Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.

Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated ß-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.

Skeletal muscle reprogramming enhances reinnervation after peripheral nerve injury.

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. The onset of PNI is characterized by nerve degeneration distal to the nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generated a mouse model in which NANOG, a pluripotency-associated transcription factor can be expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulated the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression led to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation, and downregulation of key muscle atrophy genes. Further, NANOG mice demonstrated extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice showed greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming the muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI.

Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle.

We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy.

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.

Inhibition of glutaminolysis restores mitochondrial function in senescent stem cells.

Mitochondrial dysfunction, a hallmark of aging, has been associated with the onset of aging phenotypes and age-related diseases. Here, we report that impaired mitochondrial function is associated with increased glutamine catabolism in senescent human mesenchymal stem cells (MSCs) and myofibroblasts derived from patients suffering from Hutchinson-Gilford progeria syndrome. Increased glutaminase (GLS1) activity accompanied by loss of urea transporter SLC14A1 induces urea accumulation, mitochondrial dysfunction, and DNA damage. Conversely, blocking GLS1 activity restores mitochondrial function and leads to amelioration of aging hallmarks. Interestingly, GLS1 expression is regulated through the JNK pathway, as demonstrated by chemical and genetic inhibition. In agreement with our in vitro findings, tissues isolated from aged or progeria mice display increased urea accumulation and GLS1 activity, concomitant with declined mitochondrial function. Inhibition of glutaminolysis in progeria mice improves mitochondrial respiratory chain activity, suggesting that targeting glutaminolysis may be a promising strategy for restoring age-associated loss of mitochondrial function.

Well-Defined pH-Responsive Self-Assembled Block Copolymers for the Effective Codelivery of Doxorubicin and Antisense Oligonucleotide to Breast Cancer Cells.

The worldwide steady increase in the number of cancer patients motivates the development of innovative drug delivery systems for combination therapy as an effective clinical modality for cancer treatment. Here, we explored a design concept based on poly(ethylene glycol)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(2-hydroxyethyl methacrylate-formylbenzoic acid) [PEG-b-PDMAEMA-b-P(HEMA-FBA)] for the dual delivery of doxorubicin (DOX) and GTI2040 (an antisense oligonucleotide for ribonucleotide reductase inhibition) to MCF-7 breast cancer cells. PEG-b-PDMAEMA-b-PHEMA, the precursor copolymer, was prepared through chain extensions from a PEG-based macroinitiator via two consecutive atom transfer radical polymerization (ATRP) steps. Then, it was modified at the PHEMA block with 4-formylbenzoic acid (FBA) to install reactive aldehyde moieties. A pH-responsive polymer-drug conjugate (PDC) was obtained by conjugating DOX to the polymer structure via acid-labile imine linkages, and subsequently self-assembled in an aqueous solution to form DOX-loaded self-assembled nanoparticles (DOX-SAN) with a positively charged shell. DOX-SAN condensed readily with negatively charged GTI2040 to form GTI2040/DOX-SAN nanocomplexes. Gel-retardation assay confirmed the affinity between GTI2040 and DOX-SAN. The GTI2040/DOX-SAN nanocomplex at N/P ratio of 30 exhibited a volume-average hydrodynamic size of 136.4 nm and a zeta potential of 21.0 mV. The pH-sensitivity of DOX-SAN was confirmed by the DOX release study based on the significant cumulative DOX release at pH 5.5 relative to pH 7.4. Cellular uptake study demonstrated favorable accumulation of GTI2040/DOX-SAN inside MCF-7 cells compared with free GTI2040/DOX. In vitro cytotoxicity study indicated higher therapeutic efficacy of GTI2040/DOX-SAN relative to DOX-SAN alone because of the downregulation of the R2 protein of ribonucleotide reductase. These outcomes suggest that the self-assembled pH-responsive triblock copolymer is a promising platform for combination therapy, which may be more effective in combating cancer than individual therapies.

Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo.

Senescence of myogenic progenitors impedes skeletal muscle regeneration. Here, we show that overexpression of the transcription factor NANOG in senescent myoblasts can overcome the effects of cellular senescence and confer a youthful phenotype to senescent cells. NANOG ameliorated primary hallmarks of cellular senescence including genomic instability, loss of proteostasis, and mitochondrial dysfunction. The rejuvenating effects of NANOG included restoration of DNA damage response via up-regulation of DNA repair proteins, recovery of heterochromatin marks via up-regulation of histones, and reactivation of autophagy and mitochondrial energetics via up-regulation of AMP-activated protein kinase (AMPK). Expression of NANOG in the skeletal muscle of a mouse model of premature aging restored the number of myogenic progenitors and induced formation of eMyHC+ myofibers. This work demonstrates the feasibility of reversing the effects of cellular senescence in vitro and in vivo, with no need for reprogramming to the pluripotent state.

Short-term nicotinamide riboside treatment improves muscle quality and function in mice and increases cellular energetics and differentiating capacity of myogenic progenitors.

OBJECTIVES: Nicotinamide adenine dinucleotide (NAD+), an essential cofactor for mitochondrial function, declines with aging, which may lead to impaired physical performance. Nicotinamide riboside (NR), a NAD+ precursor, restores cellular NAD+ levels. The aim of this study was to examine the effects of short-term NR supplementation on physical performance in middle-aged mice and the effects on mouse and human muscle stem cells. METHODS: We treated 15-mo-old male C57BL/6J mice with NR at 300 mg·kg·d-1 (NR3), 600 mg·kg·d-1 (NR6), or placebo (PLB), n = 8 per group, and assessed changes in physical performance, muscle histology, and NAD+ content after 4 wk of treatment. RESULTS: NR increased total NAD+ in muscle tissue (NR3 P = 0.01; NR6 P = 0.004, both versus PLB), enhanced treadmill endurance and open-field activity, and prevented decline in grip strength. Histologic analysis revealed NR-treated mice exhibited enlarged slow-twitch fibers (NR6 versus PLB P = 0.014; NR3 P = 0.16) and a trend toward more slow fibers (NR3 P = 0.14; NR6 P = 0.22). We next carried out experiments to characterize NR effects on mitochondrial activity and cellular energetics in vitro. We observed that NR boosted basal and maximal cellular aerobic and anaerobic respiration in both mouse and human myoblasts and human myotubes. Additionally, NR treatment improved the differentiating capacity of myoblasts and increased myotube size and fusion index upon stimulation of these progenitors to form multinucleated myotubes. CONCLUSION: These findings support a role for NR in improving cellular energetics and functional capacity in mice, which support the translation of this work into clinical settings as a strategy for improving and/or maintaining health span during aging.

Fast Stereolithography Printing of Large-Scale Biocompatible Hydrogel Models.

Large size cell-laden hydrogel models hold great promise for tissue repair and organ transplantation, but their fabrication using 3D bioprinting is limited by the slow printing speed that can affect the part quality and the biological activity of the encapsulated cells. Here a fast hydrogel stereolithography printing (FLOAT) method is presented that allows the creation of a centimeter-sized, multiscale solid hydrogel model within minutes. Through precisely controlling the photopolymerization condition, low suction force-driven, high-velocity flow of the hydrogel prepolymer is established that supports the continuous replenishment of the prepolymer solution below the curing part and the nonstop part growth. The rapid printing of centimeter-sized hydrogel models using FLOAT is shown to significantly reduce the part deformation and cellular injury caused by the prolonged exposure to the environmental stresses in conventional 3D printing methods. Embedded vessel networks fabricated through multiscale printing allows media perfusion needed to maintain the high cellular viability and metabolic functions in the deep core of the large-sized models. The endothelialization of this vessel network allows the establishment of barrier functions. Together, these studies demonstrate a rapid 3D hydrogel printing method and represent a first step toward the fabrication of large-sized engineered tissue models.

Fast photocurable thiol-ene elastomers with tunable biodegradability, mechanical and surface properties enhance myoblast differentiation and contractile function.

Biodegradable elastomers are important emerging biomaterials for biomedical applications, particularly in the area of soft-tissue engineering in which scaffolds need to match the physicochemical properties of native tissues. Here, we report novel fast photocurable elastomers with readily tunable mechanical properties, surface wettability, and degradability. These elastomers are prepared by a 5-min UV-irradiation of thiol-ene reaction systems of glycerol tripentenoate (GTP; a triene) or the combination of GTP and 4-pentenyl 4-pentenoate (PP; a diene) with a carefully chosen series of di- or tri-thiols. In the subsequent application study, these elastomers were found to be capable of overcoming delamination of myotubes, a technical bottleneck limiting the in vitro growth of mature functional myofibers. The glycerol-based elastomers supported the proliferation of mouse and human myoblasts, as well as myogenic differentiation into contractile myotubes. More notably, while beating mouse myotubes detached from conventional tissue culture plates, they remain adherent on the elastomer surface. The results suggest that these elastomers as novel biomaterials may provide a promising platform for engineering functional soft tissues with potential applications in regenerative medicine or pharmacological testing.

Bioengineered Skeletal Muscle as a Model of Muscle Aging and Regeneration.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

PEGylated Amine-Functionalized Poly(ε-caprolactone) for the Delivery of Plasmid DNA.

As a promising strategy for the treatment of various diseases, gene therapy has attracted increasing attention over the past decade. Among various gene delivery approaches, non-viral vectors made of synthetic biomaterials have shown significant potential. Due to their synthetic nature, non-viral vectors can have tunable structures and properties by using various building units. In particular, they can offer advantages over viral vectors with respect to biosafety and cytotoxicity. In this study, a well-defined poly(ethylene glycol)-block-poly(α-(propylthio-N,N-diethylethanamine hydrochloride)-ε-caprolactone) diblock polymer (PEG-b-CPCL) with one poly(ethylene glycol) (PEG) block and one tertiary amine-functionalized cationic poly(ε-caprolactone) (CPCL) block, as a novel non-viral vector in the delivery of plasmid DNA (pDNA), was synthesized and studied. Despite having a degradable polymeric structure, the polymer showed remarkable hydrolytic stability over multiple weeks. The optimal ratio of the polymer to pDNA for nanocomplex formation, pDNA release from the nanocomplex with the presence of heparin, and serum stability of the nanocomplex were probed through gel electrophoresis. Nanostructure of the nanocomplexes was characterized by DLS and TEM imaging. Relative to CPCL homopolymers, PEG-b-CPCL led to better solubility over a wide range of pH. Overall, this work demonstrates that PEG-b-CPCL possesses a range of valuable properties as a promising synthetic vector for pDNA delivery.

Restoring extracellular matrix synthesis in senescent stem cells.

Collagen type III (COL3) is one of the 3 major collagens in the body, and loss of expression or mutations in the COL3 gene have been associated with the onset of vascular diseases such the Ehlers-Danlos syndrome. Previous work reported a significant reduction of COL3 in tissues such as skin and vessels with aging. In agreement, we found that COL3 was significantly reduced in senescent human mesenchymal stem cells and myofibroblasts derived from patients with Hutchinson-Gilford progeria syndrome, a premature aging syndrome. Most notably, we discovered that ectopic expression of the embryonic transcription factor Nanog homeobox (NANOG) restored COL3 expression by restoring the activity of the TGF-ß pathway that was impaired in senescent cells. RNA sequencing analysis showed that genes associated with the activation of the TGF-ß pathway were up-regulated, whereas negative regulators of the pathway were down-regulated upon NANOG expression. Chromatin immunoprecipitation sequencing and immunoprecipitation experiments revealed that NANOG bound to the mothers against decapentaplegic (SMAD)2 and SMAD3 promoters, in agreement with increased expression and phosphorylation levels of both proteins. Using chemical inhibition, short hairpin RNA knockdown, and gain of function approaches, we established that both SMAD2 and SMAD3 were necessary to mediate the effects of NANOG, but SMAD3 overexpression was also sufficient for COL3 production. In summary, NANOG restored production of COL3, which was impaired by cellular aging, suggesting novel strategies to restore the impaired extracellular matrix production and biomechanical function of aged tissues, with potential implications for regenerative medicine and anti-aging treatments.-Rong, N., Mistriotis, P., Wang, X., Tseropoulos, G., Rajabian, N., Zhang, Y., Wang, J., Liu, S., Andreadis, S. T. Restoring extracellular matrix synthesis in senescent stem cells.

Efficient and high yield isolation of myoblasts from skeletal muscle.

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10-20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.