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.
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.
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.
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.
•Four weeks of supplementation with nicotinamide riboside enhances muscular performance and open-field activity in middle-aged mice.•Nicotinamide riboside increases slow-twitch fiber cross-sectional area, but 4 wk of supplementation was not sufficient to increase mitochondrial biomass.•Nicotinamide riboside treatment improves the differentiating capacity of both mouse and human myogenic progenitors to form multinucleated myofibers in vitro.•The addition of nicotinamide riboside boosts both aerobic and anaerobic respiration in mouse and human myogenic progenitors as well as in human myotubes in vitro.
Abstract We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a ...surface consisting of heparin bound to poly- l -lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2 ) to physiological (15 dyne/cm2 ). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.
Abstract
Neural crest-like stem cells resembling embryonic neural crest cells (NCs) can be derived from adult human tissues such as the epidermis. However, these cells lose their multipotency rapidly ...in culture limiting their expansion for clinical use. Here, we show that the multipotency of keratinocyte-derived NCs (KC-NCs) can be preserved by activating the Wnt and BMP signaling axis, promoting expression of key NC-specifier genes and ultimately enhancing their differentiation potential. We also show that transcriptional changes leading to multipotency are linked to metabolic reprogramming of KC-NCs to a highly glycolytic state. Specifically, KC-NCs treated with CHIR and BMP2 rely almost exclusively on glycolysis for their energy needs, as seen by increased lactate production, glucose uptake, and glycolytic enzyme activities. This was accompanied by mitochondrial depolarization and decreased mitochondrial ATP production. Interestingly, the glycolytic end-product lactate stabilized β-catenin and further augmented NC-gene expression. Taken together, our study shows that activation of the Wnt/BMP signaling coordinates the metabolic demands of neural crest-like stem cells governing decisions regarding multipotency and differentiation, with possible implications for regenerative medicine.
Graphical Abstract
Graphical Abstract
Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and ...identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate that the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG binding protein MBD2, whose isoforms play opposing roles in maintenance of and reprogramming to pluripotency. Although both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity in support of hPSC self-renewal and reprogramming.
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•The splicing factor SFRS2 supports human PSC self-renewal and is a target of OCT4•SFRS2 mediates splicing of MBD2 isoforms that play opposing roles in pluripotency•MBD2a, but not MBD2c, can interact with the NuRD chromatin remodeling complex•OCT4, SFRS2, and MBD2 comprise a positive feedback loop in human PSCs
Alternative splicing plays a central role in regulating pluripotency, and the splicing factor SFRS2, under the control of OCT4, generates splice variants of MDB2 that have opposing roles in maintaining self-renewal and reprogramming.
The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle (HF) is a readily ...accessible mini organ within the skin and contains stem cells from diverse developmental origins that were shown to have surprisingly broad differentiation potential. In this review, we discuss the biology of the HF with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative HF stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of HF stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility makes the HF an ideal source of stem cells for gene and cell therapies.
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
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.
We present a strategy to conjugate TGF- beta 1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF- ...beta 1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF- beta 1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF- beta 1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF- beta 1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF- beta 1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF- beta 1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.
Abstract Representing a new type of biodegradable cationic block copolymer, well-defined poly(ethylene glycol)- block -cationic polylactides (PEG- b -CPLAs) with tertiary amine-based cationic groups ...were synthesized by thiol-ene functionalization of an allyl-functionalized diblock precursor. Subsequently the application of PEG- b -CPLAs as biodegradable vectors for the delivery of plasmid DNAs (pDNAs) was investigated. Via the formation of PEG- b -CPLA:pDNA nanocomplexes by spontaneous electrostatic interaction, pDNAs encoding luciferase or enhanced green fluorescent protein were successfully delivered to four physiologically distinct cell lines (including macrophage, fibroblast, epithelial, and stem cell). Formulated nanocomplexes demonstrated high levels of transfection with low levels of cytotoxicity and hemolysis when compared to a positive control. Biophysical characterization of charge densities of nanocomplexes at various polymer:pDNA weight ratios revealed a positive correlation between surface charge and gene delivery. Nanocomplexes with high surface charge densities were utilized in an in vitro serum gene delivery inhibition assay, and effective gene delivery was observed despite high levels of serum. Overall, these results help to elucidate the influence of charge, size, and PEGylation of nanocomplexes upon the delivery of nucleic acids in physiologically relevant conditions.
Abstract We investigated fibrin-mediated gene transfer by embedding pDNA within the hydrogel during polymerization and using two modes of gene transfection with cells placed either on the surface (2D ...transfection) or within the hydrogel (3D transfection). Using this model, we found that cell transfection depended strongly on the local cell–pDNA microenvironment as defined by the 2D vs. 3D context, target cell type and density, as well as fibrinogen and pDNA concentrations. When cells were embedded within the fibrin matrix lipofectamine-induced cell death decreased significantly, especially at low target cell density. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner, suggesting that fibrin degradation may be necessary for efficient gene transfer. We also provided proof-of-concept that fibrin-mediated gene transfer can be used for spatially localized gene delivery, which is required in cell-transfection microarrays. When lipoplex-containing hydrogels were spotted in an array format gene transfer was strictly confined to pDNA-containing fibrin spots with no cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may be used as a biomaterial to deliver genes in an efficient, cell-controlled and spatially localized manner for potential applications in vitro or in vivo.
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.
Our results suggest that short‐term treatment of senescent myoblasts with reversine could restore insulin resistance, enhance glucose metabolism and oxidative phosphorylation, likely via reactivation of autophagy. Restoring DNA damage and the state of heterochromatin preceded restoration of proliferation, SA‐β‐Gal expression, and cell size in reversine‐treated cells, ultimately restoring the differentiation ability of myoblasts to form myofibers. Therefore, reversine may have the potential to be used as a novel, anti‐aging treatment, without the tumorigenic complications of genetic reprogramming technologies.
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