Title page for ETD etd-04042014-164139


Type of Document Master's Thesis
Author Jarazo, Javier Martin
URN etd-04042014-164139
Title Effect of Tissue Source on Adult Equine Multipotent Stromal Cell Pluripotency Induction Treatment with Synthetic mRNA
Degree Master of Science (M.S.)
Department Animal Science (Animal, Dairy, & Poultry Sciences)
Advisory Committee
Advisor Name Title
Bondioli, Kenneth Committee Member
Gentry, Glen Committee Member
Lopez, Mandi Committee Member
Keywords
  • induction of pluripotency
  • regenerative therapy
Date of Defense 2014-03-12
Availability restricted
Abstract
Autogenous and autologous adult multipotent stromal cells are applied to treat equine musculoskeletal injuries in clinical practice. However, options for autologous therapy in the equine specie are restricted due to the limited biological material obtained or MSCs tissue harvest procedure and because of their unpredictable in vivo behavior. Induced pluripotent stem cells represent an alternative to overcome these limitations.

Traditionally, iPSCs are produced by introducing DNA for early embryonic genes into cells with viruses. The method tested in this study, avoids the use of viral vectors and potential for exogenous DNA integration into the cellís genome by utilizing a lipid delivery vehicle carrying synthetic mRNA coding for the human pluripotency factors OCT4, KLF4 and SOX2. Cells from different tissue have specific epigenetic profiles that determine their phenotype and functionality; hence they might have a distinct susceptibility to induction of pluripotency. This study was designed to test the expansion capacity and potency of equine adult MSCs from adipose tissue, bone marrow, and fibroblast following chemical transfection with synthetic mRNA of human sequences for embryonic genes, OCT4, KLF4 and SOX2. Target gene mRNA and cell protein expression was compared among passage (P) 3 cells from the different tissue sources before and after 7 and 14 days of transduction. Additionally, multilineage capacity and expansion rate were compared in P10 cells. Target protein expression localized to the nucleus was higher in transduced cells at both time points compared to untreated cells based on immunocytochemistry. Based on qRT-PCR, OCT4 and SOX2 expression was significantly higher in transfected cells after 14 days of transduction compared untreated cells (Oct4: ASCs, P=0.030; BMSCs, P=0.005 and fibroblast, P<0.001; Sox-2: ASCs, P=0.012; BMSCs, P=0.001 and fibroblast, P=0.005). Multilineage differentiation was detected in transfected versus untreated cells both at passage 10 by histological staining after differentiation.

Expansion rates of transduced and untreated cells were not different with the exception of BMSCs (P<0.05). Based on these results, dedifferentiation of equine MSCs with synthetic mRNA increases in vitro potency and expansion capacity.

The ability to induce pluripotency from MSCs obtained from multiple equine tissue sources will significantly increase accessibility to highly characterized stem cells. This will in turn improve the ability to predict cell behavior for customized therapies, becoming an important model for future human applications.

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