Discussion
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 myofibers 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 distinct 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 disorders.
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+ myofibers 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 myofibers 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 myofiber-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. 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.