Effect of cellular mass on chondrogenic differentiation during embryoid body formation

One approach to cell differentiation is to use the natural capacity of pluripotent stem cells to form three germ layers via embryoid bodies (EB). However, unification of this process during in vitro culture remains challenging and many microenvironmental factors including the number of cells in the culture can influence differentiation patterns. The number of cells serves a crucial role as it determines access to nutrients, the distribution of oxygen concentration and cellular interactions, all of which influence the fate of the differentiated cells. The influence of EBs derived from human pluripotent cells on the chondrogenic potential of such cells is not well understood. For this reason, the present study sought to determine the effect of varying amounts of cells on the properties of EBs derived from human embryonic stem cells (BG01V cell line). In the present study, 500‑2,000 cells per well were cultivated from 5 to 15 days in suspension cell culture. Expression of pluripotency genes and germ layer markers were evaluated in order to determine the EBs with the greatest and least mesodermal properties. Genes associated with pluripotency and chondrogenesis were also evaluated to assess the influence of suspension culture duration and EB size on chondrogenic differentiation. Immunofluorescence staining for pluripotent and chondrocyte‑associated proteins confirmed successful differentiation into chondrocyte‑like cells. Alcian blue staining confirmed deposition of proteoglycans. These results suggested that EBs formed in 500‑cell wells possess the highest mesodermal and prochondrogenic properties. Differentiation of EBs into chondrocytes on day 5 in 500‑cell wells was more efficient than in that observed in larger and older EBs.