ons expressing b-III-tubulin were detected in 565% and 363% of colonies grown on MEF and MM55K respectively, whereas co-culture with PA6-DA cells resulted in 82613% b-III-tubulin+ colonies. The average percentages of Oct3/ 4, nestin, b-III-tubulin and TH expressing colonies cultured on MEF, MM55K and PA6-DA cells are presented as bar graphs in Analysis of gene expression By comparing 
gene expression of the PA6-DA cell line to the five cell lines lacking SDIA, we identified a set of candidate genes as potential DA-inducing elements. A complete gene list containing Z-ratios can be found in the supplemental data at ftp://137.187.144.38/freed. In total, 288 genes were preferential- 4 Dopaminergic Induction of hESC ly expressed in PA6-DA cells as compared to the PA6-X cell subtype. The majority of these genes were also significantly up-regulated in PA6-DA cells as compared to the transformed stromal cell lines, PA6-X1, MS5, 21505263 and the MM55K and MEF cells. Biological processes were available for 159 of the 288 genes analyzed using the FatiGO web tool. The functions of these genes included 50 6-Carboxy-X-rhodamine web different categories at the most basic level. In order to identify genes involved in biological processes and pathways related to CNS development among the 288 up-regulated genes, we performed a more advanced gene ontology analysis using the FatiGO web tool at specificity level six, as illustrated in identified stromal cell-derived factor 1, pleiotrophin, insulin-like growth factor 2, insulin-like growth factor binding protein 4, and ephrin B1 as factors potentially responsible for DA inducing activity. This combination of factors was termed ��SPIE”, as it was eventually determined that IGFBP4 was unnecessary. These genes were among the most up-regulated in PA6-DA cells as compared to PA6-X or MM55K cells, each having a seven-fold or greater Z-ratio. Relative expression of these genes in PA6-DA versus each cell type is presented 20032260 in Gene Ontology Biological Process at Level 6 Gene UniGene Accession Percentage of Genes Z-ratios PA6-DA MM55K 2.57 7.43 14.34 1.66 5.18 2.90 4.71 3.99 2.04 2.57 14.34 2.90 3.99 1.40 14.60 6.33 4.61 3.99 14.53 3.09 2.56 7.36 7.43 14.34 5.18 3.99 7.43 0.40 6.33 14.34 5.18 6.33 3.99 2.18 Neurogenesis IDB4 EFNB1 CXCL12 TIMP2 MYH10 THY1 RUNX1 NOTCH1 NM_031166.1 NM_010110.2 NM_013655.2 NM_011594.2 NM_009382.2 NM_175260.1 NM_009821.1 NM_008714.2 NM_011023.2 NM_031166.1 NM_013655.2 NM_175260.1 NM_008714.2 NM_008597.2 NM_008973.1 NM_011448 NM_020595 NM_008714.2 NM_013834.1 NM_009144.1 NM_011356.2 NM_080555.1 NM_010110.2 NM_013655.2 NM_009382.2 NM_008714.2 NM_010110.2 NM_011349.2 NM_011448 NM_013655.2 NM_009382.2 NM_011448 NM_008714.2 NM_033509.2 5.88 3.16 8.56 16.27 3.32 9.62 3.15 4.28 3.02 Central nervous system development OTX1 IDB4 CXCL12 MYH10 NOTCH1 3.68 3.22 3.16 16.27 3.15 3.02 Tissue development MGLAP PTN SOX9 OTOR NOTCH1 3.68 9.42 13.50 11.62 4.04 3.02 Wnt receptor signaling pathway SFRP1 SFRP2 FRZB PPAP2B 2.94 14.24 3.16 3.44 3.37 Cellular morphogenesis during differentiation EFNB1 CXCL12 THY1 NOTCH1 2.94 8.56 16.23 9.62 3.02 Neural crest cell differentiation EFNB1 SEMA3F SOX9 2.21 8.56 4.66 11.62 Regulation of cell migration CXCL12 THY1 2.21 16.23 9.62 Cell fate specification SOX9 NOTCH1 2.21 11.62 3.02 Neural tube development LTAP 0.74 3.21 Gene clusters categorized into biological processes at level six with relevance to various aspects of brain development and maintenance of central neurological processes. Expression levels of