3.4. Transcriptome Heterogeneity Analyses of Adherent Cultures
In our single-cell transcriptome analysis of suspension cultures, no
subpopulations were apparent. This finding differs from that of previous
studies, which indicated a strong correlation between genome-wide gene
expression and the cell cycle stage of adherent cells (Buettner et al.,
2015; Santos, Wernersson, & Jensen, 2015; Sasagawa et al., 2013;
Scialdone et al., 2015). Cellular heterogeneity is known to be
influenced by the cellular microenvironment in adherent, two-dimensional
cultures (Snijder et al., 2009). It is possible that cell
cycle–dependent genome-wide gene expression is an adherent
culture-specific phenomenon. In this context, the origin of cell
cycle–dependent cellular heterogeneity in adherent cultures would not
be the product of intrinsic factors but rather the product of the
extrinsic milieu, with cellular division altering genome-wide gene
expression in cells by changing the microenvironment in adherent
cultures. A previous study supports this interpretation; suspended T
cells did not show any cell cycle–dependent cellular transcriptome
heterogeneity (Mahata et al., 2014).
To further investigate cell cycle–dependent cellular transcriptome
heterogeneity, we performed single-cell SMART-Seq as previously reported
(Ziegenhain et al., 2017) on 24 G1 cells and 8 G2 cells from adherent
cultures. To compare the transcriptomes between cells in different
phases of the cell cycle, we performed PCA of all the expression data
(Figure 4). The proportion of variance of PC1 was 0.86, while that of
PC2 was 0.033. The G2 cells formed a distinct cluster. We, therefore,
rejected the null hypothesis that cell cycle–dependent subpopulations
do not appear during adherent culture, based on the Bartlett test for
the homogeneity of variances in PC1 between G1 and G2 cells (p =
0.000245). Thus, cell cycle–dependent heterogeneity in genome-wide gene
expression was found to be a phenomenon specific to adherent cultures of
CHO cells.