therefore, we tested whether hesmpcs have the same potential ( figure 2 ). adipocytic differentiation of hesmpcs was induced under conditions described previously for primary adult mscs [ 5 ]. appearance of cells harboring fat granules was observed after 10 – 14 d in culture. after 3 wk of induction, more than 70 % of the cells displayed oil red o + fat granules, and pparγ, a marker of adipocytic differentiation, was detected by rt - pcr. ( chondrocytic differentiation was achieved using the pellet culture system [ 5 ]. after 28 d in culture, more than 50 % of all cells exhibited robust staining for alcian blue, a marker specific for extracellular matrix proteoglycans. chondrocytic differentiation was confirmed by the gene expression of collagen ii and aggrecan, two components of extracellular matrix selectively expressed by chondrocytes, using rt - pcr ( figure 2b ). osteogenic differentiation was induced in the presence of β - glycerolphosphate [ 5 ]. osteogenesis was demonstrated by specific staining for calcium deposition in the matrix ( von kossa, figure 2c; or alizarin red, figure s2a ) and increased expression of bone - specific alkaline phosphatase and bone sialoprotein at day 28 of treatment ( figures 2c and s2b ). at day 28, alizarin red staining was detected in approximately 70 % of all cells. throughout these studies, human adult mscs and foreskin fibroblasts were used as positive and negative controls, respectively. in addition to adipocytic, chondrocytic, and osteogenic differentiation, reports suggested that adult mscs can form skeletal muscle [ 13 ]. although generation of skeletal muscle cells from adult mscs remains controversial, we tested whether hesmpcs exhibit this potential. under the conditions previously described [ 13 ], hesmpc - h1. 1 and - h9. 1 did not yield significant numbers of myod + cells after 15 – 20 d in culture. however, when confluent cells were maintained in culture in the presence or absence of 5 - azac without passage for more than 21 d, expression of specific skeletal muscle markers such as myod and fast - switch myosin was observed ( figure 3a ). more rapid myogenic differentiation was obtained in the presence of 24 - h - conditioned medium from the murine myoblastic cell line c2c12 previously induced to form myotubes [ 14 ]. direct coculture of hesmpcs with c2c12 cells led to the formation of hesmpc - derived myotubes, as visualized by expression of human nuclear antigen ( figure 3b ), similar to those formed by host c2c12 cells. after 1 wk of coculture, myotubes compose d of human nuclei accounted for more than 10 % of the total number of human cells present, and each human myotube was compose d of up to ten human nuclei. human cell contribution to myotubes in coculture was confirmed by expression of human muscle - specific transcripts such as myod, myosin heavy chain iia, and myogenin ( data not shown ). these data demonstrate that hesmpcs can give rise to mesenchymal derivatives typically obtained from primary adult mscs, as well as to cells expressing markers of skeletal muscle. one concern for the clinical application of hesc - derived progeny in regenerative medicine is the risk of teratoma formation due to the presence of residual undifferentiated es cells among the differentiated progeny. we did not detect markers of undifferentiated hescs, such as nanog [ 15 ] or oct - 4 [ 16 ], in any of the hesmpcs by rt - pcr ( see figure 2d ) and immunocytochemistry ( data not shown ), suggesting the lack of any undifferentiated es cells in hesmpc cultures. however, future in vivo studies are required to rule out the potential of these cells for teratoma formation. previous studies have demonstrated the derivation of neural cells [ 1 – 3 ], hematopoietic [ 17 ] and endothelial lineages [ 18 ], and cardio myoc ytes [ 19 ] from hescs. this study presents the induction of paraxial mesoderm with the generation of multipotent mesenchymal precursors. we calculate that under these conditions a single undifferentiated hesc yields an average of one cd73 + cell at day 40 of differentiation, suggesting a balance between cell proliferation and cell selection. there were no obvious differences in marker and gene - expression profile or in differentiation behavior among the five hesmpc lines generated. however, some of the lines ( e. g., hesmpc9. 1 ) exhibited a tendency of spontaneous osteogenic differentiation after long - term propagation. directed differentiation of hescs into somatic stem - cell - like precursors represents a substantial advancement in harnessing the developmental potential of hescs. the high purity, unlimited availability, and multipotentiality of hesmpcs will provide the basis for future therapeutic efforts using these cells in preclinical animal models of disease. such in vivo studies will also be required to properly assess the safety profile of these cells. furthermore, our system also offers a novel platform to study basic mechanisms of mesodermal induction and differentiation during early human development. backgroundin order to compare the gene expression profiles of human embryonic stem cell ( hesc ) lines and their differentiated progeny and to monitor feeder contaminations, we have examined gene expression in seven hesc lines and human fibroblast feeder cells using illumina® bead arrays that contain probes for 24, 131 transcript probes. resultsa total of 48 different samples ( including duplicates ) grown in multiple laboratories under different conditions were analyzed and pairwise comparisons were performed in all groups. hierarchical clustering showed that blinded duplicates were correctly identified as the closest related samples. hesc lines clustered together irrespective of the laboratory in which they were maintained. hescs could be readily disting uished from embryoid bodies ( eb ) differentiated from them and the karyotypically abnormal hesc line bg01v. the embryonal carcinoma ( ec ) line ntera2 is a useful model for evaluating characteristics of hescs. expression of subsets of individual genes was validated by comparing with published databases, mpss ( massively parallel signature sequencing ) libraries, and parallel analysis by microarray and rt - pcr. conclusionwe show that illumina ' s bead array platform is a reliable, reproducible and robust method for developing base global profiles of cells and identifying similarities and differences in large number of samples. embryonic stem cells ( escs ), derived from the inner cell mass of pre - implantation embryos, have been recognized as the most pluripotent stem cell population. human es cells ( hescs ) can be maintained and propagated on mouse or human fibroblast feeders for extended periods in media containing basic fibroblast growth factor ( bfgf ) [ 1 - 4 ] while retaining the ability to differentiate into ectoderm, endod erm and mesoderm as well as trophoectoderm and germ cells. gene expression in hesc has been investigated by a variety of techniques including massively parallel signature sequencing ( mpss ), serial analysis of gene expression ( sage ), expressed sequence tag ( est ) scan, large scale microarrays, focused cdna microarrays, and immunocytochemistry [ 5 - 7 ]. markers for hescs that may also contribute to the " stemness " phenotype have been established and markers that disting uish escs from embryoid bodies ( eb ) have been developed. novel stage - specific genes that disting uish between hescs and ebs have been identified and allelic differences between esc have begun to be recognized [ 8 - 10 ]. as the potential of hescs and their derivatives for regenerative medicine is being evaluated, it has become clear that the overall state of the cells, degree of contamination and comparisons of the more than a hundred different newly derived lines will need to be performed. it will be necessary to develop methods to monitor and assess hesc and their derivatives on a routine basis.