• The Isolation and Differentiation of Adipose Derived Mesenchymal Stem Cells

      Merilovich, Max; Department of Biological Sciences
      Pathologies concomitant with old age such as arthritis, osteoporosis, and obesity diminish the quality of life for society’s elderly. Recent advances in regenerative medicine have revealed that an abundant and readily available source of adult mesenchymal stem cells (MSCs), resides within adipose tissue. Previous research has demonstrated that MSCs can differentiate into multiple specialized cell types and may thus be capable of ameliorating symptoms of many age-associated diseases. However, several unique challenges currently limit the usefulness of adipose-derived MSCs in regenerative therapies. In this thesis I assessed novel ways of isolating and differentiating adipose-derived MSCs into desired cell types. Specifically, I assessed the capacity of a non-enzymatic sonication based method to the collagenase digestion based method of isolating MSCs from human adipose. Sonication yielded fewer total cells than collagenase. However, alizarin red staining revealed that cells obtained using sonication possessed a stronger osteogenic capacity than those obtained using collagenase. Additionally, oil red o staining showed that both methods produced cells capable of adipogenesis. Yet, alcian blue staining demonstrated that sonication was unable to yield cells capable of chondrogenisis. Lastly, rat adipose-derived stromal cells were cultured in medium containing stimulators of UCP1 expression in order to induce a brite cell phenotype. Following a 9 day treatment period, cultures displayed characteristic traits of brite cells such as increased intra-cellular lipid accumulation and increased mitochondrial content. However, when exposed to acute norepinephrine treatment, stromal cells did not display an increased rate of respiration. Furthermore, western blot analysis revealed that these cells did not express UCP1, suggesting that the differentiation treatment likely only induced a partial differentiation response. Overall, the above work provides valuable insight into addressing challenges currently limiting the usefulness of adipose-derived MSCs in regenerative therapies. Future works should assess the differentiation capacity of stromal cultures depleted of non-MSC cells and stromal cells obtained using sonication in medium containing additional inducers of differentiation. Moreover, future efforts based on the brite cell differentiation method used here should assess whether extending treatment induction/differentiation periods is capable of producing brite cell phenotype.