Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers

Background: Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripot ent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganiz ed. The recent isolation of human ES cells calls for exploring their plurip otential nature. Materials and Methods: Human ES cells were grown in suspension to induce th eir differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression patt ern of several lineage-specific molecular markers using reverse transcripti on polymerase chain reaction (RT-PCR) and in situ hybridization. Results: Here we report the induction in vitro of cystic embryoid bodies fr om human ES cells. Our findings demonstrate induction of expression of cell -specific genes during differentiation of the human ES cells into EBs. In t he human EBs, we could show a characteristic regional expression of embryon ic markers specific to different cellular lineages, namely, zeta-globin (me soderm), neurofilament 68Kd (ectoderm), and alpha-fetoprotein (endoderm). M oreover, we present a synchronously pulsing embryoid body that expresses th e myocardium marker a-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologie s, among them cells with neuronal appearance that express neurofilament 68K d chain. Conclusion: Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formatio n of human embryoid bodies that contain cells of neuronal, hematopoietic an d cardiac origins will be useful in studying early human embryonic developm ent as well as in transplantation medicine.