chondrocytic differentiationdifferentiation of hesmpcs was induced in pellet culture [ 5 ] by exposure to 10 ng / ml tgf - β3 ( r & d systems, minneapolis, minnesota, united states ) and 200 μm ascorbic acid ( sigma ) in alpha mem medium containing 10 % fbs for 3 – 4 wk. data were confirmed in hesmpc - h1. 1, - h1. 3, and - h9. 1 ( hesmpc - h1. 2 and - h1. 4 were not tested ). osteogenic differentiationhesmpcs were plated at low density ( 1 × 103 to 2. 5 × 103 cells / cm2 ) on tissue - culture - treated dishes in the presence of 10 mm β - glycerol phosphate ( sigma ), 0. 1 μm dexamethasone, and 200 μm ascorbic acid in alpha mem medium containing 10 % fbs for 3 – 4 wk. data were confirmed in hesmpc - h1. 1, - h1. 3, and - h9. 1 ( hesmpc - h1. 2 and - h1. 4 were not tested ). myogenic differentiationconfluent hesmpcs were maintained for 2 – 3 wk in alpha mem medium with 20 % heat - inactivated fbs. more rapid induction was observed in the presence of medium conditioned for 24 h by differentiated c2c12 cells. coculture of hesmpcs and c2c12 cells was carried out in alpha mem with 3 % horse serum and 1 % fbs [ 8 ]. data were confirmed in hesmpc - h1. 3, - h1. 4, and - h9. 1 ( hesmpc - h1. 1 and - h1. 2 were not tested ). cytochemistryimmunocytochemistry for all surface markers was performed on live cells. monoclonal antibod ies vcam, stro - 1, icam - 1 ( cd54 ), cd105, cd29, and mf20 were from developmental studies hybridoma bank ( university of iowa, iowa city, iowa, united states ); cd73, cd44, and alcam ( cd166 ) were from bd biosciences pharmingen ( san diego, california, united states ). all other immunocytochemical analyses were performed after fixation in 4 % paraformaldehyde and 0. 15 % picric acid, followed by permeabilization in 0. 3 % trit on x100. polyclonal antibod ies used were myod ( santa cruz biotechnology, santa cruz, california, united states ) and nestin ( gift from r. mckay ); monoclonal antibod ies were vimentin, alpha smooth muscle actin, fast - switch myosin, pan - cytokeratin ( all from sigma ), and human nuclear antigen ( chemicon, temecula, california, united states ). alkaline phosphatase reaction was performed using a commercially available kit ( kit - 86; sigma ) and the mineral was stained with silver nitrate according to the von kossa method. fat granules were visualized by oil red o staining solution ( sigma ). alcian blue ( sigma ) was used to detect extracellular matrix proteoglycans in chondrogenic cultures. gene - expression analysesrt - pcr analysistotal rna was extracted by using the rneasy kit and dnase i treatment ( qiagen, valencia, california, united states ). total rna ( 2 μg each ) was reverse transcribed ( superscript; invitrogen, carlsbad, california, united states ). pcr conditions were optimized and linear amplification range was determined for each primer by varying annealing temperature and cycle number. pcr products were identified by size, and identity was confirmed by dna sequencing. primer sequences, cycle numbers, and annealing temperatures are provided in table s1. affymetrix analysistotal rna ( 5 μg ) from primary mscs, from hesmpc - h9. 1, hesmpc - h1. 2, and three samples of undifferentiated hescs ( h1; passages 42 – 46 ), were processed by the memorial sloan - kettering cancer center genomics core facility and hybri diz ed on affymetrix ( santa clara, california, united states ) u133a human oligo nucleot ide arrays. data were analyzed using mas5. 0 ( affymetrix ) software. transcripts selectively expressed in each of the mesenchymal cell populations ( msc, hesmpc - h9. 1, and hesmpc - h1. 2 ) were defined as those called “ increased ” by the mas5. 0 algorithm in each of three comparisons with independent samples of undifferentiated hescs. a venn diag ram was generated to visualize overlap in gene expression. further statistical analyses were performed as described below. mesenchymal differentiation of hescs ( lines h1 [ wa - 01 ] and h9 [ wa - 09 ] ) [ 9 ] was induced by plating undifferentiated hescs on a monolayer of murine op9 stromal cells [ 10 ], in the presence of 20 % heat - inactivated fbs in alpha mem medium. op9 cells have been previously shown to induce blood cell differentiation from mouse es cells [ 11 ]. after 40 d of coculture, cells were harvested and sorted by facs for cd73, a surface marker expressed in adult mscs [ 5 ] ( figure 1a ). an average of 5 % cd73 + cells was obtained from the mixed culture of op9 and differentiated hesc progeny. cd73 + cells were replated in the absence of stromal feeders on tissue culture plates and expanded in alpha mem medium with 20 % fbs for 7 – 14 d. we next established the membrane antigen profile of the resulting population of flat spindle - like cells. the h1 - and h9 - derived cd73 + cells expressed a comprehensive set of markers that are considered to define adult mscs, including cd105 ( sh2 ), stro - 1, vcam ( cd106 ), cd29 ( integrin β1 ), cd44, icam - 1 ( cd54 ), alcam ( cd166 ), vimentin, and alpha smooth muscle actin ( figure 1b and 1c ). the cells were negative for hematopoietic markers such as cd34, cd45, and cd14. they were also negative for neuroectodermal, epithelial, and muscle cell markers including nestin, pancytokeratin, and desmin ( data not shown ). the human identity of these presumed mesenchymal cells ( termed hesmpc - h1. 1, - h1. 2, - h1. 3, - h1. 4, and - h9. 1 ) was confirmed for all experiments by immunocytochemistry for human nuclear antigen to rule out the possibility of contamination with op9 cells ( figure s1 ). to further characterize hesmpcs, we performed genome-wide expression analysis using oligo nucleot ide arrays ( affymetrix u133a ). the expression profiles of hesmpc - h1. 2 and hesmpc - h9. 1 were compared with that of human primary adult mscs. housekeeping genes for each of the mesenchymal cell populations were eliminated by subtracting those transcripts also expressed in at least one of three independent samples of undifferentiated hescs. based on this analysis, 1, 280 transcripts were selectively expressed in hesmpc - h1. 2, 932 transcripts in hesmpc - h9. 1, and 1, 218 transcripts in primary adult mscs. a remarkable overlap of 579 transcripts shared among the three mesenchymal populations was observed ( figure 1d ). using the genes that were selected in the initial filter, we performed a statistical analysis on the expression levels to determine whether the genes were expressed significantly differently in the two cell types. we used a bayesian extension to the standard t - test [ 12 ] to assess this difference. of the 579 genes, 412 of them were significantly different, at a false discovery rate cutoff of 0. 05. the relative fold changes were also extremely large in many of the cases. we also looked at the variance of the expression levels within the cell types. for the mscs, 94 % had a coefficient of variation less than 20 % for the expression ( log transformed ); for the es - derived cells, 72 % had a coefficient of variation less than 20 %. numerous known msc markers were included in the list of 412 genes, such as the mesenchymal stem cell protein dsc54 ( 13. 9 - fold increase, p < 0. 001 ), neuropilin 1 ( 30. 4 - fold increase, p < 0. 001 ), hepatocyte growth factor ( 48. 1 - fold increase, p < 0. 001 ), forkhead box d1 ( 14. 8 - fold increase, p < 0. 001 ), and notch homolog 2 ( 2. 9 - fold increase, p < 0. 001 ). table s2 lists the p - values from the test, the mean and standard deviation of the expression levels, and the relative fold change of all 412 genes between the two types. known markers of mscs, such as mesenchymal stem cell protein dsc54, were all included within the 579 shared transcripts. these findings support the immunocytochemical data and suggest that hesmpcs and primary mscs are highly related. mscs are characterized functionally by their ability to differentiate into mesenchymal tissues, such as fat, cartilage, and bone.