  The maintenance of the pluripotent state in human embryonic stem cells
(hESCs) is critical for further application in regenerative medicine, drug
testing and studies of fundamental biology. Currently, the selection of the
best quality cells and colonies for propagation is typically performed by eye,
in terms of the displayed morphological features, such as prominent/abundant
nucleoli and a colony with a tightly packed appearance and a well-defined edge.
Using image analysis and computational tools, we precisely quantify these
properties using phase-contrast images of hESC colonies of different sizes (0.1
-- 1.1$\, \text{mm}^2$) during days 2, 3 and 4 after plating. Our analyses
reveal noticeable differences in their structure influenced directly by the
colony area $A$. Large colonies ($A > 0.6 \, \text{mm}^2$) have cells with
smaller nuclei and a short intercellular distance when compared with small
colonies ($A < 0.2 \, \text{mm}^2$). The gaps between the cells, which are
present in small and medium sized colonies with $A \le 0.6 \, \text{mm}^2$,
disappear in large colonies ($A > 0.6 \, \text{mm}^2$) due to the proliferation
of the cells in the bulk. This increases the colony density and the number of
nearest neighbours.
  We also detect the self-organisation of cells in the colonies where newly
divided (smallest) cells cluster together in patches, separated from larger
cells at the final stages of the cell cycle. This might influence directly
cell-to-cell interactions and the community effects within the colonies since
the segregation induced by size differences allows the interchange of
neighbours as the cells proliferate and the colony grows. Our findings are
relevant to efforts to determine the quality of hESC colonies and establish
colony characteristics database.
