Title page for ETD etd-04032008-211137


Type of Document Dissertation
Author Vidal, Martin Andreas
Author's Email Address mvidal@vetmed.lsu.edu
URN etd-04032008-211137
Title Characterization and Comparison of Cell Frequency, Growth, and Multipotential Differentiation of Adult Mesenchymal Stromal Cells Derived from Equine Bone Marrow and Adipose Tissue
Degree Doctor of Philosophy (Ph.D.)
Department Veterinary Clinical Sciences
Advisory Committee
Advisor Name Title
Jill R. Johnson Committee Co-Chair
Rustin M. Moore Committee Co-Chair
Jeffrey M. Gimble Committee Member
Mandi J. Lopez Committee Member
Lee L. Southern Dean's Representative
Keywords
  • growth factor
  • stem cells
  • horse
  • fat
Date of Defense 2007-11-05
Availability unrestricted
Abstract
Equine bone marrow-derived mesenchymal stromal cells (MSCs) and adipose tissue-derived stromal cells (ASCs) were compared for frequency within their respective tissues, cell doubling characteristics and differentiation multipotential in culture based on histochemical staining and compositional analysis. Equine MSCs and ASCs from young adult horses were harvested and isolated from sternal bone marrow and supragluteal subcutaneous adipose tissue, respectively, and grown up to passage 10 (P10) to determine cell doubling characteristics. Limit dilution assays were performed on primary and passaged (P2, P4) MSCs and ASCs to determine the frequency of colony forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). Pellet cultures of MSCs and ASCs at P2 were performed in chondrogenic media with or without transforming growth factor (TGF-3) and bone morphogenic protein (BMP-6). Collagen type II expression, glycosaminoglycan and DNA content and pellet size were measured.

Primary MSCs doubled more slowly than subsequent MSC passages (DT = 4.9 1.6 days compared to 1.4 0.22 days). Doubling time of ASCs (2.1 0.9 days) was significantly slower than that of MSCs. Primary MSC frequency was 1 in 4,224 3,265 nucleated BM cells while the frequency of ASC was 1 in 2.3 0.4 nucleated stromal vascular fraction cells. Primary and subcultured MSCs showed robust adipogenic and osteogenic differentiation potential. MSC pellet cultures developed collagen type II expression by Day 7 and hyaline matrix by Day 14. ASC pellets only exhibited mild matrix or collagen formation under electron microscopic examination but showed no immunohistochemical expression of collagen type II. MSC pellets supplemented with growth factors were larger (p <0.0033) and showed significant increases in GAG concentration by Day 14 compared with all other pellets of both cell types (P<.0001).

The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of young adult horses are similar to those documented for MSCs of other species, whereas in vitro growth rate of ASCs differs from human ASCs. MSCs show earlier osteogenesis compared with ASCs and more robust chondrogenesis in the presence or absence of human recombinant TGF3 and BMP6.

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