While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the “anti-aging” protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.

Adult stem cells grow poorly in vitro compared to embryonic stem cells, and in vivo stem cell maintenance and proliferation by tissue niches progressively deteriorates with age. We previously reported that factors produced by human embryonic stem cells (hESCs) support a robust regenerative capacity for adult and old mouse muscle stem/progenitor cells. Here we extend these findings to human muscle progenitors and investigate underlying molecular mechanisms. Our results demonstrate that hESC-conditioned medium enhanced the proliferation of mouse and human muscle progenitors. Furthermore, hESC-produced factors activated MAPK and Notch signaling in human myogenic progenitors, and Delta/Notch-1 activation was dependent on MAPK/pERK. The Wnt, TGF-β and BMP/pSmad1,5,8 pathways were unresponsive to hESC-produced factors, but BMP signaling was dependent on intact MAPK/pERK. c-Myc, p57, and p18 were key effectors of the enhanced myogenesis promoted by the hECS factors. To define some of the active ingredients of the hESC-secretome which may have therapeutic potential, a comparative proteomic antibody array analysis was performed and identified several putative proteins, including FGF2, 6 and 19 which as ligands for MAPK signaling, were investigated in more detail. These studies emphasize that a “youthful” signaling of multiple signaling pathways is responsible for the pro-regenerative activity of the hESC factors.

It is well recognized that in vitro differentiation of embryonic stem cells (ESC) can be best achieved by closely recapitulating the in vivo developmental niche. Thus, implementation of directed differentiation strategies has yielded encouraging results in the area of pancreatic islet differentiation. These strategies have concentrated on direct addition of chemical signals, however, other aspect of the developmental niche are yet to be explored. During development, pancreatic progenitor (PP) cells grow as an epithelial sheet, which aggregates with endothelial cells (ECs) during the final stages of maturation. Several findings suggest that the interactions with EC play a role in pancreatic development. In this study, we recapitulated this phenomenon in an in vitro environment by maturing the human ESC (hESC)-derived PP cells in close contact with ECs. We find that co-culture with different ECs (but not fibroblast) alone results in pancreatic islet-specific differentiation of hESC-derived PP cells even in the absence of additional chemical induction. The differentiated cells responded to exogenous glucose levels by enhanced C-peptide synthesis. The co-culture system aligned well with endocrine development as determined by comprehensive analysis of involved signaling pathways. By recapitulating cell-cell interaction aspects of the developmental niche we achieved a differentiation model that aligns closely with islet organogenesis.

Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing 'disorderliness' of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular 'order' and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.

FORTRAN codes for the PI3K/AKT pathway and the RS-HDMR implementation are available from the authors upon request.

Aging is accompanied by a disrupted information flow, which results from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders such as skeletal muscle wasting, or sarcopenia. To estimate the growing “disorderliness” of the aging muscle system, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Although the most prominent structural and functional alterations were observed in the oldest old mice (27-29 months), we found that the escalating network entropy reached an inflection point at old age (22-24 months). To probe the potential for restoration of molecular “order” and reversal of the sarcopenic phenotype, we overexpressed the longevity protein, α-Klotho. Klotho overexpression modulated genes representing all hallmarks of aging in both old and oldest-old mice. However, whereas Klotho improved strength in old mice, intervention failed to induce a benefit beyond the entropic tipping point.

Loss of muscle stem cell (MuSC) self-renewal with aging reflects a combination of influences from the intracellular (e.g., post-transcriptional modifications) and extracellular (e.g., matrix stiffness) environment. Whereas conventional single cell analyses have revealed valuable insights into factors contributing to impaired self-renewal with age, most are limited by static measurements that fail to capture nonlinear dynamics. Using bioengineered matrices mimicking the stiffness of young and old muscle, we showed that while young MuSCs were unaffected by aged matrices, old MuSCs were phenotypically rejuvenated by young matrices. Dynamical modeling of RNA velocity vector fields in silico revealed that soft matrices promoted a self-renewing state in old MuSCs by attenuating RNA decay. Vector field perturbations demonstrated that the effects of matrix stiffness on MuSC self-renewal could be circumvented by fine-tuning the expression of the RNA decay machinery. These results demonstrate that post-transcriptional dynamics dictate the negative effect of aged matrices on MuSC self-renewal.

No abstract