Method and Techniques for Myogenic Differentiation of Human Adipose-Derived Stem Cells

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Human adipose-derived stem cells (hASCs) are multipotent stem cells that have the potential to self-renew and differentiate. In an effort to optimize conditions for the myogenic differentiation of adult stem cells and improve their potential in the clinic, we cultured hASCs on collagen-coated tissue culture plates using two different medias with varying amounts of fetal bovine serum (FBS). Following six weeks of myogenesis, cells were characterized by cellular morphology, protein expression, and transcription of myogenic specific genes. Immunofluorescence (IF) is used to determine the success of each culture's surface and media by allowing for the visual and qualitative evaluation of myogenic protein expression. Immunofluorescence using the antibodies for MYOD and myosin will be used to qualitatively assess the expression of these myogenic transcription factors and proteins respectively to evaluate differentiation. Phalloidin is used to visualize actin filaments, while DAPI binds to adenine-thymine rich areas in DNA to visualize cell nuclei. To quantitatively analyze our cultured hASCs, we use quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Here, RNA is extracted from cells and reverse-transcriptase is used to synthesize complementary DNA (cDNA). The cDNA is then used as a template for quantitative PCR which utilizes fluorescent dyes to record amplification and provide more accurate data of transcript levels of myogenic markers such as myod, myf5, and myogenin. We are able to differentiate hASCs and characterize the efficiency of myogenesis under various conditions. Conclusions drawn from this research contribute to regenerative medicine research currently seeking improved methods for the generation of functional muscle tissue.