Conceived and designed experiments: JBV SDT MJB MS JH. Performed experiments: JBV SDT MJB MS. Analyzed data: JBV SDT MJB MS. Contributed reagents/materials/analysis tools: JH. Wrote the manuscript: JBV SDT JH.
The authors have read the journal's policy and have the following conflicts: The authors wish to disclose that Dr. Johnny Huard received remuneration as a consultant with Cook MyoSite, Inc. during the performance period of this project. None of the other authors have any potential conflicts of interest to disclose. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.
Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs.
The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.
Duchenne muscular dystrophy is a degenerative muscle disease caused by a mutation of the gene encoding dystrophin, a protein that anchors the myofiber cytoskeleton to the basal lamina, resulting in muscle fiber necrosis and progressive weakness
Numerous myogenic progenitors have been isolated from post-natal murine and human skeletal muscle for cell therapy such as satellite cells, myoblasts, MDSCs, side-population cells, Sk-DN/Sk-34 cells, pericytes, mesangioblasts, human SMALD+ cells, and myo-endothelial cells
Previously we demonstrated the central role that the intracellular antioxidant glutathione (GSH) plays in the increased survival and muscle regenerative capacity of MDSCs. Increased levels of GSH in MDSCs compared to myoblasts was correlated with the increased rates of survival, proliferation, and myogenic differentiation in conditions of oxidative and inflammatory stress
In the current study, we sought to further validate this hypothesis through the isolation of myogenic progenitors with enhanced stress resistance using elevated expression of cytosolic aldehyde dehydrogenase (ALDH1A1) as a marker for this trait. ALDH represents a family of intracellular enzymes that regulate retinoic acid (RA) concentration, which plays an important role in embryonic myogenesis, by driving the expression of multiple myogenic regulatory factors (MRFs) in murine and human embryonic stem cells
Subpopulations of skeletal muscle derived cells that expressed high and low levels of ALDH (ALDHhi and ALDHlo) were isolated from cultured murine and human skeletal muscle by fluorescence activated cell sorting (FACS) as depicted in
Cells with elevated ALDH levels become fluorescent when exposed to boron-dipyrromethene (BODIPY) labeled amino acetaldehyde (Aldefluor, StemCell Technologies) and can be isolated using fluorescence activated cell sorting (FACS). The non-polar BODIPY-aminoacetaldehyde diffuses freely into the cytoplasm and is converted by cytosolic ALDH1A1 to the negatively charged BODIPY-aminoacetate, which accumulates in the cytoplasm and causes the cell to fluoresce with an emission peak at 513 nm
ALDHhi cell subpopulations were isolated from rapidly adhering preplate myoblasts, slowly adhering MDSCs as well as unfractionated hMDCs
Isolation of ALDHlo and ALDHhi subpopulations from cultured myoblasts was performed using FACS. (a) Dead cells were excluded from the isolation by detection of nuclear propidium iodide fluorescence. (b, c) ALDHhi SCClo (high ALDH activity, low side scatter) cells were isolated from a heterogeneous population of myoblasts using DEAB, a potent inhibitor of ALDH, as a gating control. (d) Measurement of the FITC channel signal intensity of Aldefluor stained murine myoblasts and MDSCs demonstrated a shift in the distribution of signal intensity between the two populations, suggesting an increase in the median ALDH activity in MDSCs compared to myoblasts. (e) ALDH sorted murine muscle derived cells were preplated to demonstrate the increased yield of cells in later preplate cycles from ALDHhi cells (up to PP5) compared to ALDHlo cells. Cells were isolated from three mice labeled m1, m2, and m3. (* indicates p<0.05).
We observed an elevated median Aldefluor fluorescence in murine MDSCs compared to myoblasts, despite the absence of a difference in autofluorescence in their untreated controls (data not shown). In addition, MDSCs appear to have a more homogenous elevated ALDH activity than myoblasts, indicating less heterogeneity in ALDH expression. The red overlay in
To verify this trend of increased ALDH activity in slowly adhering MDSCs, the preplate technique was performed using ALDHlo and ALDHhi cells isolated from dissociated murine skeletal muscle (
Myoblasts isolated from rapidly adhering cells (RACs) using the preplate technique have been previously characterized as a heterogeneous population in various states of quiescence, activation, and differentiation
(a, b) ALDHhi murine myoblasts were isolated via fluorescence activated cell sorting (FACS) from a heterogeneous population of skeletal muscle derived cells using DEAB, a potent inhibitor of ALDH, as a gating control. (c, d) A significantly increased rate of proliferation of ALDHhi myoblasts compared to ALDHlo myoblasts was observed in conditions of oxidative stress (H2O2, 250 µM) (* indicates p<0.05) and inflammatory stress conditions (TNF-α, 2.5 ng/ml) (n = 9). (e) ALDHlo and ALDHhi myoblasts underwent myogenic differentiation by fusing into MHC+ myotubes (red) under oxidative stress conditions (H2O2, 250 µM). Nuclei were stained with DAPI (blue). (f) Significantly increased myogenic differentiation indices (MDI) were observed in ALDHhi cells at several concentrations of oxidative stress (0, 250 and 500 µM) when compared to ALDHlo (n = 3). (g) Similarly increased MDIs of ALDHhi cells were observed under all inflammatory stress conditions when compared to ALDHlo myoblasts. (h) An increased number and density of dystrophin positive myofibers (stained in red) were observed in
Following FACS isolation of ALDHlo and ALDHhi subpopulations from the RACs, we quantified the oxidative (hydrogen peroxide or H2O2) and inflammatory (TNF-α) stress resistance of these subpopulations during proliferation and myogenic differentiation
Significant differences in stress resistance capacity were observed between ALDHlo and ALDHhi subpopulations of murine myoblasts in terms of proliferation and myogenic differentiation. The quantification of proliferation rates of ALDHlo and ALDHhi myoblasts was performed using live cell imaging microscopy in conditions of oxidative and inflammatory stress. This study showed a significant proliferation advantage by the ALDHhi cells compared to ALDHlo cells (
ALDHhi and ALDHlo murine myoblasts were injected intramuscularly into the gastrocnemius of
To further characterize the increased stress resistance capacity of the ALDHhi myoblasts, we examined two major intracellular antioxidants, GSH and SOD. Significantly increased concentrations of GSH and increased activity of SOD was observed in ALDHhi myoblasts compared to ALDHlo myoblasts using spectrophotometric assays (
To further elucidate the role of intracellular antioxidant levels in the stress resistance capacity of ALDH sorted myoblasts, we altered the cells antioxidant levels prior to oxidative stress challenge. ALDHlo myoblasts were treated with a mitochondrial targeted antioxidant, XJB-5-131 (XJB) prior to exposure to hydrogen peroxide to determine whether the proliferation rate could be elevated to that of ALDHhi cells
Alteration of the antioxidant levels of these ALDH subpopulations had a significant impact on their proliferation capacities in oxidative stress. In contrast to the decreased proliferative capacity of ALDHlo murine myoblasts compared to ALDHhi myoblasts seen in
(a) When the antioxidant levels of ALDHhi myoblasts were decreased by treatment with diethyl maleate (DEM), their proliferation rate was decreased to that of ALDHlo myoblasts (n = 5). (* indicates p<0.05 at a given timepoint.) (b) By increasing the antioxidant levels of ALDHlo cells with XJB prior to oxidative stress (H2O2, 250 µM) exposure, the proliferation rate in oxidative stress was increased to the levels observed in ALDHhi myoblasts. (c) DEAB (50 µM) treatment of ALDHhi murine myoblasts significantly decreased their proliferative rate in the absence of oxidative stress compared to untreated ALDHhi myoblasts. (d) However, DEAB treatment had no statistically significant effect on the oxidative stress (H2O2, 250 µM) resistance of ALDHhi myoblasts in terms of proliferation with the exception of an isolated data point at 24 hrs (p = 0.034).
Given the role of antioxidant activity in the stress resistance of ALDHhi myoblasts, we questioned whether the ALDH enzyme was directly participating in the oxidative stress resistance of ALDHhi myoblasts. ALDH has been directly implicated in the mitigation of oxidative damage by converting aldehyde by-products of lipid peroxidation such as malenaldehyde to non-reactive carboxylic acids
It is well known that RA, a product of ALDH mediated oxidation of retinal, is required for embryonic myogenesis and can accelerate differentiation in postnatal derived myoblasts
We studied the chondrogenic and osteogenic differentiation of ALDH sorted myoblasts and found that, ALDHhi myoblasts demonstrate a greater capacity to undergo chondrogenic and osteogenic differentiation in vitro when compared to ALDHlo myoblasts (
(a–c) Chondrogenic differentiation in ALDH sorted myoblasts was induced via a pellet system containing TGF. Rapid proliferation and chondrogenic pellet formation was observed in ALDHhi myoblasts (24 hrs) with robust production of glycosaminoglycans (GAGs), visualized via Alcian blue staining. This is in contrast to the poor proliferation and GAG production of ALDHlo myoblasts. This result suggests that the chondrogenic differentiation capacity of unsorted myoblasts is dominated by ALDHhi myoblasts. (d–f) Osteogenic differentiation of ALDH sorted myoblasts was induced in vitro by BMP-4 stimulation. Alkaline phosphatase levels (ALP, shown in blue), an early marker of osteogenic differentiation were significantly increased in ALDHhi myoblasts compared to ALDHlo myoblasts after a period of 4 d of BMP-4 stimulation.
Osteogenic differentiation of murine myoblasts was induced via bone morphogenic protein-4 (BMP-4) stimulation. The unsorted myoblast population increased their alkaline phosphatase expression, an early marker of osteogenic differentiation, in response to BMP-4 stimulation (
While the differences in osteogenic differentiation in ALDH sorted myoblasts may either be attributed to increased osteogenic capacity or increased media stress resistance, the case of chondrogenic differentiation capacity suggests the latter mechanism. It seems clear that ALDHlo myoblasts experienced some form of growth retardation in chondrogenic media compared to ALDHhi myoblasts to the extent that ALDHlo myoblasts did not form a condensed pellet. Furthermore increased RA production by the ALDHhi cell would not be expected to increase the chondrogenic differentiation capacity of these cells. The inability of ALDHlo myoblasts to survive and proliferate in chondrogenic media impaired their capacity for chondrogenic differentiation, as measured by pellet size and amount of GAG production. Consequently, we believe that the increased proliferation potential of ALDHhi myoblasts may not only improve the myogenic differentiation but also facilitate increased chondrogenic and osteogenic differentiation capacities.
Given the heterogeneity of ALDH activity and stress resistance in myoblasts, we hypothesized that such heterogeneity may not be observed in MDSC populations since we have previously reported that MDSCs are highly resistant to stress
(a) A schematic of the isolation of ALDHlo and ALDHhi subpopulations of MDSCs is depicted here. (b) As discussed previously, ALDHlo and ALDHhi MDSCs were isolated, by excluding propidium iodide stained dead cells, and forming appropriate ALDH gates using DEAB inhibited controls. (c, d) No statistically significant difference in proliferative capacity between ALDHlo and ALDHhi MDSCs in oxidative stress (H2O2, 250 µM) or in inflammatory stress (TNF-α, 2.5 ng/ml) was observed (n = 9). (e) ALDH sorted MDSCs underwent myogenic differentiation in oxidative stress (H2O2, 250 µM) by expressing myosin heavy chain (MHC) and fusing into MHC+ clusters (red) rather than fusiform myotubes. (f, g) Significant differences in myogenic differentiation indices (MDI) were not observed in ALDHhi MDSCs compared to ALDHlo MDSCs except at one very high oxidative stress (H2O2, 500 µM) and at an intermediate inflammatory stress level (TNF-α, 1 ng/ml) (n = 9,* indicates p<0.05). (h) Robust engraftment of ALDHlo and ALDHhi MDSCs was observed following intramuscular injection into the gastrocnemius of
In contrast to myoblasts, MDSCs did not display a high degree of heterogeneity in stress resistance behavior when sorted into ALDHlo and ALDHhi subpopulations. No significant differences in proliferation (
When injected intramuscularly into the
Given the clinical relevance of working with human muscle derived cells, we proceeded to study unfractionated or primary hMDCs to determine whether their ALDH sorted subpopulations would exhibit similar behavior to murine muscle derived cells. We sought to identify a similar ALDHhi subpopulation of cells from cultured human primary cells using the same methodology described for murine myoblasts and MDSCs (
(a) ALDHhi hMDCs were isolated using FACS. (b, c) Significantly increased rates of proliferation of ALDHhi hMDCs compared to ALDHlo hMDCs were observed in conditions of oxidative (H2O2, 250 µM) and inflammatory stress (TNF-α, 2.5 ng/ml) (n = 6, * indicates p<0.05). (d) Myogenic differentiation capacity was measured in low serum conditions by myosin heavy chain (MHC, shown in red) expression. ALDHhi hMDCs generated dense networks of MHC+ myotubes in oxidative stress conditions (H2O2, 250 µM) in contrast to ALDHlo hMDCs. (e, f) Significantly increased myogenic differentiation indices (MDI) were calculated in ALDHhi hMDCs in oxidative (H2O2) and inflammatory (TNF-α) stress conditions compared to ALDHlo hMDCs. (g, h) Dystrophin (DYS, red) positive myofiber regeneration was observed following intramuscular injection of ALDH sorted hMDCs. A significantly increased regeneration index was observed in ALDHhi hMDC transplanted
A dramatic increase in oxidative and inflammatory stress resistance was observed in terms of proliferation and myogenic differentiation in ALDHhi hMDCs compared to ALDHlo hMDCs. A significant proliferation advantage of ALDHhi hMDCs compared to ALDHlo hMDCs was observed (
In vivo, a significantly increased RI was observed in the gastrocnemius muscles of
To determine if the increased stress resistance capacity of the ALDHhi hMDCs may be conferred by elevated intracellular antioxidants like ALDHhi myoblasts, we examined the GSH and SOD levels in ALDHlo and ALDHhi hMDCs. Although a trend of increased GSH was observed in ALDHhi hMDCs compared to ALDHlo hMDCs, this trend was not statistically significant (
Our research on the muscle regenerative capacity of various populations of myogenic progenitors suggests that stress resistance is predictive of muscle regeneration capacity
In the current study, ALDHhi muscle progenitor cells isolated by FACS from murine and human skeletal muscle demonstrated an enhancement in cell proliferation and myogenic differentiation capacities, when cultivated in conditions of oxidative (H2O2) and inflammatory stress (TNF-α). Vauchez et al. and Jean et al. have previously isolated ALDHhi progenitors from human muscle
While ALDH has been shown to mitigate oxidative damage such as lipid peroxidation, our results indicate that increased intracellular antioxidant levels observed in ALDHhi cells are primarily responsible for the increased oxidative stress resistance. This conclusion is not entirely consistent with the findings of Jean et al. who observed a dramatic loss in viability of human ALDHhi myoblasts in oxidative stress when ALDH activity was inhibited by DEAB
ALDH mediated production RA has been shown to promote myogenic differentiation but play a more nuanced or synergistic role in osteogenic and chondrogenic differentiation
RA has been shown to promote osteogenesis synergistically with bone morphogenic protein
We observed an enrichment of ALDHhi cells in late preplate cells (slowly adhering cells), a population enriched with MDSCs which exhibit elevated oxidative stress resistance
No improvement in stress resistance was observed in ALDHhi MDSCs compared to ALDHlo MDSCs in terms of proliferation and myogenic differentiation. This result is not surprising given the increased homogeneity of MDSCs in addition to previous observations of increased stress resistances in MDSCs in general compared to myoblasts
The improved proliferative capacity of murine ALDHhi myoblasts and hMDCs over that of their ALDHlo counterparts, in the presence of H2O2 and TNF-α in vitro suggests that the ALDHhi cells may have an increased proliferative capacity under conditions of post-transplantation inflammation in vivo. Similarly, an increased differentiation capacity in conditions of oxidative and inflammatory stress is likely to translate to an improvement in the differentiation capacity when the cells are exposed to post-transplantation inflammation. Indeed we observed improved muscle regeneration in muscles injected with both murine or human ALDHhi muscle cells compared to their ALDHlo counterparts, which appears to be a function of improved survival, proliferation, and myogenic differentiation.
The source of this improved regeneration capacity of ALDHhi myogenic cells may be increased stress resistance, due to the elevated intracellular levels of antioxidants such as GSH and SOD in addition to the stress resistance role of ALDH, an observation that was also made previously in MDSCs
While numerous myogenic cells have been identified for muscle cell therapies, identification of definitive or even desirable markers for myogenic progenitor isolation remains elusive
Although it has been known for many years that stem and progenitor cells display unique behaviors that are advantageous to applications of cell therapy such as self-renewal, long-term proliferation, and multipotent differentiation, our results suggest that resistance to stress should be included among these vital attributes. Stem cell behavior such as improved survival may even represent a more important “marker” for stem cell therapy than the use of traditional surface marker profiles. This particular hypothesis is supported by our extensive studies of MDSCs, which are isolated by their slow adhesion behavior and their remarkable survival capacity in vitro and in vivo
All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publications 85-23) as promulgated by the Committee of Care and Use of Laboratory Animals of the Institute of Laboratory Sciences, the National Academy of Sciences, and the National Research Council. The University of Pittsburgh's Institutional Care and Use Committee (IACUC) reviewed and approved all the procedures performed in these studies (IACUC protocol #: 0902596A-2).
Murine myoblasts and MDSCs were isolated from the skeletal muscle of 3-wk-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) as previously described using a modified preplate technique
The preplate technique segregates muscle derived cell populations by how quickly they adhere to collagen-coated plates as described elsewhere
Myoblasts were isolated from rapidly adhering cell fractions of the early preplate cycles while MDSCs were isolated from the slowly adhering cell fraction of the late preplate cells (PP6). All cells were cultured and expanded in PM in collagen-coated flasks at 37°C and 5% CO2 as described previously
Human gastrocnemius muscle biopsies, 9 in total, were obtained (mean age 58 years; range 25 to 75 years, both male and female) from the National Disease Research Interchange (NDRI) and the Center for Organ Recovery and Education (CORE). All biopsies were taken from patients with no history of neuromuscular/skeletal disease, sepsis, chemotherapy, drug abuse, or ventilatory support greater than 1 month. Furthermore, NDRI and CORE obtain written informed consent from the patient's family prior to harvesting any organs or tissues. Acquisition of muscle from the NDRI and CORE, and subsequent muscle stem cell isolation, was performed in accordance to the protocol reviewed and approved by the University of Pittsburgh's Institutional Review Board (Protocol #: PRO09030265, entitled: Isolation of Human Muscle-Derived Stem Cells). Tissue was finely minced then digested for 60 min at 37°C with 2.4 U/mL dispase (GIBCO), type-I and type-IV collagenases (both at 0.5 mg/ml; Sigma-Aldrich). The digested tissue was pelleted and resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S), then passed through a 100 µm filter followed by a 70 µm filter to obtain a single cell suspension. Cells were then treated with red cell lysis buffer for 15 minutes before being cultured in PM in collagen-I coated flasks at 37°C and 5% CO2. PM consists of 10% horse serum (GIBCO), 10% FBS (GIBCO), 1% chick embryo extract (Accurate Chemical Company), and 1% penicillin–streptomycin (GIBCO) in DMEM high glucose (Invitrogen). Following enzymatic dissociation, human muscle cells were cultured for three to four days in the flasks of their original seeding and were passaged one to two times as necessary prior to FACS. This protocol is illustrated in
Cultured murine and human cells were trypsinized, washed in cold PBS, and counted using a hemocytometer. Cells (106) of each population were resuspended in Aldefluor buffer, which contains an ABC transport inhibitor that prevents efflux of the Aldefluor dye, and incubated at 37°C with BAAA according to the manufacturer's instructions (Aldagen Inc, Durham, NC). Cells were washed in Aldefluor buffer and maintained in 4°C throughout the cell sorting process. ALDH activity was assessed using the FL1 channel of a BD FACSAria Cell Sorting System and FACSDiva software (version 6.1.2) (Becton, Dickinson and Company, San Jose, CA). Collected cells were gated on their fluorescence intensity, which corresponds to their ALDH activity levels, as well as low side scatter (SCClo). Sorted cells were recaptured in cold (4°C) PM and immediately plated in collagen-I coated flasks and normal incubation conditions (5% CO2 at 37°C).
Time-lapse live cell microscopy was employed to monitor the rate of proliferation under conditions of oxidative and inflammatory stress
The capacity of ALDH sorted cell populations to differentiate into MHC expressing cells under varying conditions of oxidative and inflammatory stress was quantified in vitro using the MDI metric. Cells were plated at 1000 cells/cm2 in 24-well plates for 2 d in PM or until near confluence. PM was then exchanged for differentiation media (DM; 2% HS, 1% P/S in DMEM) treated to simulate oxidative stress (25 µM, 100 µM, 250 µM, and 500 µM, H2O2, Sigma-Aldrich) and inflammatory stress (1 ng/ml, 2.5 ng/ml, and 5 ng/ml, TNF-α, R&D Systems). The media was exchanged daily to maintain constant stress levels
The antioxidant capacity was measured in terms of the activity of the major intracellular antioxidant molecules: GSH and SOD. Levels of GSH were measured colorimetrically (Calbiochem, 354102) using a spectrophotometer (TECAN Infinite M200, Männedorf, Switzerland). GSH detection is mediated by capture of all thiol mercaptans (RSH) from mechanically homogenized cells into thioethers by 4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate followed by the formation of a chromophoric thione in those GSH specific thioethers using NaOH. GSH levels were quantified by chromophoric thione absorbance at 400 nm. Total activity of SOD was measured using a colorimetric assay (Chemicon, Temecula, CA; APT290). SOD levels of chemically lysed cells (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, and 0.5% Triton X-100) were measured using a xanthine/xanthine oxidase system in which superoxide-chromagen absorbance (490 nm) is lowered by the presence of SOD.
The intracellular antioxidant levels of ALDHlo and ALDHhi myoblasts were modified by a chemical antioxidant (XJB-5-131, generously donated by Prof. Peter Wipf at the University of Pittsburgh) and DEM, a pro-oxidant (Sigma-Aldrich). XJB is a membrane permeable, mitochondrial targeted nitroxide antioxidant that has proven to be cytoprotective in disease states associated with oxidative stress, such as hemorrhagic shock, and mitigates apoptotic cell death in vitro
To examine the role of ALDH in the antioxidant capacity of ALDH sorted myoblasts a potent ALDH inhibitor, DEAB, was used. Pretreatment of ALDHhi myoblasts with 50 µM of DEAB in PM was used 24 hrs prior to oxidative stress exposure. A concentration of 50 µM DEAB was maintained during the oxidative stress exposure to maintain ALDH inhibition throughout the experiment. The rates of proliferation and survival were quantified using a live cell imager and ImageJ software (NIH).
Skeletal muscle regeneration capacity of ALDH sorted myogenic cells was quantified using the regeneration index metric as previously described
Differentiation of myogenic cells into MHC expressing cells in vitro was quantified using the MDI metric described previously. Briefly, samples were blocked with 5% HS and incubated with monoclonal antibodies for MHC (Sigma; 1∶300) followed by Cy3-conjugated anti-mouse antibodies. Nuclei were stained blue with 4, 6-diamidino-2-phenylindole (DAPI, Sigma; 100 ng/ml, 1∶1000). At least 200 cell nuclei were counted for each MDI measurement.
All murine skeletal muscle tissue samples were frozen in 2-methylbutane cooled in liquid nitrogen, then stored at –80°C. 8 µm cryosections were fixed in 5% formalin for 2 min and blocked in 5% donkey serum for 1h. Skeletal muscle sections were stained for dystrophin (DYS) using an anti-dystrophin primary antibody (1∶300, Rabbit Anti-DYS, Abcam Ab15277) and a secondary anti-rabbit antibody (1∶500, Donkey anti-Rabbit, Molecular Probes A21207) using a protocol previously described
ALDHhi, ALDHlo and unsorted cells were prepared in pellet form (2.5×105 cells) or as a monolayer (1.5×104 cells/well in 12 well plates). For osteogenesis, murine myoblasts were plated on collagen-I coated 12 well plates and maintained for 4 d in osteogenic media (Lonza, Walkersville, MD), which included dexamethasone, glutamine, ascorbate, penicillin/streptomycin (P/S), β-glycerophosphate and bone morphogenic protein-4 (50 ng/ml). The cells were stained for alkaline phosphatase following the manufacturer's instructions (Sigma-Aldrich, 86C-1KT). Chondrogenesis of murine myoblasts was studied by culturing the cells in a pellet culture by incubating the cell pellets for 21 d in chondrogenic media (Lonza, PT3003), which contains dexamethasone, ascorbate, ITS+, P/S, sodium pyruvate, proline, glutamine and TGF-beta-3 (10 ng/ml) (Lonza, PT4124). All pellets were fixed in 10% formalin for 24 hrs followed by dehydration, paraffin embedding, sectioning, and Alcian blue staining.
Data are expressed as a mean with its standard deviation. Direct comparisons between two cell populations were made using an unpaired, two-tailed Student's
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The authors wish to thank Alison Logar (Rangos Research Center of Children's Hospital of Pittsburgh) and Lynda Guzik (McGowan Institute for Regenerative Medicine at the University of Pittsburgh) for their technical assistance with flow cytometry, and James Cummins (Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh) for his editorial assistance. The authors would like to thank Professor Peter Wipf of the University of Pittsburgh for generously donating the XJB-5-131 antioxidant compound synthesized in his lab.