Single embryonic stem cell-derived embryoid bodies for gene screening.

BioTechniques 349 Embryonic stem (ES) cells were established from the inner cell mass of blastocysts and have been shown to spontaneously differentiate into multiple cell types representative of all three embryonic germ layers, both in teratomas after grafting or in vitro under appropriate conditions (1,2). ES cells can be maintained in their pluripotent state if cultured in the presence of leukemia inhibitory factor (LIF) or cultured on feeder layers, which inhibit their differentiation. When LIF is removed, ES cells spontaneously differentiate into cyst-like structures termed embryoid bodies (EBs), which can give rise to any cell type in the body including germ cells and terminally differentiated stages of the cardiogenic, neurogenic, hematopoietic, adipogenic, and chondrogenic lineages. The pluripotent properties of ES cells are the basis of gene targeting technologies used to create mutant mouse strains (transgenic and knockout) for gene function studies in vivo. The in vitro differentiation of ES cells allows investigators to study cell differentiation and gene regulation, as well as pharmacological effects on functionally active cells (e.g., cardiomyocytes). Potentially, the greatest importance is to establish strategies for cell therapy and gene therapy. The differentiation of ES cells can also provide a very useful system for the identification of genes involved in the development of a specific cell line. Commonly used methods including hanging drop and culture in suspension, result in the initial formation of multi-ES cell aggregates and further differentiation into EBs (2,3). With these methods, it is difficult Single embryonic stem cell-derived embryoid bodies for gene screening