Derek Milner

We explored the involvement of the muscle-specific intermediate filament protein desmin in the model of tumor necrosis factor alpha (TNF-alpha)-induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-alpha in the heart (alpha-myosin heavy chain promoter-driven secretable TNF-alpha [MHCsTNF]), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and beta-catenin show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms, we examined whether desmin is a substrate for caspase-6 in vivo as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin, ...

Regenerative medicine has long sought to develop therapies for articular cartilage repair and for enhancing endochondral ossification to address complications of long bone healing. The objective of this study was to determine the chondrogenic potential of porcine primary cell cultures for possible utility in orthopedic tissue engineering applications. Adipose-derived mesenchymal stem cell (ASC), chondrocyte (positive control), periosteal cell, and fibroblast (negative control) primary cell cultures from 8- to 12-month-old Yorkshire pigs were plated at 5000 to 10000cellscm(-2) in 75-cm(2) cell culture flasks using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with NaHCO3, 10% fetal bovine serum (FBS), and antimicrobials (penicillin-streptomycin, gentamicin sulfate, and amphotericin B), then incubated at 37°C, 5% CO2, and 18% O2. Cells were trypsinized at ~80% confluency and transferred into 15-mL conical tubes at 500000 cellstube(-1). Suspensions were wa...

Mesenchymal stem cells (MSC) have been shown to be useful in regenerative medicine with their capability to give rise to various different cell types, including osteoblasts, adipocytes, chondrocytes, muscle cells, and neurons. Among MSC, bone marrow-derived stem cells (BMSC) are considered the primary source. Recently, many studies have shown the regenerative capabilities of adipose-derived stem cells (ASC). The ASC, with their greater abundance and ease of harvest, provide clear advantages over BMSC. We have previously demonstrated the myogenic differentiation of porcine ASC when co-cultured with differentiating C2C12 myoblasts in a myogenic differentiation-promoting medium. In this study, we sought to examine the myogenic potential of porcine ASC when co-cultured with fully differentiated murine myotubes. For the present study, we used porcine ASC isolated from the back fat of a transgenic gree fluorescent protein (GFP)-expressing pig at passage 3. The ASC were added to mouse C2C12 myotube cultures that had been induced towards myogenesis for 72h. As controls, we co-cultured, in the same conditions, GFP-expressing endothelial cells (ENDO) from the aorta of the same pig. Additionally, we cultured ASC, ENDO, and C2C12 cells alone in myogenic-differentiation medium. Cultures were harvested at 12, 24, and 48h after addition of porcine cells to myotube cultures for measurement of mouse- or porcine-specific myogenic markers by quantitative RT-PCR and immunohistochemistry. We were able to observe fusion of ASC GFP-expressing cells with pre-formed mouse myotubes by detection of myotubes expressing GFP. Additionally, immunofluorescent staining of co-cultures with an antibody specific for porcine nuclear Lamin A demonstrated the presence of ASC nuclei incorporated into myotubes. We observed large increases in gene expression of porcine-specific myogenin (MYOG; >900-fold) and desmin (DES; 8-fold). Unexpectedly, ENDO in co-culture with myotubes also had increased expression of DES (4-fold) and MYOG (400-fold), possibly indicating their de-differentiation and adaptation to a myogenic phenotype. In addition, expression of mouse-specific DES and MYOG were boosted in C2C12 myotubes when co-cultured for 48h compared with C2C12 alone, suggesting enhanced myogenesis or prolonged survival of myotubes in co-culture. Cultures of ASC and ENDO alone did not display increased expression of myogenic markers. These results provide support for the use of ASC for muscle regeneration strategies, as in the case of damaged muscles and muscular dystrophy. In addition, compared with our previous observations, where ASC were co-cultured with undifferentiated C2C12, the ASC co-cultured with myotubes appeared to have an enhanced fusion and expression of myogenic markers. Finally, the capacity of ENDO to fuse and actively expressed muscle-specific genes deserves further investigation.