Title page for ETD etd-07142009-134947


Type of Document Master's Thesis
Author Picou, Alicia A
Author's Email Address apicou2@tigers.lsu.edu
URN etd-07142009-134947
Title The Isolation and Characterization of Bovine Adult Derived Adipose Stem Cells for the Use in Nuclear Transfer
Degree Master of Science (M.S.)
Department Animal Science (Animal, Dairy, & Poultry Sciences)
Advisory Committee
Advisor Name Title
Bondioli, Keneth Committee Chair
Godke, Robert Committee Member
Lopez, Mandi Committee Member
Keywords
  • Stem Cells
  • Cloning
  • Adipose Tissue
Date of Defense 2009-06-24
Availability unrestricted
Abstract
Since the cloning of Dolly there has been little change in the efficiency of nuclear

transfer (NT). Research is beginning to investigate the characteristics of donor cells. Adiposetissue is an abundant source of adult-derived cells that have displayed “stemness” in-vitro(Gimble et al., 2003). The overall goal of this research was to define the in-vitro characteristicsof bovine adipose-derived adult stem cells (ADAS) for the use in NT. Isolation methods weredetermined by a 3 x 3 factorial design. 1 g of subcutaneous fat was collected and subjected to0.10%, 0.25% or 0.50% collagenase type I solution for 1, 2 and 3 h. Nucleated cells werecounted using heochst stain. There was no significant difference (P>0.05) in number of

nucleated cells released during the incubation period or collagenase concentrations. Viable

cells were determined by those that remained adherent 24 h post plating. Incubation in 0.25%

collagenase for 2 h had the consistently highest percentage of viable cells (45%). The lifespan

and growth characteristics were determined by in a 2 x 2 factorial experiment of DMEM or DMEM:F12 supplemented or not supplemented with growth factors. DMEM with growth factors supplementation was significantly shorter lifespan (P>0.05) than DMEM:F12. The averagelifespan was ~30 population doublings (PDs), with 1 cell cycle every two days until passage 8(P8). Two bovine ADAS cell lines were differentiated into adipocytes, chondrocytes and osteoblasts at middle and late passages along side of adult derived skin fibroblasts. Differentiation was confirmed by histological staining resulting in early passage ADAS cells

staining more intensely compared to late passage ADAS cells and skin fibroblasts. Global levels

of DNA methylation and histone acetylation were analyzed from P1 to P6 in ADAS and skin fibroblasts from three animals. There was no significant difference (P>0.05) between cell types

for DNA methylation or histone acetylation. The percentage of cleaved and developing blastocyst embryos from the ADAS cells (62% and 8%) and skin fibroblasts cells (42% and 8%)were not different (P>0.05). Interspecies nuclear transfer utilized eland ADAS cells into enucleated bovine oocytes. A total of 3 interspecies embryos (1%) developed to blastocyst.

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