h & e analysis reveals newly formed myof ibers, based on their smaller size and centrally located nuclei. additionally, de novo myof ibers in the damaged area appear positive for emyhc, while undamaged myof ibers remain negative. as shown in fig. 6a and quantified in 6b, injection of hescs significantly enhanced regeneration of skeletal muscle. remarkably, this positive embryonic effect was especially pronounced in old tissue. fig. 6skeletal muscle regeneration following hesc transplantation is a balance between the inhibitory influence of aged niches and the rejuvenating effects of hescs. young and old tibialis anterior and gastrocnemius muscles were injured by cardiotoxin injection. hescs or hmscs were transplanted at the site of injury and were analyzed by cryosectioning at day 5 after injury ( as described in experimental procedures ). ( a ) newly regenerated myof ibers were detected using emyhc - specific antibod y ( green ) and staining with h & e. in h & e staining, newly regenerated areas contain smaller, immature myof ibers with centrally located nuclei. uninjured myof ibers are much larger, by comparison, with peripherally restricted nuclei. poorly regenerated areas lack new myof ibers and contain areas of fibrosis and inflammation. emyhc immunode tection is specific for regenerating areas of muscle only. both assays showed dramatic enhancement of muscle regeneration in ‘ old + hesc ’ vs. ‘ old + hmsc ’. regeneration improvement was also seen in young + hesc, as compared to young + hmsc. ( b ) quantification of muscle regeneration was performed by analyzing the density of newly formed myof ibers per mm2 of injury site, which is the volume that typically covers the whole injured area. multiple, 10 µm h & e sections were examined through the entire volume of injury in multiple, independently injured muscles. n = 20; * indicates p < 0. 001 ( ‘ old + hmsc ’ compared to young + hmsc and ‘ old + hmsc ’ compared to ‘ old + hesc ’. ( c ) h & e and immunofluoresence staining for oct4, and a human - specific antibod y to numa, revealed the failure of hescs to expand or persist in old, but the presence of hescs in young muscle at 5 days post - transplantation. importantly, such enhanced and rejuvenated muscle repair stems from an indirect induction, as hescs themselves ( or control hmscs ) did not physically contribute to the mouse myof ibers, as judged by near absence ( less than 0. 1 % ) of human - specific numa + nuclei in de novo desmin + myof ibers, analyzed through multiple injury sites. an example of one regenerated myof iber from young muscle injected with hescs, with numa + nucleus in a field of numa− / desmin + mouse myof ibers, is shown in supplementary fig. s6. no such numa + / desmin + myof ibers were detected in aged regenerated muscle ( not shown ). in agreement with the in vitro data, establishing that aged systemic and local niches inhibit hesc proliferation and oct4 expression ( figs 2 and 4 and supplementary fig. s2 ), hescs failed to expand or even persist in old muscle, as judged by the absence of numa + / oct4 + hesc - derived cells in the aged tissue. in contrast, colonies of numa + / oct4 + hesc - derived cells that did not undergo myogenic differentiation were easily detected in young regenerating muscle ( fig. 6c ). this finding validates several technical aspects of these experiments, and confirms the contrasting effects of young and old systemic and local organ niches on hesc self - renewal. these data further confirm and extrapolate our findings and demonstrate that when exposed to both aged systemic and local organ niches, hescs fail to persist and do not contribute to tissue repair directly. at the same time, these embryonic cells indirectly but significantly improve the repair of aged injured muscle in vivo. the data presented here establish for the first time that both the local environment of old differentiated organ, e. g., skeletal muscle and the systemic milieu dramatically affect the regenerative potential of both hescs and mouse post - natal myogenic progenitor cells. not only are the factors promoting myogenic differentiation and proliferation of hescs likely to become depleted with age, but the aged systemic and local organ niches are likely to contain dominant inhibitors of asc and hesc regenerative potential ( figs 1, 2, and 4, summarized in tables 1 and 2 ). importantly, the similar inhibitory effects of old mouse serum and old myof ibers on satellite cell ( figs 1 and 4c ) and hesc ( figs 2 and 4a ) proliferation and regenerative capacity suggest the conservation of elements in age - specific extrinsic regulatory mechanisms between evolutionarily distinc t species and stem cell types. additionally, a similarity in the inhibitory properties between systemic and local organ niches is also of interest and may indicate that molecules produced by old tissues have circulatory / endocrine activity; and / or that age - specific systemic inhibitory components become deposited in the old tissues. humans display broad genetic polymorphisms and behavioral variations, which makes the identification of age - specific molecular changes complicated. in contrast, laboratory mice are genetically and environmentally controlled. establishing that age - specific signals, regulating stem cell responses, are evolutionarily conserved and soluble enables the formation of rational approaches for the identification and characterization of the inhibitors involved, and for revealing the precise timing of their first appearance in serum and differentiated tissues with advancing age. significantly, these experiments have also revealed that not only are hescs able to protect themselves against the negative influences of aged mouse sera ( fig. 3 ), but these cells also produce factors that dramatically enhance the myogenic capacity of primary myoblasts and young and old satellite cells ( fig. 5 ), and also significantly improve repair of young and old injured muscle in vivo ( fig. 6 ). identification of these embryonic factors would allow us to potentially enrich the arsenal of therapeutic tools for combating age - specific degenerative disor ders. the interactions between hescs and heterochronic differentiated niches, initially identified in vitro, have been confirmed by in vivo experiments. namely, while the regenerative capacity, or presence, of hescs is greatly restricted in aged, as compared to young skeletal muscle ( where transplanted cells experience both old systemic and local environments ), embryonic cells indirectly enhance and rejuvenate muscle repair when introduced at the time of muscle stem cell activation in the host, e. g., at day 1 after the injury ( fig. 6 ). it remains to be determined whether the percentage of hescs direct contribution to desmin + myof ibers in young muscle will be increased by transplanting these cells at a different time - point after muscle injury, e. g., at days 3 – 5 ( as in co - cultures with myof ibers pre - injured for 3 days, fig. 4a ). in any case, the virtual lack of hesc and hmsc direct contribution to the newly regenerated skeletal muscle, when small numbers of these cells were injected into injured tissue, is completely consistent with the body of previous data demonstrating that myof iber - associated satellite cells conduct rapid and robust muscle repair and greatly outnumber injected human cells ( collins et al., 2005; wagers & conboy, 2005 ); that compared to muscle - specific satellite cells, the myogenic differentiation of hescs in vitro remains very small ( fig. 5, table 2 ), and that control hmscs are not normally myogenic unless these cells overexpress exogenous constitutively active domain of notch ( dezawa et al., 2005 ). intriguingly, the failure of hescs to strive in old skeletal muscle might represent a therapeutically desirable outcome.