Myo/Nog cells are identified by their expression of the skeletal muscle specific transcription factor MyoD and the bone morphogenetic protein inhibitor noggin, and binding of the G8 monoclonal antibody. Their release of noggin is critical for morphogenesis and skeletal myogenesis. In the adult, Myo/Nog cells are present in normal tissues, wounds and skin tumors. Myo/Nog cells in the lens give rise to myofibroblasts that synthesize skeletal muscle proteins. The purpose of this study was to screen human lens tissue, rhabdomyosarcoma cell lines, and tissue sections from rhabdomyosarcoma, Wilms and tumors lacking features of skeletal muscle for co-localization of antibodies to Myo/Nog cell markers and the lens beaded filament proteins filensin and CP49. Immunofluorescence localization experiments revealed that Myo/Nog cells of the lens bind antibodies to beaded filament proteins. Co-localization of antibodies to G8, noggin, filensin and CP49 was observed in most RC13 and a subpopulation of RD human rhabdomyosarcoma cell lines. Western blotting with beaded filament antibodies revealed bands of similar molecular weights in RC13 and murine lens cells. Human alveolar, embryonal, pleomorphic and spindle cell rhabdomyosarcomas and Wilms tumors contained a subpopulation of cells immunoreactive for G8, noggin, MyoD and beaded filaments. G8 was also co-localized with filensin mRNA. Staining for beaded filament proteins was not detected in G8 positive cells in leiomyosarcomas, squamous and basal cell carcinomas, syringocarciomas and malignant melanomas. Lens beaded filament proteins were thought to be present only in the lens. Myo/Nog-like cells immunoreactive for beaded filaments may be diagnostic of tumors related to the skeletal muscle lineage.

Osteoarthritis (OA), a progressive and degenerative disease, affects millions of aging adults and results in the loss of cartilage. In healthy joints, articular cartilage provides a smooth and cushioned surface that allows bones to glide over each other with little friction. However, when OA begins to develop, degradation of the cartilage extracellular matrix begins to outweigh its synthesis. Production of growth factors, cytokines, and matrix‐degrading enzymes increases. Development of OA therapeutics is the gold standard in orthopedic medicine, but the complexity of the joint makes it a difficult system to manipulate. In this study, we optimize our novel, serum‐free, three‐dimensional culture system for human osteoarthritic articular chondrocytes (HOACs) obtained from patients with end‐stage osteoarthritis who have undergone a total knee arthroplasty. We altered the parameters of culture size, tissue harvest sites and the number of cultures pooled to determine whether clinically‐relevant endpoints are within measurable ranges. In previous studies, sides of greatest and least pathology, as determined from gross examination of the femoral condyles and tibial plateau, were separated and cells were isolated and plated within serum‐free, three‐dimensional alginate cultures on 35 mm plates at a plating density of 2.5×106 cells/milliliter. In this study, we combine both sides of the joint tissue (greatest and least pathology) together to increase the number of cells and cultures obtained from each patient. In addition, we scaled down the size of the cultures to 22 mm plates at a plating density of 1.8×106 cells/milliliter. The scaled‐down, serum‐free, three‐dimensional alginate cultures were maintained for five days, media fractions collected on the second and fifth day of the culture and the alginate‐associated matrix collected at termination of the cultures on day five. Intact and degraded collagens were measured by immunoassay. Both collagens type I and type II, intact and degraded, were detectable within the scaled‐down culture system. Pooling three cultures together provided adequate measurable material of the clinically‐relevant endpoints. Collagen I intact and degraded values were lower than collagen II intact and degraded regardless of the number of cultures pooled (n= 2, 3 or 4). In summary, our optimization techniques have provided more cultures per patient thus enabling measurement of clinically‐relevant endpoints of OA and a screening system to test potential OA therapeutics.Support or Funding InformationNew Jersey Health Foundation Research Award, Center for Chronic Disorders of Aging Small Grant, and PCOM's Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Osteoarthritis (OA) is a common chronic condition which involves the loss of articular cartilage, the cushion between joints that provides a smooth surface for the bones to glide. Three compounds that make up most of the articular cartilage are type II collagen, proteoglycans, and water. In OA there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage. The composition of the extracellular matrix changes in OA as evidenced by production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free alginate culture but measurable extracellular matrix endpoints of the OA profile are dampened when chondrocytes isolated from sides of greater and least pathology in the joint are pooled together in culture. In this study, we obtained HOACs from patients undergoing total knee arthroplasty. Femoral condyles and the tibial plateau were labeled greater or least pathology, determined by gross anatomical observation, the cells were isolated and plated separately in three‐dimensional, twelve‐well, alginate cultures at a density of 1.8 × 106 cells per 0.5 mL. Samples were treated for 48 hours from day of plating with either 250nM Extracellular Matrix Protective Factor‐1 (ECPF‐1) or hyaluronic acid conjugated ECPF‐1 (HA‐ECPF‐1). Collagen I degradation and sulfated proteoglycan synthesis were measured in alginate‐associated matrix of the treated cultures. HOACs isolated from the side of greatest pathology responded to treatment by reducing collagen type I degradation (8.42ng/culture Control, 7.25ng/culture ECPF‐1 and 4.25ng/culture HA‐ECPF‐1) and increasing retention of sulfated proteoglycans in the extracellular matrix (0.36ug/culture Control versus 0.56ug/culture ECPF‐1 treated). These data confirm that 48 hour treatment in serum free HOAC culture is sufficient to measure the therapeutic efficacy of an OA drug intervention and that both formulations of ECPF‐1 can slow the progression of OA by altering production of sulfated proteoglycan and collagen type I.Support or Funding InformationThis work was supported in part by a grant from the New Jersey Health Foundation and Philadelphia College of Osteopathic Medicine's Center for Chronic Disorders of Aging and the Division of Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myo/Nog cells, named for their expression of the skeletal muscle specific transcription factor Myo D, and the bone morphogenic protein inhibitor noggin, are critical regulators of normal development, progenitors of myofibroblasts in the ocular lens and neuroprotective in the retina. They also rapidly respond to wounding of the skin. The role of Myo/Nog cells in the modulation of tissue injury and fibrosis in the liver has not been studied. We examined the response of Myo/Nog cells to acetaminophen induced acute liver toxicity and their relationship to human hepatic stellate cells already known to play a role in acute and chronic liver injury responses. Liver tissue was obtained from mice following acetaminophen overdose over a time course of 0 to 96 hours. Additionally, LX‐2 human hepatic stellate cells were grown to confluence and subjected to a mechanical injury (scratch assay) and examined at several time points 0–24 hours after wounding. Immunofluorescent staining with the G8 monoclonal antibody was used to identify Myo/Nog cells in tissue sections of liver and stellate cell cultures. Preliminary analyses showed that in the mouse model of acetaminophen toxicity, there is a resident population of Myo/Nog cells in the liver that decline in number during the first 24 hours after the acetaminophen overdose and then increase, particularly in the region near the central vein (zone 3) where hepatocyte necrosis was greatest. The highest number of Myo/Nog cells was seen at 48 hours post acetaminophen administration, the time of maximal liver injury as previously documented by serum biomarkers. With the in vitro human stellate cell scratch assay, we found that a subset of these cells express the Myo/Nog marker G8, and the proportion of cells that express this marker increases after wounding, peaking at 24 hours after the scratch. G8 positive cells consistently clustered in proximity to the margins of the wound, with the greatest densities counted within the first high‐power field of the scratch. These studies suggest that Myo/Nog cells respond to acute liver injury by increasing in number and/or migrating toward the site of injury. Whether these cells play a role in mediating progression of liver injury or promote recovery via liver regeneration needs to be further evaluated in the model. The in vitro studies show that Myo/Nog cells may represent a subpopulation of human stellate cells in the liver, and this relationship should be further explored as a potential target for future therapeutics for acute and chronic liver injury. Support or Funding Information Anonymous donation from a private funder.

Osteoarthritis (OA) is a common condition involving the loss of articular cartilage which is primarily made up of type II collagen, proteoglycans, and water. In OA, there is an increase in collagenase activity that degrades collagens and proteoglycans necessary for healthy cartilage and leads to the abnormal production of type I collagen. Previous studies in our lab have shown that primary cultures of human osteoarthritic chondrocytes (HOACs) can be reared in serum‐free, three‐dimensional alginate culture and components of the extracellular matrix (ECM) can be measured. However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. In this study, we obtained HOACs from the femoral condyles and the tibial plateau of patients undergoing total knee arthroplasty. HOACs of greater or least pathology, determined by gross observation, were isolated and plated in 12‐well cultures at a density of 1.8 × 106 cells/0.5 mL alginate. Collagens I and II degradation and proteoglycan synthesis were measured in conditioned media and alginate‐associated matrix of the cultures at days 2, 5, 8 and 11. During long term HOAC culture, collagen degradation was reduced (p<0.001 for coll I) while proteoglycans were retained in the ECM. This trend suggests that long term, three‐dimensional, serum‐free culture of HOACs may revert to a healthier phenotype. The largest changes in extracellular matrix production were demonstrated between days 2 and 5 in HOAC culture giving insight of a treatment window to test possible therapeutics. Support or Funding Information Support was provided by New Jersey Health Foundation Research Award, Cooper Foundation Research Grant and the Division of Research, PCOM.