All procedures involving human tissues were approved by IRB and where with the consent of adult donors. 1-2 grams of muscle tissue were obtained from rectus abdominus of obese adult female subjects with no recorded history of diabetes. Donor lot demographics were as follows: Lot-1 was composed of cells derived from one donor: 44 year-old female, BMI 36.4. Lot-2was composed of satellite cells derived from three donors: 29 year-old female, BMI 42.9; 30 year-old female, BMI 46; 29-year old female, BMI 39.3. Assay development/validation studies and pilot screening were performed on cells derived from Lot-1 and the EC50 confirmatory studies were performed on Lot-1 and Lot-2 to yield insight into donor-to-donor variability. The satellite cells were isolated based on the modified procedure utilized by Blau and Webster, 1981 [30Blau HM, Webster C. Isolation and characterization of human muscle cells Proc Natl Acad Sci USA 1981; 78(9): 5623-7.]. Briefly, a sample of rectus abdominus muscle tissue was excised and transferred to Hank’s Balanced Salt solution (HBSS). After removal of visible adipose and connective tissues, the muscle tissues were minced and washed in HBSS. Subsequently, the minced muscle tissue was digested in 37°C HBSS containing 0.2% Trypsin-EDTA, 0.1% type IV collagenase, and 1% BSA. Following tissue digestion, the isolated cells were collected by centrifugation in skeletal muscle culture media (ZenBio; DMEM, 10% FBS, 0.05% BSA, 0.05% fetuin, 20 ng/ml hEGF, 0.4 µg/ml dexamethasone and antibiotics). After minimal expansion the cells were stained for desmin and shown to be ≥90% desmin positive; suggesting homogenous myoblast population. These preparations of myoblasts were also ≥ 85% CD56+. The satellite cells were expanded (passage 4) and cryogenically preserved in skeletal muscle media supplemented with 5%DMSO.

Active in receptor-like kinase 5 (Alk5) inhibitor (LY364947) was utilized as a positive control in the present cellular proliferation assay. It has been demonstrated that signaling via the TGF-β pathway results in the reduction in proliferation and healing of skeletal muscle [31Carlson ME, Conboy MJ, Hsu M, et al. Relative roles of TGF-beta 1 and Wnt in the systemic regulation and aging of satellite cell responses Aging Cell 2009; 8(6): 676-89.]. ALK5, also known as TGF-β R1, is a receptor for TGF-β, and small molecule inhibitors have been developed that inhibit kinase activity associated with Alk5 [32Gellibert FO, Woolven J, Fouchet MH, et al. Identification of 1,5-naphthyridine derivatives as a novel series of potent and selective TGF-beta type I receptor inhibitors J Med Chem 2004; 47(18): 4494-506.]. Treatment of cells with the Alk-5 inhibitor, LY364947 (1 uM), significantly reduced the doubling times of satellite cells from both young and, to a greater extent, old donors (data not shown). These preliminary data confirm the ability of the assay to serve as a relevant phenotypic drug discovery platform to discover agents that can modulate proliferative capacity of skeletal muscle satellite cells.

To establish the optimal concentration of LY364947 as the positive control, satellite cells from Lot-1 were screened in a 7-point, 2-fold dilution series between 100-0.5µM of LY364947 with 48 replicates per condition. Detailed cell plating conditions are described in Satellite Cell Proliferation Assay section. Based on the analysis of assay window and replicate variability, it was determined that 25 µM of LY364947 results in maximum proliferation rate in satellite cells derived from obese subjects and was selected as the positive control concentration.

Two commercially available compound libraries consisting of 1120 FDA-approved drugs (Prestwick Chemical Library®) and 502 purified natural products (Enzo Life Sciences) were screened on satellite cells derived from obese female donors. The compound plates were stored in deep-well polypropylene blocks, at 4 °C with no humidity control. All compounds were resuspended in 100% DMSO. The compound libraries were plated in Axygen384-well rigid PCR plates (Cat no321-67-051) at 1000X concentration for both single-concentration screening and dose-response confirmation.

Satellite cells were thawed and plated directly from cryopreservation and diluted to the final concentration of 0.075x10⁶ viable cells/ml. The satellite cells were then plated in 384 well collagen-coated flat bottom imaging plates (Greiner Cell Coat Clear Cat-No:781956). 30uL of pre-warmed growth media was added to each well, followed by 20µl of cells (total 1500 cells/well). Cells were observed to attach at approximately four hours post plating and compounds were added shortly thereafter. Cells were allowed to incubate with compounds for 72 hrs in humidified 37°C, 5% CO₂ incubator.

In the pilot screen, compounds were applied to Lot-1 satellite cells with the Biomek NX workstation (Beckman-Coulter, Brea, CA) equipped with a 50nL pin tool array (V&P Scientific, San Diego, CA) for a direct 1000-fold dilution, thereby avoiding an intermediate dilution step. The final concentration of compounds derived from Prestwick library was 1µM and the final concentration of compounds derived from Natural Product library was 5 µM. Controls were added using the pin tool array in a separate plate where columns 1-2 and 23-24 were negative (DMSO) and positive (25 µM of LY364947) controls, respectively. Plate-based normalization was performed using the mean of controls, yielding activity scores between 0-100% (Fig. 3). Compounds with 40% proliferative activity or above (approximately µ+3σ) were selected for dose response analysis.

Fig. (3) High content screening data for satellite cell proliferators. (A) Scatterplot for 1600 test compounds screened (red circles), positive control (LY364947, blue triangles), and negative control (DMSO, green-filled triangles); (B) histogram portraying normal statistical distribution; (C) box plot showing distribution statistics and outlier compounds.

Compounds selected from the pilot screen were serially diluted in a 10-point/2-fold format for an EC50 dose-response confirmation derived from Lot-1 and Lot-2 (mixed donor lot). The serial dilution of compounds was performed in Axygen conical 384-well PCR plates. A Biomek® 3000 was utilized to perform a 10-point 2-fold serial dilution series. Serially diluted compounds were applied to the cell plate by four subsequent pin tool transfers, which resulted in Natural Product concentrations ranging from 20 µM to 0.039 µM and Prestwick concentrations ranging from 4 µM to 0.0078 µM. As in the pilot screen, the controls were in columns 1-2 (DMSO) and 23-24 (25 µM of LY364947). The dose response compounds were tested in intra-plate triplicates and the EC50 values were calculated based on the average of each concentration.

Following the 72-hr incubation period, media was removed with Biomek NX workstation. Subsequently 30 µL of IX PBS (pH 7.4) containing fresh 4% Formaldehyde and 10 µg/mL Hoechst-33342 was added to the cells and then incubated for 30min at 37°C. 30µL of stain and fixative-containing PBS was aspirated out and replaced with 40µL of PBS. All the liquid additions were performed at the slowest Biomek NX dispense speed to prevent cell detachment. Following PBS addition, plates were sealed with Thermo-ABgene plate sealer and imaged on BD Pathway 855 bioimager (Becton Dickenson, San Jose CA).

Two passes of image acquisition were performed using a BD Pathway 855 with either an Olympus UPlanSApo4X (entire-well cell counting) or an Olympus UPlanSApo10X (cell morphology) objective lens. To assure an ideal signal-to noise ratio, the exposure times and laser-based autofocus were adjusted for each plate prior to image collection using the BD Attovision software. A 2X2 montage image was acquired for each well of 384-well plate at 4X magnification. The 2X2 montage image area at 4X magnification assured an entire well coverage.

We found that satellite cell morphology was easily distinguishable with either Hoechst-33342 alone or multiplexed with Cell Tracker Red CMTPX. Therefore, we selected Hoechst-33342 alone to visualize both nuclei and cytoplasm. To establish compound-related changes in cellular phenotype, satellite cells were imaged with 10X objective lens. The 10X magnification was found to be ideal to visualize changes in cell shape parameters.

Based on the high-replication dose response analysis in our initial positive control screen run, we established that 25 µM of LY364947 results in a maximum proliferation rate in satellite cells derived from both donor lots (Fig. 1 and 2). Over 72-hrs, LY364947 increased cell count by 17% in a single donor lot (Lot-1) and by 37% in a mixed donor lot (Lot-2). Next, plating density was optimized with respect to the assay window (cell count of negative control/DMSO vehicle vs.25 µM of LY364947). Our initial assumption was that by plating fewer cells-per-well, we would achieve a wider assay window given the same amount of proliferation time without approaching contact inhibition. After examining 1000, 1500, and 2000 cells per well, we determined that 1500 cells per well provided the most robust assay window. After careful examination of the cellular phenotype, we realized that by plating more cells, they were up against a natural differentiation/proliferation checkpoint. However, in untreated wells, proliferation was rapidly declining due to contact stimulated differentiation and elongation of cells into myotubes. With the addition of LY364947, the cells were able to push past the differentiation checkpoint and continue proliferating, resulting in higher cell counts and smaller cells, which is indicative of stimulating satellite cell proliferation. In addition to increased cell counts, the LY364947 had an effect on factors that define cellular phenotype such as cell perimeter, major axis length, cell area, eccentricity, and form factor (Fig. 1). Strong correlation between cell number and cell size is indicative of compounds that increase satellite cell proliferation and, at the same time, delay differentiation.

Fig. (1) Comparison of Lot 1 (A-D) satellite cells treated for 72 hrs with 0.1% DMSO (negative control: A,C) and with 25µM LY364947 (positive control: B,D). Cells were seeded at 1500 cells/well. Images portray satellite cells stained with Hoechst 33342 (A, B) and corresponding two phase detection showing nuclei (red) and cytoplasmic outline (green) (C, D). Images are at 100X magnification, scale bar =50µm. (E) Quantification of cellular parameters that were most distinct between negative and positive controls. Cellular dimension values are expressed in pixels (pixel size=0.61µM).

Fig. (2) Dose response curve for the positive control compound, LY364947(Lot 2).

After conducting a pilot screen of Prestwick and Natural product libraries, we were able to identify and cluster compounds into discrete categories based on toxicity, proliferative capacity, potential myogenic properties and intermediate activity (Fig. 3, 4). In this pilot screen, we were liberal in selecting potential hits for compound validation, since we only screened a limited number of compounds. The main aim for the pilot screen was to identify compounds that cause a significant increase in satellite cell proliferation. The single-concentration pilot screen of Lot-1 cells with Prestwick and Natural Product libraries resulted in 22 compounds with 40% or higher activity above the negative control mean (Fig. 3).

Fig. (4) Screening dot-plot showing the relationship between cell size and satellite cell proliferation.

The selected compounds were screened in triplicate dose/response for both donor lots. In Lot-1, 15 out of 22 compounds were dose responsive (68% confirmation rate). However, only two compounds were found to be dose responsive in Lot-2 (Geraldol and Bromopride, Fig. 5). Geraldol originated from the Natural Product library and Bromopride originated from the Prestwick library. The calculated EC50 values for Geraldol and Bromopride were 0.65µM and 0.66 µM, respectively and were nearly identical in both lots. Since donor to donor variability in satellite cell proliferation is often observed in response to compound treatment, the goal was to identify compounds that significantly increased satellite cell proliferation rate in both donor lots. Additionally, the aim was to find compounds that cause increases in satellite cell proliferation in the highest number of donors despite donor to donor variability. Using lots composed of cells derived from multiple donors (such as our Lot-2), helps to ensure that compounds selected are effective on multiple donors.

Fig. (5) Log-transformed dose response curves for two compounds that significantly increased proliferation rate in satellite cells (Lot 2).

There are key morphological features that indicate-changes in the proliferative state of satellite cells. Principal Component Analysis (PCA) of approximately 100 different cellular output parameters resulted in 6 factors that correlated best with the positive control treatment (Fig. 1E). These parameters allow for identification of distinct modes of action of analyzed compounds through natural clustering (Fig. 4). Parameters including small satellite cell cytoplasmic area, small cellular length and perimeter, decreased eccentricity, and increased form factor all indicate increased proliferation. After screening two highly-annotated small molecule libraries, we were able to detect different modes of action through visualization of key phenotypic parameters discovered in the PCA analysis across the compounds screened. We observed natural clustering of compound effects and were able to identify common modalities/cell fates. For example, one cluster of compounds resulted in large cytoplasmic area, increased cellular length and perimeter, increased eccentricity and decreased form factor, all signaling that satellite cells are differentiating into myotubes and are entering the process of myogenesis. Once satellite cells enter myogenesis, they terminally differentiate, stop proliferating and begin to fuse and form long chains of nuclei. From our pilot screen performed on Lot-1, we identified a number of compounds that may have a myogenic potential including: Pratol, Limonin, and Idirubin. The activity of these compounds will be confirmed by EC50 studies in multiple donor lots in future studies.

The pilot screen also revealed a large number of compounds that cause satellite cell toxicity. Some of the most toxic compounds identified were: Camptothecin, Parthenolide, and Cantharidin. Because these compounds were screened at a single concentration, it is unknown if these compounds have activity at lower concentrations.

Satellite cells are indispensable to the recovery and maintenance of skeletal muscle. Under normal physiological conditions, satellite cells contribute to muscle growth and repair by proliferating and donating nuclei to pre-existing myofibers. Because satellite cells function as adult muscle stem cells, they are able to restock their own population via a process of self-renewal [27Collins CA, Partridge TA. Self-renewal of the adult skeletal muscle satellite cell Cell Cycle 2005; 4(10): 1338-41.]. Cultured satellite cells encompass many phenotypic characteristics of quiescent satellite cells present in in vivo systems and therefore present a great model for studying satellite cell stimulation, renewal, and proliferation [28Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Bio 2004; 166(3): 347-57., 35Yablonka-Reuveni Z, Anderson JE. Satellite cells from dystrophic (mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers Dev Dyn 2006; 235(1): 203-12.].

Despite their ability to self-renew, satellite cell proliferative and regenerative capacity can be altered by many physiological and environmental stressors. Inactivity-associated muscle disuse, metabolic disorders, physical trauma, denervation, muscle wasting diseases, and sarcopenia have detrimental effects on satellite cell population and function. During mild injury or mild loss of muscle function, satellite cells migrate to the site of injury and begin the process of repair. As a result of severe physical myotrama, basal lamina may break apart and satellite cells begin to migrate to neighboring myofibers instead of the site of the injury, and the repair process does not proceed [36Schultz E, McCormick KM. Skeletal muscle satellite cells Rev Physiol, Biochem Pharmacol 1994; 123: 213-57.]. Prolonged decrease in muscle activity caused by factors such as muscle unloading, malnutrition, obesity and denervation results in significant reduction in myonuclear number and muscle atrophy [37Carlson BM, Faulkner JA. Reinnervation of long term denervated rat muscle freely grafted into an innervated limb Exp Neurol 1988; 102(1): 50-6.-39Borisov AB, Dedkov EI, Carlson BM. Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture Histochem Cell Biol 2005; 124(1): 13-23.]. Progressive decrease in satellite cell reserve/quiescent population results in myonuclear apoptosis and in decreased input of growth factors, such as IGF-1, which stimulate satellite cell proliferation. Additionally, satellite cell proliferative potential in atrophic muscle is reduced by replacement of muscle tissue with fibrotic tissue and confinement of satellite cells within endomysial tubes [39Borisov AB, Dedkov EI, Carlson BM. Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture Histochem Cell Biol 2005; 124(1): 13-23.].

The physical age of an individual also plays an important role in satellite cells’ ability to self-renew and proliferate. Satellite cell-stimulated skeletal muscle repair process gradually decreases with age [10Allbrook DB, Han MF, Hellmuth AE. Population of muscle satellite cells in relation to age and mitotic activity Pathology 1971; 3(3): 233-43., 11Mozdziak PE, Schultz E, Cassens RG. Satellite cell mitotic activity in posthatch turkey skeletal muscle growth Poult Sci 1994; 73(4): 547-5., 17Gibson MC, Schultz E. Age-related differences in absolute numbers of skeletal muscle satellite cells Muscle Nerve 1983; 6(8): 574-80.]. Along with a decrease in satellite cell mitotic activity, the satellite cell quiescent population also decreases with age. These changes are major reasons for the development of sarcopenia and decreased quality of life in the elderly. Therefore, discovery of small molecule therapeutics that aid in increasing satellite cell number is crucial in preventing and possibly treating muscle atrophy in aging individuals.

In the current study, we utilized satellite cell cultures derived from obese human subjects. Obesity-related inactivity and resulting metabolic imbalance cause phenotypic alterations in skeletal muscle. Obesity leads to reduced satellite cell proliferative capacity [12Purchas RW, Romsos DR, Allen RE, Merkel RA. Muscle growth and satellite cell proliferative activity in obese (ob/ob) mice J Anim Sci 1985; 60(3): 644-51.] and significant decrease in fast-twitch myofiber size [14Kemp JG, Blazev R, Stephenson DG, Stephenson GMM. Morphological and biochemical alterations of skeletal muscles from the genetically obese (ob/ob) mouse Int J Obes 2009; 33(8): 831-41.]. Furthermore, obese subjects suffering from Type II Diabetes have higher ratio of insulin-resistant muscle fibers, which are more prone to oxidative damage and atrophy [9Aguiari P, Leo S, Zavan B, et al. High glucose induces adipogenic differentiation of muscle-derived stem cells Proc Natl Acad Sci USA 2008; 105(4): 1226-31., 13Sishi B, Loos B, Ellis B, Smith W, du Toit EF, Engelbrecht A-M. Diet-induced obesity alters signalling pathways and induces atrophy and apoptosis in skeletal muscle in a prediabetic rat model Exp Physiol 2011; 96(2): 179-93., 40Schuler M, Ali F, Chambon C, et al. PGC1α expression is controlled in skeletal muscles by PPARβ, whose ablation results in fiber-type switching, obesity, and type 2 diabetes Cell Metabol 2006; 4(5): 407-14.-42Costford SR, Crawford SA, Dent R, McPherson R, Harper ME. Increased susceptibility to oxidative damage in post-diabetic human myotubes Diabetologia 2009; 52(11): 2405-15.]. Consequently, our in vitro satellite cell platform offers an excellent model for studying potential satellite cell proliferators in metabolically compromised individuals and presents promise for treatment of atrophic muscles affected by serious diseases such as Type II Diabetes

Depending on the environmental and metabolic cues, satellite cells either remain quiescent, proliferate, or enter myogenesis. In our pilot screen, we were able to identify compounds that induce satellite cell proliferation, stimulate myoblast differentiation, or cause cellular death. Compounds that induced proliferation in the pilot screen play many different biochemical roles that can possibly be tied to increased satellite cell activity. A number of positive hits from this study have hypotensive activities (from literature searching from highly annotated compounds), increase activity of intercellular insulin, and mimic caloric restriction. Recent research demonstrated that drugs lowering blood pressure may be beneficial in treatments of muscular dystrophy [43D'Angelo MG, Gandossini S, Boneschi FM, et al. Nitric oxide donor and non steroidal anti inflammatory drugs as a therapy for muscular dystrophies: Evidence from a safety study with pilot efficacy measures in adult dystrophic patients Pharmacol Res 2012; 65(4): 472-9.]. Furthermore, many studies show that insulin signaling and caloric restriction stimulate satellite cell mitotic activity [9Aguiari P, Leo S, Zavan B, et al. High glucose induces adipogenic differentiation of muscle-derived stem cells Proc Natl Acad Sci USA 2008; 105(4): 1226-31., 18Nierobisz LS, Felts V, Mozdziak PE. The effect of early dietary amino acid levels on muscle satellite cell dynamics in turkeys Comp Biochem Phys B 2007; 148(3): 286-94., 44Haddad F, Adams GR. Exercise effects on muscle insulin signaling and action - Selected contribution: Acute cellular and molecular responses to resistance exercise J Appl Physiol 2002; 93(1): 394-403.-49Lee S, Barton ER, Sweeney HL, Farrar RP. Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats J Appl Physiol 2004; 96(3): 1097-4.]. In future studies we will extend screening efforts to larger small molecule libraries and confirm our primary compound hits on satellite cells derived from a variety of new human donor lots with muscular atrophies caused by aging and disease.

Our dose response study confirmed two compounds (Geraldol and Bromopride) to be active in single donor satellite cell lot and triple donor satellite cell lot. Geraldol(3,4',7-trihydroxy-3'-methoxyflavone) is a metabolite of fisetin [50Touil YS, Auzeil N, Boulinguez F, et al. Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite Biochem Pharmacol 2011; 82(11): 1731-9.], a flavonoid that mimics calorie restriction process [51Howitz KT, Bitterman KJ, Cohen HY, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan Nature 2003; 425(6954): 191-6.]. Geraldol has shown activity in three confirmatory potency bioassays for inhibitors of human lipoxygenase, hydroxysteroid dehydrogenase, and Caspase-1 (PubchemAID: 887, 893, 923). Bromopride (4-amino-5-bromo-N-[2-(diethylamino)ethyl]-2-methoxybenzamide) has been shown to function as a dopamine-antagonist and anti-emetic [52Nasello AG, Vanzeler MLA, Felicio LF. A comparison of bromopride and domperidone effects on rat conditioned avoidance and motor activity Pharmacol Toxicol 1991; 68(1): 46-50., 53Niemegeers CJE. Anti-emetic specificity of dopamine antagonists Psychopharmacology 1982; 78(3): 210-3.]. Bromopride was active in one confirmatory assay for P45-cyp1a2 activators (Pubchem AID: 410), and in one potency assay for antagonists of the human M1 muscarinic receptor (Pubchem AID: 602250). Further studies are necessary to determine the exact mechanism by which Geraldol and Bromopride stimulate satellite cell mitotic activity.

The single point pilot screen resulted in a number of compounds that induced myogenesis. The compounds that significantly increased cellular size and halted proliferation were Pratol, Limonin, and Idirubin. The activity of these compounds still needs to be confirmed by EC50 studies in multiple donors. Data on these compounds is very limited and further experiments may be needed to confirm their myogenetic function.

The pilot screen also revealed a large number of compounds that caused satellite cell toxicity. Some of the most toxic compounds identified were: Camptothecin, Parthenolide, and Cantharidin. Camptothecin is an anti-tumor agent that inhibits the DNA topoisomerase I [54Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mamalian topoisomerase-I J Biol Chem 1985; 260(27): 4873-8., 55Slichenmyer WJ, Rowinsky EK, Donehower RC, Kaufmann SH. The current status of camptothecin analogs as antitumor agents J Natl Cancer Inst 1993; 85(4): 271-91.]. Parthenolide also has anti-cancer properties. It has been shown to induce apoptosis in acute myelogenous leukemia cells [56Guzman ML, Rossi RM, Karnischky L, et al. The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells Blood 2005; 105(11): 4163-9.]. Cantharidinis a toxic agent that causes burns and blisters [57Honkanen RE. Cantharidin, another natural toxin that inhibits the activity of serine threonine protein phosphatases type-1 and type-2a FEBS Lett 1993; 330(3): 283-6.]. Because of the truly toxic biological nature of these compounds, it is likely that even lower concentrations will cause satellite cell death.

The pilot screen resulted with 22 compounds that had high proliferative activity (at least 40% above negative control) in a single donor lot. Out of 22 selected compounds,15 were dose responsive in a single donor lot and 2 had confirmed activity in a satellite cell lot that was composed of three donors. Although the satellite cells were derived from subjects with very similar demographics, we still observed large donor-to-donor variability in response to the positive control compound and in response to test compounds. Satellite cells readily change their proliferative capacity in response to many physiological and environmental factors [58Matsakas A, Patel K. Skeletal muscle fibre plasticity in response to selected environmental and physiological stimuli Histol Histopathol 2009; 24(5): 611-29.]. Consequently, sample selection based on donor sex, age, tobacco use, presence of diabetes, or BMI may not provide an adequate assessment of the proliferative state of muscle satellite cells. Since the BMI does not provide an accurate measure of a total body fat, evaluating lean to fat ratio of an individual prior to sample acquisition may decrease donor to donor variability in response to tested compounds. However, we have determined it necessary to adjust assay timing (compound exposure period) to reflect substantial differences in satellite cell doubling time observed in donors with similar demographics. Normalizing the compound exposure period to a static number of cell doublings will provide the most robust and unbiased analysis of proliferative potential across widely variable donor lots. Additionally, we observed that optimal positive control concentration for one donor results in cell toxicity in another donor. Therefore, it is necessary to perform dose response analysis of the positive control for each cell lot to determine the positive control concentration that will result in maximum activity.

While high content screening/image cytometry provides a very accurate method for counting cells and observing cellular phenotype, additional confirmatory experiments are necessary to verify changes in cell proliferation status. We will perform pulse-chase EdU incorporation secondary/orthogonal assays to directly measure the percent of the population in S-phase in the treatment group relative to negative control (DMSO vehicle) using the Click-iT® EdU cell proliferation kit (Life Technologies, Carlsbad, CA). Mitotic activity will be assessed in this assay using either imaging or flow cytometry with similar performance. Future studies can be optimized by determining the exact point of proliferation activation and may shorten the time required for compound treatment. Follow up studies will involve in vivo treatment with selected compounds utilizing atrophic/or dystrophic and healthy mice models, and subsequent estimation of muscle satellite cell proliferative status by EdU profiling.

The novel high content, high throughput skeletal muscle satellite cell platform developed herein was validated for assay performance through screening of 1600 highly-annotated compounds. These data are highly suggestive that this platform can be readily adapted to a significantly larger screening effort (>100,000 compounds). In the present study we developed a screening assay for satellite cells derived from obese adult female subjects. Because obesity often results in inactivity and significant changes in cellular metabolism, satellite cells derived from obese individuals present a very good model for studying muscular atrophy. The current screening system will be utilized in experiments involving satellite cells derived from elderly individuals and individuals suffering from muscle wasting diseases. Our robust high content phenotypic assay provides a fast and effective screen for compounds that will have wide applications in muscle research and will be beneficial in the treatment of muscular disorders.