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Aging and age‐associated disease are a major medical and societal burden in need of effective treatments. Cellular reprogramming is a biological process capable of modulating cell fate and cellular age. Harnessing the rejuvenating benefits without altering cell identity via partial cellular reprogramming has emerged as a novel translational strategy with therapeutic potential and strong commercial interests. Here, we explore the aging‐related benefits of partial cellular reprogramming while examining limitations and future directions for the field. Partial cellular reprogramming is able to improve or restore multiple age‐related phenotypes including lifespan, healthspan, epigenetic age, aging hallmarks, and tissue regeneration.

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity. Aging involves gradual loss of tissue function, and transcription factor (TF) expression can ameliorate this in progeroid mice. Here the authors show that transient TF expression reverses age-associated epigenetic marks, inflammatory profiles and restores regenerative potential in naturally aged human cells.

Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML. Volumetric muscle loss leads to functional muscle impairment, and current stem cell-based treatments show limited efficacy. Here, the authors generate a stem cell scaffold, implant it in mice, and show that an exercise regimen enhances innervation and restoration of muscle function in mice.

The dedifferentiation of somatic cells into a pluripotent state by cellular reprogramming coincides with a reversal of age-associated molecular hallmarks. Although transcription factor induced cellular reprogramming has been shown to ameliorate these aging phenotypes in human cells and extend health and lifespan in mice, translational applications of this approach are still limited. More recently, chemical reprogramming via small molecule cocktails have demonstrated a similar ability to induce pluripotency in vitro, however, its potential impact on aging is unknown. Here, we demonstrated that chemical-induced partial reprogramming can improve key drivers of aging including genomic instability and epigenetic alterations in aged human cells. Moreover, we identified an optimized combination of two reprogramming molecules sufficient to induce the amelioration of additional aging phenotypes including cellular senescence and oxidative stress. Importantly, in vivo application of this two-chemical combination significantly extended C. elegans lifespan and healthspan. Together, these data demonstrate that improvement of key drivers of aging and lifespan extension is possible via chemical-induced partial reprogramming, opening a path towards future translational applications. Synopsis This study reveals that partial chemical reprogramming with defined small-molecule cocktails rejuvenates aged human cells and significantly extends lifespan and healthspan in C. elegans, offering a non-genetic strategy to reverse aging phenotypes. A seven-compound (7c) reprogramming cocktail was shown to reverse multiple aging hallmarks in human dermal fibroblasts. A reduced two-compound (2c) cocktail was identified that retained rejuvenating effects in vitro and was sufficient to ameliorate additional aging phenotypes, including senescence, heterochromatin loss, genomic instability, and oxidative stress. 2c treatment in C. elegans improved stress resistance, thermotolerance, reproductive and healthspan markers, and extended median lifespan by over 42%. The results suggest that partial chemical reprogramming could modulate the underlying mechanisms of aging and reproductive aging, offering a potential strategy for extending both healthspan and overall lifespan in aging populations. This study reveals that partial chemical reprogramming with defined small-molecule cocktails rejuvenates aged human cells and significantly extends lifespan and healthspan in C. elegans, offering a non-genetic strategy to reverse aging phenotypes.

Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation. Karina Nakayama et al. bioengineer endothelialized mouse skeletal muscle using parallel-aligned nanofibrillar scaffolds. They find that muscle produced using the aligned scaffolds developed a spatially patterned structure and showed improved endothelial interaction compared to muscle engineered with randomly-oriented scaffolds.

Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.

Despite the regenerative capacity of muscle, tissue volume is not restored after volumetric muscle loss (VML), perhaps due to a loss-of-structural extracellular matrix. We recently demonstrated the structural and functional restoration of muscle tissue in a mouse model of VML using an engineered “bioconstruct,” comprising an extracellular matrix scaffold (decellularized muscle), muscle stem cells (MuSCs), and muscle-resident cells (MRCs). To test the ability of the cell-based bioconstruct to restore whole-muscle biomechanics, we measured biomechanical parameters in uninjured muscles, muscles injured to produce VML lesions, and in muscles that were injured and then treated by implanting either the scaffolds alone or with bioconstructs containing the scaffolds, MuSCs, and MRCs. We measured the active and passive forces over a range of lengths, viscoelastic force relaxation, optimal length, and twitch dynamics. Injured muscles showed a narrowed length-tension curve or lower force over a narrower range of muscle lengths, and increased passive force. When treated with bioconstructs, but not with scaffolds alone, injured muscles showed active and passive length-tension relationships that were not different from uninjured muscles. Moreover, injured muscles treated with bioconstructs exhibited reduced fibrosis compared to injured muscles either untreated or treated with scaffolds alone. The cell-based bioconstruct is a promising treatment approach for future translational efforts to restore whole-muscle biomechanics in muscles with VML lesions.

Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

The dedifferentiation of somatic cells into a pluripotent state by cellular reprogramming coincides with a reversal of age-associated molecular hallmarks. Although transcription factor induced cellular reprogramming has been shown to ameliorate these aging phenotypes in human cells and extend health and lifespan in mice, translational applications of this approach are still limited. More recently, chemical reprogramming via small molecule cocktails have demonstrated a similar ability to induce pluripotency in vitro, however, its potential impact on aging is unknown. Here, we demonstrated that partial chemical reprogramming is able to improve key drivers of aging including genomic instability and epigenetic alterations in aged human cells. Moreover, we identified an optimized combination of two reprogramming molecules sufficient to induce the amelioration of additional aging phenotypes including cellular senescence and oxidative stress. Importantly, in vivo application of this two-chemical combination significantly extended C. elegans lifespan. Together, these data demonstrate that improvement of key drivers of aging and lifespan extension is possible via chemical induced partial reprogramming, opening a path towards future translational applications.

Unlike aged somatic cells, which exhibit a decline in molecular fidelity and eventually reach a state of replicative senescence, pluripotent stem cells can indefinitely replenish themselves while retaining full homeostatic capacity. The conferment of beneficial-pluripotency related traits via in vivo partial cellular reprogramming (IVPR) significantly extends lifespan and restores aging phenotypes in mouse models. Although the phases of cellular reprogramming are well characterized, details of the rejuvenation processes are poorly defined. To understand whether epigenetic reprogramming can ameliorate DNA damage, we created reprogrammable accelerated aging mouse model with an ERCC1 mutation. Importantly, using enhanced partial reprogramming by combining small molecules with the Yamanaka factors, we observed potent reversion of DNA damage, significant upregulation of multiple DNA damage repair processes, and restoration of the epigenetic clock. In addition, we present evidence that pharmacological inhibition of ALK5 and ALK2 receptors in TGFb pathway is able to phenocopy some benefits including epigenetic clock restoration suggesting a role in the mechanism of rejuvenation by partial reprogramming.