for example, while in young tissue hesc derivatives putatively would go on to produce teratomas, it is unlikely that teratoma formation would occur after hesc transplantation into aged skeletal muscle. thus, the indirect beneficial effects of hescs on tissue repair are unlikely to be compromised by the oncogenic properties of these embryonic cells in the context of old skeletal muscle. comprehensively, the results of this work increase our understanding of aging as a process, reveal evolutionary conserved age - specific interactions between stem cells and their differentiated niches, and suggest novel therapeutic approaches for improving the regenerative responses of endogenous or transplanted stem cells in old individuals. animal strainsyoung ( 2 – 3 months ), c57 - bl / 6 male mice were obtained from pathogen - free breeding colonies at jackson laboratories ( bar harbor, me, usa ). aged 22 – 24 months c57 - bl / 6 male mice were obtained from the national institute on aging ( nih ). animals were maintained in the north - west animal facility of the university of california, berkeley, ca, usa, and handled in accordance with the administrative panel on laboratory animal care at uc berkeley. muscle injury and isolation myof iber cultures, in which satellite cells were activated by in vivo injury, were set up as previously described ( conboy & rando, 2002; conboy et al., 2005 ). briefly, mice were injured by direct injection with 5 ng cardiotoxin ( ctx - 1 ) ( sigma, st louis, mo, usa ) into the tibialis anterior and gastrocnemius muscles using a 28 - gauge needle. after 1 – 5 days post - injection, injured or uninjured muscle tissue was dissected out. once isolated, whole muscle was prepared for cryosectioning ( see below ) or myof iber fragments were obtained from hind limb muscles by enzymatic digestion ( see below ), trit uration, and multiple sedimentation and washing procedures. additionally, blood was collected from mice for the isolation of sera. briefly, blood cells were coagulated at 37 °c for 15 ’ and then were centrifuged repeatedly at 5900 g, 4 °c in a microfuge for 3 ’ to isolate sera. mixtures of young and old sera were made 1: 1. for example, in 5 % + 5 % conditions, 50 µl of young and 50 µl old serum were added to 900 µl of culture medium ( opti - mem or mcm, see co - culture procedures below ). myof iber explant culturesexplant and primary cell cultures were generated from c57 - bl / 6 mice, as previously described ( conboy & rando, 2002; conboy et al., 2003 ). dissected gastrocnemius and tibialis anterior muscles underwent enzymatic digestion at 37 °c in dmem ( invitrogen ) / pen - strep ( invitrogen ) / 0. 2 % collagenase type iia ( sigma ) solution. isolated fibers were resuspended in gm ( ham ' s f10 nutrient mixture ( mediatech, inc., herndon, va, usa ), 20 % fbs ( mediatech ), 5 ng ml−1 bfgf ( chemicon, temecula, ca, usa ) and 1 % pen - strep, and cultured on ecm - coated ( bd biosciences, san jose, ca, usa ) plates ( diluted 1: 500 in pbs ). cultures of primary myoblasts were derived from isolated fibers, through repeated passaging, and were maintained in gm. myoblast differentiation medium [ dmem, supplemented with 2 % horse serum ( mediatech ) ] was used to promote rapid formation of myotubes from cultured myoblasts ( morgan & partridge, 2003 ). human embryonic and mesenchymal stem cell culturethe federally approved hesc line, h7 ( nih no. wa07, obtained from wicell research institue, madison, wi, usa ), was used in accordance with the uc berkeley and uc san francisco committee on human research guidelines, and in accordance with nih guidelines. to propagate hescs, routine culturing and maintenance was performed using standard in vitro conditions for both feeder - dependent and feeder - free cultures ( geron corporation, 2002 ). briefly, hescs grown on mefs were cultured in standard hesc medium [ knockout™ dmem, 20 % ksr, 1 % neaa, 1 mm l-glutamine ( invitrogen ), 0. 1 mmβ - mercaptoethanol ( sigma ) ] and were supplemented with 4 ng ml−1 hbfgf ( invitrogen ). feeder - free hesc cultures were maintained in mef - conditioned hesc medium ( mcm ), 4 ng ml−1 hbfgf. differentiation medium for hescs ( dmem / fbs ) was made by replacing ksr with 20 % fbs ( hyclone, logan, uh, usa ). hmscs were maintained in mesenchymal stem cell gm, msc - gm™ and were cultured according to supplier recommendations ( cambrex walkersville, md, usa ). hescs and hmscs were typically seeded onto chambered slides coated with a 3 % gfr matrigel™ ( bd biosciences ) substrate in pbs. cells were typically incubated for 48 h at 37 °c, 5 % co2, under the various experimental conditions employed, then were fixed with 70 % etoh / pbs at 4 °c. hescs and hmscs were analyzed 24 – 48 h after experimental treatments, during which no apoptosis - related differences in cell numbers were observed. heterochronic co - culture systemsheterochronic systemic cultures were established by culturing myof iber explants ( in gm ) or hescs ( in mcm ) in the presence of young, old or young + old sera for 48 h ( figs 1 and 2 and supplementary figs s1 – 3 ). in such cultures, hescs were passaged immediately prior to sera exposure. in contrast, preculturing of hescs for 24 h in mcm, prior to replacing mcm with mcm + 10 % old mouse sera was done for embryonic microniche experiments ( fig. 3 ). for heterochronic local organ niche cultures, hescs were co - cultured directly with myof iber explants for 48 h in gm, or were cultured in the presence of supernatants derived from cultured myof iber explants for 48 h ( figs 4a and 5 ). specifically, 1 × 105 hescs or control hmscs were co - cultured with identical volume, e. g., 100 µl, of young or old myof iber fragments with their associated satellite cells ( fig. 5 ). in experiments shown in supplementary fig. s5, culture - conditioned supernatant produced by hescs grown in mcm was used as a medium in which 1 × 105 of myof iber - associated young or old satellite cells were cultured for 48 h. in direct co - cultures, mouse vs. human cells were disting uished by immunode tection with human - specific / hesc - specific and mouse - specific antibod ies ( supplementary fig. s4 and see below ). to prepare muscle supernatants, explants were cultured for 24 h in gm and cellular debris was removed from conditioned media by multiple rounds of centrifugation. the absence of cells was confirmed by microscopic examination. to mimic the local organ niche for satellite cell assays ( fig. 4b ), 1. 0 µm transwell ( corning, ny, usa ) co - cultures of uninjured explants with activated satellite cells were established. activated - by - injury ( 24 h post - injury ) satellite cells were seeded onto ecm - coated 12 - well plates in opti - mem ( invitrogen ) and 5 % fbs. transwells were placed over satellite cells and contained isolated myof iber explants from uninjured young or old muscle ( i. e., resting muscle ). satellite cells were cultured for 72 – 96 h in the presence of myof iber explants and were fixed for immunode tection, as described above. cell transplantationhescs were grown on mefs and expanded in 6 - well plates. cells were treated with 1 mg ml−1 collagenase type iv ( invitrogen ) for 5 – 10 min, were washed and then incubated with 0. 5 mg ml−1 dispase ( invitrogen ) to lift only human cell colonies. isolated hescs were washed several times and resuspended in 100 µl hesc medium. similarly, hmscs were expanded in 6 - well plates, lifted with trypsin / edta ( invitrogen ), washed and resuspended in 100 µl hesc medium. approximately 5 × 105 hescs or hmscs were injected into 24 h post - injured gastrocnemius and tibialis anterior muscles of young and old mice, using a 21 - gauge needle. immunosupp ression of animals was achieved by intraperitoneal injection of 1 mg kg−1 fk506 ( sigma ) at 48 h prior to cell transplantation, and on each day following transplantation.