additionally, in this experimental set - up there was no general inhibitory effect of sera per se on hesc proliferation and oct4 expression, as 10 % young mouse sera ( young ) and 10 – 20 % of fbs ( growth medium and dmem / fbs ) allowed for a high rate of cell proliferation and for uniformly high oct4 levels ( fig. 2 ). when instead of immediate exposure to aged mouse serum, hescs were first cultured overnight in mcm, these cells were no longer susceptible to the negative effects of old systemic milieu ( fig. 3 ), suggesting that hesc - produced factors established an embryonic microniche that may provide temporary protection from the aged environment. it appears that satellite cells do not have such anti - aging ability, because despite an initial activation in entirely young environments, e. g., after muscle injury to young muscle, isolated satellite cells remain susceptible to inhibition by the old mouse serum ( figs 1 and 4c ). similarly, culturing satellite cells isolated from noninjured muscle in growth - promoting medium for 1 – 2 days does not protect against the inhibitory affects of aged systemic milieu ( not shown ). fig. 3embryonic stem cells produce youthful microniche in culture. ( a ) as opposed to immediate exposure to old mouse serum after passaging ( 10 % old ), preculturing of hescs for 24 h in feeder - free conditions, e. g., matrigel™ + mcm, prior to replacing mcm with mcm + 10 % old mouse sera, resulted in continuously high brdu incorporation and oct4 expression ( embryonic microniche + 10 % old ). brdu was added for the last 2 h of culture to measure the rate of cell proliferation. immunode tection of brdu and oct4 ( both in red ) was performed as described in experimental procedures. hoechst ( blue ) labels nuclei. ( b ) three independent experiments yielded similar results and were quantified as percentage of brdu + / oct4 + for each condition. * indicates p < 0. 001 as compared to ‘ old + mcm ’. fig. 4aged muscle niche inhibits the regenerative potential of hescs and satellite cells. ( a ) immunode tection of a mouse - specific m - cadherin ( green ) or desmin ( red; both human and mouse proteins are detected ) revealed that hescs underwent muscle lineage differentiation when co - cultured with young, but not old myof ibers. the myogenic progeny of hescs appears m - cadherin− / desmin + ( white arrow in young ), as opposed to m - cadherin− / desmin− hescs that lack myogenic commitment ( white arrow in old ). m - cadherin + / desmin + cells are the myogenic progeny of mouse satellite cells ( yellow arrows ). to assess the effects of secreted factors produced by young vs. old myof ibers on the rate of hesc proliferation, transient, 2 h brdu incorporation was examined in hescs cultured for 48 h with supernatants produced by heterochronic myof iber explants ( see experimental procedures for details ). as compared to young myof iber - derived supernatants ( young myof iber supernant ), exposure to old myof iber - derived supernatants ( old myof iber supernant ) inhibited hescs proliferation, as judged by brdu immunode tection ( red ). as expected, the rate of hescs proliferation was high in control media ( shown in fig. 2 ). hoechst ( blue ) labels nuclei in all experiments. quantification of desmin + / brdu + hescs in direct myof iber cocultures, or with muscle supernatants, is shown in ( b ). * indicates p≤ 0. 001 as compared to young. ( c ) transwell co - cultures between purified young satellite cells and myof ibers isolated from uninjured young ( young myof iber ) and old ( old myof iber ) muscle demonstrated that satellite cell regenerative myogenic capacity was inhibited by the aged differentiated muscle. myogenic potential was determined by the ability of satellite cells to generate proliferating desmin + myoblasts ( immunode tection shown in green ) and by rate of proliferation ( 2 h brdu incorporation; immunode tection shown in red ). ( d ) satellite cell regenerative potential was quantified as percentage of desmin + / brdu + cells for transwell co - cultures with young or old uninjured myof ibers ( i. e., rm, resting muscle ). n = 3; * indicates p≤ 0. 05 as compared to young. comprehensively, these data establish that the inhibition of stem cell regenerative potential by the aged systemic milieu is conserved between species ( mouse vs. human ) and cell types ( adult vs. embryonic stem cells ). as summarized in table 1, aged mouse sera similarly affected the expression of key molecular identifiers of both embryonic and adult stem cells, e. g., oct4 in hescs and myf5 in mouse ascs. as expected, adult mouse stem cells did not express oct4, and hescs did not express myf5 in these experimental conditions ( not shown ). moreover, aged systemic milieu had similar inhibitory effects on proliferation of hescs and ascs, suggesting that not only the regenerative capacity, but also the presence and expansion of stem cells will be significantly restricted in aged organs. intriguingly, prolonged culturing of hescs in their preferred in vitro conditions enables generation of an embryonic microniche that antagonizes the inhibitory influences of aged circulatory factors. table 1conservation of stem cell aging in the systemic environmentrate of proliferation esc / asc ( percentage of brdu ) call - fate identifier esc ( percentage of oct4 ) / asc ( percentage of myf5 ) 10 % young59. 5 ± 0. 8, 59. 3 ± 4. 099. 0 ± 0. 1, 50. 7 ± 9. 510 % old32. 7 ± 2. 1, 27. 3 ± 3. 517. 6 ± 3. 2, 18. 1 ± 5. 95 % young + 5 % old31. 0 ± 2. 6, 38. 0 ± 2. 020. 6 ± 3. 5, 17. 1 ± 4. 2quantified results from figs 1, 2 are summarized and presented as mean percentages from experimental replicates ± se. rate of proliferation ( brdu ) and cell - fate identifier ( oct4 or myf5 ) are shown for both escs and ascs cultured in heterochronic systemic conditions of 10 % young ( young ), 10 % old ( old ) or in 5 % + 5 % mouse sera combination ( young + old ). results for 5 % young mouse sera are very similar to those for 10 % young mouse sera and are shown in fig. 1 ( ascs ) and supplementary fig. s3 ( hescs ). the regenerative potential of hescs and ascs is inhibited by aged differentiated muscleafter establishing that the aged systemic niche negatively affects the regenerative capacity of hescs and of ascs, we then assessed whether myogenic potential and the rate of cell proliferation would be inhibited in hescs and ascs by the aged local muscle niche. myof ibers with associated satellite cells were isolated from young and old injured muscle, and were directly co - cultured with hescs in typical hesc differentiation medium ( dmem / fbs ). similar to fig. 1, the myogenic potential in these co - cultures was assayed by the expression of desmin, which is present in both fusion - competent myoblasts and newly formed myotubes. to analyze whether hescs, mouse myogenic progenitor cells or both could express desmin in direct co - cultures, we costained these cells with a mouse - specific antibod y to a myogenic marker, m - cadherin, which does not react with human protein, and a desmin - specific antibod y that recognizes both mouse and human proteins. as shown in fig. 4a, hescs underwent myogenic differentiation in co - cultures with young myof ibers ( m - cadherin− / desmin + mononucleated cells, white arrow in young ). these myogenic progeny of hescs in co - cultures with young myof ibers could be of skeletal, smooth or cardiac muscle lineages ( debus et al., 1983; fischman & danto, 1985; schultz & mccormick, 1994 ). as expected, the young mouse muscle progenitor cells ( m - cadherin + / desmin + ) were more advanced in their degree of myogenic differentiation, which was of skeletal muscle lineage, as judged by the formation of large, multinucleated de novo myotubes ( yellow arrow in young ).