The utilization of Stromal-Derived Inducing Activity (SDIA) is one of the most efficientmethods in generating dopaminergic (DA) neurons from embryonic stem cells (ESC).Neuronal and dopaminergic induction can be achieved by co-culturing ESC with mousestromal cell lines, PA6 or MS5. Although it is clear that SDIA has neural inducing andmidbrain patterning activity, the molecular nature of SDIA is so far unknown. There arecontrary reports that either cell surface activity or factors secreted by PA6 cells areresponsible for SDIA. Recently, we found that PA6 cell surface and extracellular matrixmolecules primarily promoted cell survival and general neurogenesis of hESC. Incontrast, factors secreted by PA6 cells provided lineage-specific instructions in thepresence of a stabilizing factor, heparin. In the present study, in an attempt to identify thefactors responsible for dopaminergic induction of hESC, we performed cell wholegenome expression analysis to search for soluble factors produced by PA6 cells bycomparing them to various cell lines lacking the SDIA effect. Among the soluble secretedfactors differentially expressed by PA6 cells were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1(EFNB1). The combination of these factors, which we termed SPIE, was sufficient toproduce dopaminergic neuronal differentiation of the hESC line BG01V2 and otherkaryotypically normal hESC lines in vitro.