Sparse plating increases the heterogeneity of proliferative potential of fibroblasts

High heterogeneity of proliferative potential in the cultures of diploid human fibroblasts was reported in many studies. It was generally believed that the heterogeneity of proliferative potential of human fibroblasts reflects the unevenness of their senescence. However we show here that immortalized (telomerized) human fibroblasts obey the same rule. Up to 50% of these cells rapidly ceased to proliferate when plated at low density in contrast to usual conditions of mass culture where at least 98% of these cells keep on proliferating. Initially, we proposed that the appearance of non-dividing or slow-dividing cells in low-density cell culture experiments could be caused by cell damage due to the experimental setup. Indeed, lowering of oxygen level and addition of conditioned medium improved colony formation, but there were a large number of non-proliferating cells (13–20%). When we sparsely plated cells on a feeder layer of cells of certain density, the portion of non-proliferating cells decreased to 2%, i.e. became the same as in mass culture. Thus, the heterogeneity of proliferative potential is partially a result of the adverse effect of low cell density.