entiated on PA6 cells. Expression of ephrin B1 receptors EPHB2 and EPHB4 was also detected in undifferentiated hESC. Differentiation increased expression of EPHB2 and EPHB3. The PTN receptor N-syndecan was expressed in undifferentiated hESC as well as in hESC differentiated on PA6 cells. The SDF-1 receptor, CXCR4, was undetectable or minimally expressed in undifferentiated hESC, although it should be noted that the form of MPSS used at that time failed to detect certain transcripts such as human TH. In a previous study by our group, gene expression profiling by a focused human stem cell microarray showed expression of CXCR4 in neurons generated from hESC by the SDIA method. In addition, previous focused gene expression array revealed that IGFR1, IGFR2 and CXCR4 were expressed in undifferentiated BG03 hESC. A summary of the MPSS data can be found at ftp://137.187.144.38/freed in the file entitled ��MPSS detection of SPIE receptors”. The varying levels of expression 7510950 of these receptors suggests the possibility that the various components of SPIE 
may be required at different stages of differentiation, a possibility that we have not yet tested. Induction of a midbrain DA phenotype by SPIE was confirmed by the expression of midbrain specific markers including Lmx1b, which is present during early specification of DA precursors, Pitx3 and En1. In addition, expression of receptors GFR1 and c-RET for glial cell line-derived neurotrophic factor, which is a selective neurotrophic factor for midbrain DA neurons, was upregulated by SPIE. Increased expression of the brainderived neurotrophic factor receptor, TrkB, and the SMO receptor that is involved in SHH signaling, both of which are highly expressed in midbrain DA neurons, was also detected in SPIE-treated cultures. Furthermore, neurons differentiated from hESC by SPIE exhibited electrical excitability, generated action potentials, and spontaneous, miniature postsynaptic ML 176 chemical information currents which reflects establishment of functional synaptic networks. It is interesting that there are two apparently unrelated procedures for differentiating mesencephalic DA neurons from hESC; techniques involving SDIA or SPIE, and the method of Yan and coworkers. Although both SPIE and the Yan et. al. techniques employ an EB formation phase, the treatment protocols and growth factors which are employed are quite different. In the present study, the morphology of SPIE-derived cells 12182951 was similar to that of cells differentiated by SDIA and to that of DA neurons differentiated by the procedure described by Yan and coworkers. Cells produced by the two methods are also similar in that they express markers of mesencephalic DA neurons. There may, however, be other or more subtle differences between the cells derived by the two methods, which would require additional studies to identify. Growth and soluble factors produced by PA6 cells Heparan-sulfate glycosaminoglycans such as heparin exist at the cell surface and also in the extracellular matrix of many different cell types are known to interact strongly with several growth factors and modulate their biological activity during development. PTN is among the factors reported to have a high affinity association with heparin-like molecules. PTN has trophic effects on DA neurons and increases the yield of TH+ neurons differentiated from mouse ESC. Serpin peptidase inhibitor, clade E, also known as SERPINE2, was also identified among the heparin binding factors that were differentially expr