Regenerative responses of adult muscle stem cells and hESCs are dominantly inhibited by the aged systemic milieu
Previous 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). Myofiber 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 myofiber 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. 1  The 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 immunofluorescence microscopy, using anti-BrdU (red), antidesmin (green) or anti-Myf5 antibodies (green, small panels). Similar results are shown for Pax7 immunodetection (Supplementary Fig. S1). Hoechst (blue) labeled nuclei. (B) Three independent experiments were quantified [300 young myofibers 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. 2  The 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. Immunodetection assays were performed for BrdU (red), Oct4 (red), and Ki67 (Supplementary Fig. S2). Hoechst (blue) labels nuclei. 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). 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. 3  Embryonic 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. Immunodetection 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. 4  Aged muscle niche inhibits the regenerative potential of hESCs and satellite cells. (A) Immunodetection 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 myofibers. 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 myofibers on the rate of hESC proliferation, transient, 2 h BrdU incorporation was examined in hESCs cultured for 48 h with supernatants produced by heterochronic myofiber explants (See Experimental procedures for details). As compared to young myofiber-derived supernatants (young myofiber supernant), exposure to old myofiber-derived supernatants (old myofiber supernant) inhibited hESCs proliferation, as judged by BrdU immunodetection (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 myofiber 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 myofibers isolated from uninjured young (young myofiber) and old (old myofiber) 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 (immunodetection shown in green) and by rate of proliferation (2 h BrdU incorporation; immunodetection 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 myofibers (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 1  Conservation of stem cell aging in the systemic environment   Quantified 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).

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