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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.

No MeSH data available.


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 Initiative11. (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.
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f2: 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 Initiative11. (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.

Mentions: First, the expression of the representative undifferentiated stem cell markers NANOG, OCT-3/4, SOX2, C-MYC, TRA-1-60 and SSEA4 and the early differentiated cell markers SSEA1 and VIMENTIN was determined from global gene profiles and compared between clusters (Fig. 2A). Among both the 201B7 and 201B7-1A cluster-A colonies, there were large variations in the gene expression levels of NANOG, SOX2 and VIMENTIN. Greater variation was observed for 201B7-1A clusters compared with 201B7 clusters. Large variations in OCT-3/4, C-MYC and TRA-1-60 expression were observed in cluster-A 201B7-1A colonies. Conversely, the gene expression levels of NANOG, OCT-3/4, SOX2 and TRA-1-60 were comparable between cluster-I, cluster-J, cluster-D and cluster-B for both 201B7 and 201B7-1A. These results reveal greater variations in the gene expression levels of a proportion of undifferentiated or differentiated markers among cluster-A colonies, indicating that cells in cluster-A colonies are unstable and in a dysregulated undifferentiated state.


Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control
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 Initiative11. (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.
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f2: 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 Initiative11. (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.
Mentions: First, the expression of the representative undifferentiated stem cell markers NANOG, OCT-3/4, SOX2, C-MYC, TRA-1-60 and SSEA4 and the early differentiated cell markers SSEA1 and VIMENTIN was determined from global gene profiles and compared between clusters (Fig. 2A). Among both the 201B7 and 201B7-1A cluster-A colonies, there were large variations in the gene expression levels of NANOG, SOX2 and VIMENTIN. Greater variation was observed for 201B7-1A clusters compared with 201B7 clusters. Large variations in OCT-3/4, C-MYC and TRA-1-60 expression were observed in cluster-A 201B7-1A colonies. Conversely, the gene expression levels of NANOG, OCT-3/4, SOX2 and TRA-1-60 were comparable between cluster-I, cluster-J, cluster-D and cluster-B for both 201B7 and 201B7-1A. These results reveal greater variations in the gene expression levels of a proportion of undifferentiated or differentiated markers among cluster-A colonies, indicating that cells in cluster-A colonies are unstable and in a dysregulated undifferentiated state.

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.