in addition to the myogenically differentiated human cells, co - cultures with young myof iber explants also contained some small undifferentiated hesc colonies, as determined by immunoreactivity to a human - specific antibod y to the nuclear mitotic apparatus protein, numa and oct4 expression ( supplementary fig. s4 ). in contrast, when co - cultured with the aged mouse myof ibers, only mouse cells appeared desmin + ( fig. 4a, yellow arrow in old ). these aged myogenic cells were of skeletal muscle lineage, based on spontaneous generation of multinucleated myotubes ( see fig. 5b ) and based on induced differentiation into myotubes in dmem + 2 % horse serum ( not shown ). importantly, the myogenic differentiation of hescs failed in the aged co - cultures ( fig. 4a, white arrow in old ). furthermore, colonies of hescs in co - cultures with aged myof ibers typically differentiated into cells with fibroblast morphology, which lacked oct4 expression ( not shown ). spontaneous production of desmin + myogenic cells in control hesc cultures without myof ibers, or with young / old mouse sera was less than 0. 1 % ( not shown ). fig. 5in vitro co - culture with hescs enhanced myogenesis of mouse cells. ( a ) 1 × 105 hescs or control hmscs were co - cultured with 5 × 106 primary mouse myoblasts. hescs expressing oct4 ( immunode tection shown in red ) dramatically enhanced myotube formation of co - cultured mouse myoblasts ( immunode tection of emyhc is shown in green ), as compared to co - cultures between mouse myoblasts and human mesenchymal stem cells ( mb + hmscs ) or myoblasts alone ( mb alone ). experiments were carried out in myoblast differentiation medium. hoechst ( blue ) labels nuclei throughout this figure. ( b ) 1 × 105 hescs or control hmscs were co - cultured with young or old myof iber - associated satellite cells, as described in experimental procedures. co - culture with hescs ( myof iber + hesc ), but not hmscs ( myof iber + hmsc ) or control medium ( dmem / fbs ), greatly enhanced the myogenic potential of both young and old myof iber - associated satellite cells, based on immunode tection of percentage of desmin + de novo generated myoblasts and multinucleated myotubes. these experiments were carried out in gm. shown are myogenic responses of mouse cells only, judged by lack of immunoreactivity to human - specific / hesc - specific antigens, such as numa and oct4; and presence of mouse - specific immunoreactivity, e. g., m - cadherin ( not shown ). both young and old myof iber associated satellite cells exhibited considerable myogenic improvement over control conditions. n = 3. in concert with the conservation of inhibitory affects of aged systemic niche, the negative influence of local muscle niche was also found to be conserved in its inhibition of hesc and asc regenerative responses. specifically, the myogenic capacity ( generation of desmin + myoblasts ) was inhibited in young satellite cells co - cultured in a transwell system with aged myof ibers ( fig. 4b ). in addition, hesc and asc proliferation ( brdu incorporation ) was also inhibited by aged differentiated muscle ( fig. 4a, c ). these data suggest that not only systemic but also local organ niches would inhibit key stem cell properties, e. g., myogenic capacity and the rate of proliferation in the aged organism. the conserved inhibitory influences of the differentiated muscle niche on hesc and asc regenerative responses are summarized in table 2. table 2conservation of stem cell aging in the local organ nicherate of proliferation esc / asc ( percentage of brdu ) myogenic differentiation esc / asc ( percentage of desmin ) young myof iber60. 2 ± 2. 5, 40. 5 ± 2. 67. 4 ± 0. 9, 47. 6 ± 5. 0old myof iber30. 1 ± 4. 3, 21. 5 ± 4. 11. 3 ± 0. 7, 19. 7 ± 4. 7quantified results from fig. 4 are summarized and presented as mean percentages from experimental replicates ± se. rate of proliferation ( brdu ) and myogenic differentiation ( desmin ) are shown for both escs and ascs, in the presence of young vs. old differentiated muscle environments ( young myof iber or old myof iber ). hescs indirectly enhance and rejuvenate the regeneration of skeletal musclewhile hesc properties were inhibited by aged differentiated muscle, the myogenic potential of aged satellite cells seemed to be enhanced by co - cultures with hescs ( fig. 4a ). therefore, we further explored the enhancing and rejuvenating effects of hescs on myogenic potential in vitro and in vivo, using human mesenchymal stem cells ( hmscs ) as a negative control. first, we examined the effects of hescs on myotube generation by co - culture with primary myoblasts freshly derived from activated - by - injury satellite cells ( conboy et al., 2003 ). as shown in fig. 5a ( mb + hesc ), primary myoblasts underwent very rapid and robust nascent myotube formation, when co - cultured with hescs for 48 h in myoblast differentiation medium. namely, remarkably large fused myotubes containing approximately 50 – 70 nuclei formed around hescs colonies ( fig. 5a ). in contrast, when co - cultured with hmscs, myotube formation was no greater than in myoblast cultures alone ( fig. 5a, mb + hmsc and mb alone ). encouraged by these data, we analyzed the myogenic potential of young and old satellite cells co - cultured with hescs for 48 h. as shown in fig. 5b, hescs conferred a much - enhanced myogenic capacity on both young and, importantly, old myof iber - associated satellite cells ( rapid formation of desmin + myogenic cells, many of which formed de novo multinucleated myotubes ). control co - cultures of these satellite cells with hmscs displayed no enhanced myogenicity. in summary, while the myogenic potential ( production of desmin + fusion - competent cells ) was more pronounced in young vs. old myof iber - associated satellite cells under all experimental conditions, a finding that is consistent with previous data ( conboy et al., 2003 ), a clear increase in myogenic potential of old satellite cells was noted in co - cultures with hescs, as compared to control cultures devoid of hescs ( fig. 4a, b ). interestingly, in addition to the rejuvenating effects of direct co - cultures shown in fig. 5, soluble factors present in hesc - conditioned culture supernatants were also able to enhance myogenesis of aged satellite cells ( supplementary fig. s5 ). thus, in agreement with the notion that an established embryonic microniche antagonizes the inhibitory effects of the aged environment on stem cell responses ( fig. 3 ), the hesc - produced factors enhanced myogenic capacity of even old mouse satellite cells. establishing that hesc - produced factors enhance adult myogenesis and rejuvenate the regenerative capacity of even aged satellite cells in vitro prompted us to examine whether the regeneration of old injured muscle will be improved by hesc transplantation in vivo. additionally, based on the data shown above, we speculated that even if the host ' s repair capacity is improved, hescs themselves will not be efficiently maintained or expanded in the context of old systemic and local organ environments, and will not directly contribute to the repair of aged skeletal muscle. to test these hypotheses, we injected 5 × 105 hescs or control hmscs into the tibialis anterior ( ta ) and gastrocnemius muscles of young and old mice at 24 h after cardiotoxin - induced injury, when activation / proliferation of endogenous satellite cells normally begins ( conboy et al., 2003, 2005; wagers & conboy, 2005 ). to avoid immune response against hesc antigens, mice were immunosuppressed using fk506 ( ito & tanaka, 1997; dumont, 2000 ). muscle was isolated 5 days post - injury, when nascent differentiated myof ibers normally replace the damaged tissue ( conboy et al., 2003 ), and 10 µm cryosections were analyzed for the success in tissue repair using hematoxylin and eosin ( h & e ) histochemistry and emyhc immunode tection.