies categorised into cluster-A (27.3%) was higher than that of 201B7 colonies (13.3%), whereas the proportions of 201B7-1A colonies categorised into the other major categories were lower or approximately equal to those of 201B7 colonies. The proportion of 253G1-B1 colonies in cluster-A (40.5%) was higher than that of 253G1 colonies (4.8%). The actual morphologies of the representative clusters were manually verified (Fig. 1B, Supplementary Fig. S4 and Supplementary Table S6). Cluster-A colonies were characterised by the loss of clear colony edges, a comparatively flatter cytoplasm and a low nucleus-to-cytoplasm ratio. Although the colonies in cluster-B and cluster-D were comparatively smaller or partially surrounded by flatter cells, they exhibited a central area with hESC-like morphology. The morphologies of colonies in cluster-C and cluster-E resembled typical hESC-like morphologies of growing colonies. Thus, the morphology of 201B7-1A colonies in cluster-A may constitute a major signature morphology that is distinguishable from 201B7 colony morphology.


Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5036041&req=5

f1: 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.
Mentions: Second, colony morphology in the database was analysed by performing hierarchical clustering using the open source clustering software Cluster 3.0 and sorting colonies into morphological categories depending on the patterns exhibited by multiple parameters (Supplemetary Fig. S2, Step 1–3). The following clusters comprising >5% of total colonies in the database were defined as ‘major clusters’: cluster-A, cluster-B, cluster-C, cluster-D and cluster-E (Fig. 1A and Supplementary Table S5). This analysis eliminated arbitrary interference by selecting specific thresholds, parameters or categories and automatically determining ‘commonly observed morphology’. The proportion of 201B7-1A colonies categorised into cluster-A (27.3%) was higher than that of 201B7 colonies (13.3%), whereas the proportions of 201B7-1A colonies categorised into the other major categories were lower or approximately equal to those of 201B7 colonies. The proportion of 253G1-B1 colonies in cluster-A (40.5%) was higher than that of 253G1 colonies (4.8%). The actual morphologies of the representative clusters were manually verified (Fig. 1B, Supplementary Fig. S4 and Supplementary Table S6). Cluster-A colonies were characterised by the loss of clear colony edges, a comparatively flatter cytoplasm and a low nucleus-to-cytoplasm ratio. Although the colonies in cluster-B and cluster-D were comparatively smaller or partially surrounded by flatter cells, they exhibited a central area with hESC-like morphology. The morphologies of colonies in cluster-C and cluster-E resembled typical hESC-like morphologies of growing colonies. Thus, the morphology of 201B7-1A colonies in cluster-A may constitute a major signature morphology that is distinguishable from 201B7 colony morphology.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus