Persistent Existence of ES-like Cell Population in Long-term Cultured Embryoid Bodies

Undifferentiated embryonic stem (ES) cells can be maintained in vitro if cultured in the presence of the cytokine leukemia inhibitory factor (LIF). Embryonic stem cells can also differentiate in vitro. A particularly efficient method for inducing ES cell differentiation is to culture ES cells as aggregates in the absence of LIF. Under these conditions, they form structures known as embryoid bodies (EBs). However, the current protocols for EB formation are still diverse. In order to facilitate further study, we carefully controlled the culture conditions for EB formation, and here we report an efficient protocol by which uniformly differentiated EBs were obtained and were monitored by measuring the differentiation of beating cardiomyocytes. Furthermore, by using this protocol, we observed in long-term cultured plating EBs (>60 days) that there still exist cell colony with pluripotency. This observation raised a potential possibility that ES cells may keep pluripotent in a niche provided by differentiated cells.