thus, a complex interplay between negative regulation of hescs and adult muscle stem cells by the aged niche, and positive regulation of the host ' s regenerative responses by hescs will likely determine the success of hesc - based cell - replacement therapies in the old. regenerative responses of adult muscle stem cells and hescs are dominantly inhibited by the aged systemic milieuprevious work established that the upregulation of repair - specific molecular signaling mechanisms, such as notch, and successful engagement of resident muscle stem cells in tissue repair are largely determined by the age of the systemic milieu, rather than by the cell - autonomous age of muscle cells, or by the differences in their numbers ( conboy & rando, 2005; conboy et al., 2005 ). intriguingly, these experiments also hinted at a small but persistent inhibitory effect of the aged systemic milieu on the performance of young stem cells. exploring this further, we found that young serum permits satellite cells to be myogenic, while old serum inhibits the satellite cell regenerative potential not only alone, but also when mixed with young serum, suggesting a dominant over - riding of ‘ young ’ serum factors ( fig. 1 ). myof iber cultures, in which satellite cells have been activated by injury in vivo, were established from young ( 2 – 3 months ) and old ( 22 – 24 months ) c57 - bl / 6 male mice, as previously described ( conboy & rando, 2002; conboy et al., 2005 ). as previously shown, this method is well suited for the assessment of satellite cell regenerative myogenic capacity ( conboy & rando, 2002; wagers & conboy, 2005 ). isolated myof iber explants with associated satellite cells were cultured overnight in the presence of young or old serum ( alone at 5 % and 10 %, and mixed at 5 % young + 5 % old ); bromodeoxyuridine ( brdu ) was added for the last 2 h of culture to measure the rate of cell proliferation. the effects of heterochronic systemic milieu on myogenic potential were examined as generation of proliferating myoblasts that express desmin and myf5, and that spontaneously form multinucleated nascent myotubes. as shown in fig. 1a and quantified in fig. 1b, the age of sera clearly determined satellite cell regenerative potential and old serum strongly inhibited the myogenic potential of young satellite cells either when present alone, or when mixed with young sera. similar data was obtained by using another myogenic marker, pax7 ( supplementary fig. s1 ). additionally, there were two to three times fewer total cells generated in the presence of aged serum ( not shown ). fig. 1the age of sera determined the regenerative potential of satellite cells. ( a ) young satellite cells were cultured either in 5 % or 10 % young ( young ), 10 % old ( old ), or in a 5 % + 5 % mouse sera combination ( young + old ). cells were analyzed by immunoflu orescence microscopy, using anti - brdu ( red ), antidesmin ( green ) or anti - myf5 antibod ies ( green, small panels ). similar results are shown for pax7 immunode tection ( supplementary fig. s1 ). hoechst ( blue ) labeled nuclei. ( b ) three independent experiments were quantified [ 300 young myof ibers per experiment ] as percentage of desmin + / myf5 + / brdu + de novo generated cells for each age and culture condition. on average, two to three fewer cells were generated when cultured in the presence of old. shown are identical microscope fields at ×40 magnification. at least three independent experiments produced similar results. ( * ) indicates p≤ 0. 001 as compared to young sera. importantly, it was not simply the dilution of young serum factors that resulted in diminished myogenic capacity when young and old sera were mixed, because young sera promoted robust myogenesis both at 10 % and 5 %. thus, old serum factors dominantly inhibited the myogenic capacity of young satellite cells even in the presence of young serum. this observation suggests that satellite cells of young mice engage in efficient myogenic responses, in part, because the inhibitory influence of old circulatory milieu is absent. these data reveal that the regenerative potential of young muscle stem cells is determined by the age of the systemic milieu, prompting us to investigate whether hescs would similarly succumb to inhibitory factors present in the aged circulation. to determine the effects of aged serum on stem cell self - renewal / pluripotency, we analyzed hesc expression of oct4 and studied the rate of hesc proliferation, by assessing brdu incorporation ( fig. 2 ) and ki67 expression ( supplementary fig. s2 ). specifically, these determinants of hesc regenerative potential were examined in the presence of heterochronic ( young vs. old ) mouse sera added to typical hesc medium, e. g., mef - conditioned medium ( mcm ). oct4 is expressed by self - renewing, pluripotent escs in culture, by the totipotent inner cell mass of the blastocyst and by the germ cells ( nichols et al., 1998; pesce et al., 1999 ). most cells in control cultures or young conditions expressed high levels of this marker of ‘ stemness ’, and maintained their normal phenotype and morphology throughout the various co - culture experiments performed in this study ( see below ). fig. 2the regenerative potential of embryonic stem cells was negatively affected by aged mouse sera. ( a ) hescs were cultured in mcm with 10 % young ( young ) or old ( old ) mouse serum, or in three control media: mcm without mouse sera; gm ( myoblast medium of ham ' s f10 with 20 % fbs ) and dmem / fbs ( hesc differentiation medium of dmem with 10 % fbs ). brdu was added for the last 2 h of culture to measure the rate of cell proliferation. immunode tection assays were performed for brdu ( red ), oct4 ( red ), and ki67 ( supplementary fig. s2 ). a high rate of hesc proliferation and oct4 expression is displayed in all control media and in the presence of young mouse serum. in contrast, hesc proliferation and oct4 expression are inhibited in the presence of old mouse serum, either alone or when mixed with young serum. mcm with mouse sera at 5 % gave results similar to those observed with 10 % young mouse sera or in control media ( supplementary fig. s3 ). ( b ) three independent experiments yielded similar results and were quantified as percentage of brdu + and oct4 + cells for each culture condition. * indicates p < 0. 001 as compared to young serum. importantly, at 10 % aged serum dramatically inhibited the self - renewal and proliferative potential of hescs, as judged by highly diminished oct4 expression and a lack of brdu incorporation. again, the inhibitory factors in the aged milieu were dominant over the young, as evidenced by a decline in oct4 expression, the low rate of brdu incorporation, and ki67 expression in young and old mixed environments ( 5 % young + 5 % old sera in mcm ). similar to the data shown for adult stem cells ( ascs ) ( fig. 1 ), it was not simply a dilution of young serum factors as hescs robustly proliferated and expressed high levels of oct4 when cultured with 5 % young sera in mcm ( supplementary fig. s3 ). quantification of multiple independent experiments has demonstrated that hesc expression of oct4 and brdu incorporation have been reduced by two - to threefold in the aged milieu ( fig. 2b ). as expected, hescs cultured in control media, including mcm alone that does not contain either young or old serum, also displayed a high rate of proliferation and oct4 expression ( fig. 2, control medium ).