Abstract
Canine adipose derived stem cells (ASCs) hold a great promise for the therapy of osteoarthritis in veterinary medicine. Current therapy is an autologous, stromal vascular fraction. Allogeneic ASCs provide many advantages, including efficient, cost-effective treatments while eliminating a surgical procedure in a diseased animal. Cultured ASCs can be expanded and characterized, allowing selection of desirable qualities. Use of allogeneic ASCs requires selection of a culture medium that provides consistent, desirable cellular products. The supplements within a medium can greatly influence cellular phenotypes. We hypothesized that medium type influenced cellular phenotype, allowing selection of a specified cellular product for clinical applications. We evaluated ASCs derived from adipose tissue of six dogs, assessing mRNA expression of proinflammatory: interleukin-1b, cyclooxygenase-2, and anti-inflammatory mediators: tissue inhibitor metalloproteinase-2 and interleukin -1 receptor antagonist, via quantitative RT-PCR prior to, and following culture in five cell culture media: basic cell growth medium (BGM), Keratinocyte N acetyl-L-cysteine supplemented (KNAC) medium, Multipotent Adult Progenitor Cell (MAPC) medium, serum free medium (SFM) and xeno-free medium. Major histocompatability complex I (MHCI), major histocompatability complex II (MHCII), CD44 and CD90 immunophenotypes were assessed via flow cytometry analysis. Tri-lineage differentiation (bone, adipose and cartilage tissue) was utilized to verify multipotency. SFM and xeno-free culture conditions did not produce cell expansion sufficient to assess phenotype. ASCs prior to culture had wide variability in all mediator levels, while culturing in the remaining conditions resulted in more predictable expression levels of inflammatory mediators, with a decrease in all levels. Cultured ASCs retained expression of cell surface markers MHCI, CD44 and CD90, while decreasing MHCII expression levels. KNAC and MAPC medium conditions consistently produced tri-lineage differentation; BGM, SFM and xeno-free medium did not. Culture condition will influence phenotype of ASCs, and should be selected according to the intended therapeutic effect.
Highlights
Mesenchymal stem cells have been identified and isolated from multiple adult tissues [1] [2]
Growth of adipose derived stem cells (ASCs) in serum free medium (SFM) and xeno free conditions was very poor and 80% confluency of cells was not reached in either medium condition
When comparing population doubling times within a passage, basic cell growth medium (BGM) created significantly shorter doubling times when compared to Keratinocyte N acetyl-L-cysteine supplemented (KNAC) (p-value = 0.028) and Multipotent Adult Progenitor Cell (MAPC) (p-value = 0.028) after passage two (Figure 1)
Summary
Mesenchymal stem cells have been identified and isolated from multiple adult tissues [1] [2]. They provide an ethically acceptable source of multipotent stem cells [3] [4]. They have been investigated for the therapy of many disease processes in several species. Using allogeneic stem cells may provide advantages over autologous SVF, including more efficient, cost effective treatments without the need for a surgical procedure on the patient in need of therapy. Allogeneic stem cells have been successfully and safely used to treat other disease processes in dogs [8]-[11]
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