Cancer stem cells (CSCs) has been a key target to cure cancer patients completely. Although many CSC markers have been identified, they are frequently cancer type-specific and those expressions are occasionally variable, which becomes an obstacle to elucidate the characteristics of the CSCs. Here we scrutinized the relationship between stemness elevation and geometrical features of single cells. The PAMPS hydrogel was utilized to create the CSCs from mouse myoblast C2C12 and its synovial sarcoma model cells. qRT-PCR analysis confirmed the significant increase in expression levels of Sox2, Nanog, and Oct3/4 on the PAMPS gel, which was higher in the synovial sarcoma model cells. Of note, the morphological heterogeneity was appeared on the PAMPS gel, mainly including flat spreading, elongated spindle, and small round cells, and the Sox2 expression was highest in the small round cells. To examine the role of morphological differences in the elevation of stemness, over 6,400 cells were segmented along with the Sox2 intensity, and 12 geometrical features were extracted at single cell level. A nonlinear mapping of the geometrical features by using uniform manifold approximation and projection (UMAP) clearly revealed the existence of relationship between morphological differences and the stemness elevation, especially for C2C12 and its synovial sarcoma model on the PAMPS gel in which the small round cells possess relatively high Sox2 expression on the PAMPS gel, which supports the strong relationship between morphological changes and the stemness elevation. Taken together, these geometrical features can be useful for morphological profiling of CSCs to classify and distinguish them for understanding of their role in disease progression and drug discovery. •Essential elements for morphological changes were investigated during reprogramming from non-cancer stem cells (CSCs) to CSCs.•Stemness markers increased in synovial sarcoma model cells on hydrogel, and the morphological heterogeneity was appeared.•Over 6,400 cells were segmented along with the Sox2 intensity, and 12 geometrical features were extracted at single cell level.•UMAP clearly revealed the relationship between geometrical features such as small round shape and the stemness elevation.