Pluripotent human stem cell lines can develop into any cell type in the body. In order to exploit the power of these cells for use in research or therapy, we need to be able to grow them in the laboratory in pure form and efficiently turn them into specific types of mature cell. Current methods for growing pluripotent stem cells yield mixed populations of cells, some of which have limited capacity for growth and development, others that represent true pluripotent stem cells. Thus, the stem cell cultures are like an ecosystem, with many components. We are studying the culture microenvironment of the stem cell to learn how it regulates cell growth. In order to do this we have to understand the heterogeneity in stem cell cultures. To this end, we have developed techniques to analyze the molecular makeup of single stem cells, and to track their fates. These techniques enable us to identify the most primitive cells in the culture with the greatest potential for growth and differentiation. These cells tend to be spatially segregated from other more mature cells, in a specialized microenvironment that helps them to maintain their ability to grow and turn into specialized cells. We have identified some key components of the microenvironment that keep stem cells in the primitive pluripotent state. We will use these techniques to develop means to produce pure populations of specialized cells from stem cell cultures.