Ex vivo antioxidant preconditioning improves the survival rate of bone marrow stem cells in the presence of wound fluid.

The advancement of autologous mesenchymal stem cell (MSC) therapy for the treatment of non-healing diabetic wounds is hampered by endogenous MSC dysfunction and limited viability of cells post-transplantation into the pathological wound environment. The development of effective strategies to restore the functional capabilities of these impaired MSCs prior to transplantation may be a key to their ultimate success as wound repair mediators. The current study therefore investigated whether antioxidant preconditioning [7.5 mM N-acetylcysteine (NAC) + 0.6 mM ascorbic 2-phosphate (AAP)] could restore the growth rate, migration ability and viability of impaired MSCs and whether this restored state is maintained in the presence of diabetic wound fluid (DWF). Healthy control (source: wild type, C57BL/6J mice) (n = 12) and impaired/diabetic MSCs (source: obese prediabetic, B6.Cg-Lepob/J mice) (n = 12) were isolated from the bone marrow of mice. Treatment groups post-isolation were as follow: (a) No treatment (baseline phenotype): MSCs expanded in standard growth media (SGM) (±8 days) and only exposed to growth media. (b) DWF (baseline response): MSCs expanded in SGM (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). (c) Antioxidant preconditioning (preconditioned phenotype): MSCs expanded in the presence of NAC/AAP (±8 days). (d) Antioxidant preconditioning + DWF (preconditioned response): MSCs expanded in the presence of NAC/AAP (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). The results demonstrated that expansion of MSCs (both healthy control and impaired diabetic) in the presence of combined NAC/AAP treatment improved ex vivo MSC viability and protected MSCs in the presence of DWF. Despite improved viability, AAP/NAC could however not rescue the reduced proliferation and migration capacity of impaired diabetic MSCs. The protective effect of NAC/AAP preconditioning against the toxicity of DWF could however be a potential strategy to improve cell number post-transplantation.

In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair.

Skeletal muscle repair requires the migration of myoblasts (activated satellite cells) both to the injury site and then within the wound to facilitate cellular alignment in preparation for differentiation, fusion and eventual healing. Along this journey, the cells encounter a range of soluble and extracellular matrix factors which regulate their movement and ultimately determine how successful the repair process will be. Sub-optimal migration can lead to a number of scenarios, including reduced myoblast numbers entering the wound, poor alignment and insufficient differentiation to correctly repair the damage. It is therefore critical that all aspects of myoblast migration are understood, particularly in response to the changing growth and matrix factor profile prevalent following skeletal muscle injury. Since 1962, when Boyden first introduced his chemotactic chamber, numerous in vitro migration assays have been developed to mimic the wound more closely. These have increased in complexity to account for the complex micro-environment found in vivo during muscle repair and include a range of modified cell exclusion, chemotactic and three-dimensional assays. This review describes and discusses these advances and highlights the importance they have in expanding our understanding of myoblast migration dynamics.

Satellite cell pool expansion is affected by skeletal muscle characteristics.

INTRODUCTION: We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type. METHODS: Participants completed a downhill running (DHR) intervention (5 × 8 min, 2-min rest; 80% VO2max ; -10% gradient). Muscle biopsies were taken 7 days before VO2max and 7-9 days after the DHR intervention. Delayed-onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post-DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis. RESULTS: DOMS and CK peaked on day 1 post-DHR (P < 0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO2max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7(+) cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06). CONCLUSION: Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO2max .

A regenerative approach to the pharmacological management of hard-to-heal wounds.

A wound is considered hard-to-heal when, despite the appropriate clinical analysis and intervention, the wound area reduces by less than a third at four weeks and complete healing fails to occur within 12 weeks. The most prevalent hard-to-heal wounds are associated with underlying metabolic diseases or vascular insufficiency and include arterial, venous, pressure and diabetic foot ulcers. Their common features include an abnormal immune response and extended inflammatory phase, a subdued proliferation phase due to cellular insufficiencies and finally an almost non-existent remodeling phase. Advances in wound care technology, tested in both pre-clinical models and clinical trials, have paved the way for improved treatment options, focused on regeneration. These interventions have been shown to limit the extent of ongoing inflammatory damage, decrease bacterial load, promote angiogenesis and deposition of granulation tissue, and stimulate keratinocyte migration thereby promoting re-epithelialization in these wounds. The current review discusses these hard-to-heal wounds in the context of their underlying pathology and potential of advanced treatment options, which if applied promptly as a standard of care, could reduce morbidity, promote quality of life, and alleviate the burden on a strained health system.

TGF-ß isoforms inhibit IGF-1-induced migration and regulate terminal differentiation in a cell-specific manner.

Following muscle injury, the damaged tissue and influx of inflammatory cells stimulate the secretion of growth factors and cytokines to initiate repair processes. This release of chemotactic signaling factors activates resident precursor cells and stimulates their mobilization and migration to the site of injury where terminal differentiation can occur. The three transforming growth factor-ß (TGF-ß) isoforms, and insulin-like growth factor-1 (IGF-1) are among the known regulatory factors released following muscle damage. We investigated the effect of recombinant active TGF-ß1, -ß2, -ß3 and IGF-1 on C2C12 skeletal muscle satellite cell and P19 embryonal carcinoma cell terminal differentiation and migration. C2C12 myoblast fusion as well as P19 embryoid body formation and myogenic differentiation was assessed following 72 h TGF-ß treatment (5 ng/ml), whereas the effect of the TGF-ß isoforms on migration was determined following 7 h incubation. Our results showed that TGF-ß decreases C2C12 myoblast fusion in an isoform-independent manner, whereas in the P19 cell lineage, results demonstrate that TGF-ß1 specifically and significantly increased P19 embryoid body formation, but not expression of Connexin-43 or Myosin Heavy Chain. IGF-1 significantly increased migration compared to TGF-ß isoforms, which, on their own, had no significant effect on the mobilization of either C2C12 or P19 cells. TGF-ß isoforms decreased IGF-1-induced migration of both cell lineages. By distinguishing the factors involved in, and the molecular signals required for, myoblast recruitment during repair processes, strategies can be developed towards improved cell-mediated therapies for muscle injury.

Co-culture of pro-inflammatory macrophages and myofibroblasts: Evaluating morphological phenotypes and screening the effects of signaling pathway inhibitors.

Skeletal muscle regeneration is a complex process influenced by non-myogenic macrophages and fibroblasts, which acquire different phenotypes in response to changes in the injury milieu or changes in experimental conditions. In vitro, serum stimulates the differentiation of fibroblasts into myofibroblasts, while lipopolysaccharide (LPS) stimulates the polarization of unstimulated (M0) macrophages to acquire an M1 pro-inflammatory phenotype. We characterized these phenotypes using morphology (with circularity as shape descriptor; perfect circularity = 1.0) and phenotype-specific markers. Myofibroblasts (high α-smooth muscle actin [SMA] expression) had high circularity (mean 0.60 ± 0.03). Their de-differentiation to fibroblasts (low α-SMA expression) significantly lessened circularity (0.47 ± 0.01 and 0.35 ± 0.02 in 2% or 0% serum culture media respectively (p < 0.05). Unstimulated (M0) macrophages (no CD86 expression) had high circularity (0.72 ± 0.02) which decreased when stimulated to M1 macrophages (CD86 expression) (LPS; 0.61 ± 0.02; p < 0.05). Utilizing these established conditions, we then co-cultured M1 macrophages with myofibroblasts or myoblasts. M1 macrophages significantly decreased relative myofibroblast numbers (from 223 ± 22% to 64 ± 7%), but not myoblast numbers. This pro-inflammatory co-culture model was used to rapidly screen the following four compounds for ability to prevent M1 macrophage-mediated decrease in myofibroblast numbers: L-NAME (inducible nitric oxide synthase inhibitor), SB203580 (p38 mitogen-activated protein kinase inhibitor), SP600125 (c-Jun N-terminal kinase inhibitor) and LY294002 (phosphoinositide 3-kinase [PI3K] inhibitor). We found that LY294002 rescued myofibroblasts and decreased macrophage numbers. Myofibroblast rescue did not occur with L-NAME, SB203580 or SP600125 incubation. In conclusion, these data suggest a PI3K-associated mechanism whereby myofibroblasts can be rescued, despite simulated pro-inflammatory conditions.

TGF-beta's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner.

Satellite cells are a quiescent heterogeneous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-beta (TGF-beta) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-beta on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-beta1, -beta2 and -beta3 on wound repair in other tissues. In the current study we compared the effect of TGF-beta1, -beta2 and -beta3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-beta increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-beta1, -beta2 and -beta3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-beta promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner.

Simple silicone chamber system for in vitro three-dimensional skeletal muscle tissue formation.

Bioengineering skeletal muscle often requires customized equipment and intricate casting techniques. One of the major hurdles when initially trying to establish in vitro tissue engineered muscle constructs is the lack of consistency across published methodology. Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts. Differences in cell type, number and density, variability in matrix and scaffold usage as well as inconsistency in the distance between and type of adhesion posts complicates initial establishment of the technique with confidence. We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space. Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

Potential myogenic stem cell populations: sources, plasticity, and application for cardiac repair.

The ability of unspecialized stem cells to differentiate into mature, specialized cell types has made them attractive as potential agents for enhanced tissue repair and regenerative medicine. This is especially true of diseases and disorders for which no or only partially effective treatments are currently available. Recently, increased focus has been placed on the regenerative potential of satellite cells (myogenic precursor cells found in the adult skeletal muscle) in various muscular disorders, such as dystrophy and myocardial injury following ischemia. Animal studies and clinical trials are in progress using satellite cells as cellular candidates; however, this early rollout in the clinical setting has deflected attention from the potential of other less specialized, but potentially more maliable, stem cell sources. Published data is still lacking on the best methods for identification, isolation, and further expansion or nuclear manipulation of these cells in vitro. Also, although differentiation capacity has been proven in terms of protein expression patterns characteristic of myogenesis, proof of contractile and energetic compatibility between graft and host is more difficult to establish. In this regard, although future animal model studies will be invaluable, they must be designed with short- and long-term functional outcomes in mind. This review moves beyond initial excitement regarding the acknowledged potential of cell therapy and provides a realistic exposition of the themes and specific issues that should be considered in current experimental research study designs.

The changing AMPK expression profile in differentiating mouse skeletal muscle myoblast cells helps confer increasing resistance to apoptosis.

AMP-activated protein kinase (AMPK) functions as a alpha/beta/gamma heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms alpha2 and gamma2 are constitutively expressed, while those for alpha1, beta1 and beta2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the gamma3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic alpha subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 alpha subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated alpha1/alpha2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part, the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-alpha] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into account.

Old dogmas and new hearts: a role for adult stem cells in cardiac repair?

The vast developmental repertoire of embryonic stem cells is well recognised. These primitive stem cells can differentiate in vivo and in vitro into cells of all three embryonic germ layers (endoderm, mesoderm, ectoderm), making them attractive potential agents to target for enhanced tissue repair and regeneration. Adult stem cells on the other hand are considered more restricted in their lineage differentiation capabilities. Recent research has challenged this dogma with the finding that bone marrow-derived stem cells can differentiate into a wide variety of cell types including muscle (skeletal and cardiac). Furthermore, although the myocardium has for decades been regarded as a post-mitotic organ, a series of studies has indicated that a population of stem cells exists which is capable of at least partial reconstitution of the myocardium following an ischaemic insult. It is therefore now accepted that adult stem cells could be used to enhance myocardial repair. This review discusses the current status of adult stem cell research in the light of its potential for improving myocardial repair.