Human embryonic stem cells (hESCs) hold a great promise for regenerative medicine and tissue engineering. In order to obtain uniform hESC cultures without spontaneous differentiation, which is of interest for basic investigations as well as the development of future therapeutic protocols, it is important that specific culture conditions are adhered to. Here, we describe in detail a procedure for propagation of hESCs that by virtue of exposure of the cultures to low atmospheric oxygen (5%) enables the maintenance of their undifferentiated phenotype in long term. The critical steps and impact of possible modifications on the final outcome are discussed, and useful hints are provided to streamline the troubleshooting.
