Medium containing Fetal Bovine Serum (FBS) provides a supportive environment for isolation and expansion of mesenchymal stromal/stem cells (MSCs); however, the inherent variability of FBS may ...contribute to inconsistencies in cell growth and yield between batches of stem cell products. For this reason, we set out to develop a serum-free medium capable of supporting the in vitro expansion of MSCs. First a naïve serum-free medium was formulated by Sato's approach. Once it was established that the naïve serum-free medium supported the expansion of canine adipose-derived MSCs (Ad-MSCs), the serum-free medium was optimized by addition of growth factors. Combinations of growth factors were chosen and compared by their effect on cell proliferation and colony formation. Growth characteristics of canine adipose-derived MSCs cultured in the serum-free medium were comparable to those cultured in standard FBS containing medium. In addition, cell surface marker expression and differentiation potential of serum-free and FBS-based cultures were also comparable. However, a commercial serum-free medium developed for human MSC culture did not support growth of canine Ad-MSCs. In summary, canine Ad-MSCs isolated and cultured in serum-free medium retained the basic characteristics of MSCs cultured in FBS containing medium.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The therapeutic potential of stem cells has fascinated those interested in treating diseases in both human and animal subjects. Although the exact mechanism of action and the definitive effectiveness ...of stem cell therapies remain unclear, animal owner perceptions and a desire for improved treatment options have fueled the interest of clinicians and stakeholders. Standards do not yet exist to define the critical attributes of mesenchymal stem/stromal cell (MSC)-based products derived from veterinary species such as the dog, cat, and horse. This has led veterinary stakeholders to adopt those guidelines and criteria set forth for human MSC-based products; however, these criteria are not always applicable to MSCs from dogs, cats, and horses (e.g., variability in species-specific cell surface marker expression and antibody cross reactivity). Establishing useful standards and meaningful product quality criteria as well as the understanding of full spectrum of MSC functions and preclinical evidence for safety and therapeutic efficacy for veterinary (companion and recreational animals) MSC-based-products will be critical to furthering product development, and may ultimately facilitate the availability of FDA-approved MSC-based products for use in veterinary medicine.
Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic ...benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90+CD44+MHC I+CD14−CD29−CD34−MHC II−. Canine MSCs were further characterized using real-time RT-PCR as CD105+CD73+CD14+CD29+MHC II+CD45− at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Non-steroidal anti-inflammatory drugs (NSAID) are a family of chemicals that function to reduce pain, fever, and inflammation, and they are commonly used in people and animals for this purpose. ...Currently there are no NSAIDs approved for the management of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess efficacy. A previous in vitro study examining biomarkers of inflammation identified fourteen genes that were significantly altered in response to Escherichia coli lipopolysaccharide (LPS)-induced inflammation. In the present study, five of those fourteen genes were tested in vivo to determine if the same effects observed in vitro were also observed in vivo. Plasma levels of prostaglandin E2 (PGE2), an essential mediator of fever and inflammation, were also determined. Two groups of swine were stimulated with LPS with the second group also treated with flunixin meglumine. Blood was collected at 0, 1, 3, 6, 8, 24, and 48h post LPS-stimulation. The RNA was extracted from the blood and quantitative real-time-PCR (qRT-PCR) was utilized to determine the expression patterns of CD1, CD4, serum amyloid A2 (SAA2), Caspase 1, and monocyte chemoattractant protein 1 (MCP-1). The LPS-stimulated animals demonstrated a statistically significant alteration in expression of SAA2 and CD1 at 3h post-stimulation. Flunixin meglumine treated animals’ demonstrated reduced expression of CD1 in comparison to the LPS-stimulated swine at 24 and 48h post LPS-stimulation. Flunixin meglumine treated animals exhibited reduced expression of SAA2 at 48h post-stimulation compared to LPS-stimulated swine. Swine treated with LPS demonstrated statistically significant increases in plasma PGE2 at 1h post-stimulation. Swine treated with flunixin meglumine had no increase in plasma PGE2 levels at any time. These results demonstrate that PGE2 production, along with two out of five genes (SAA2 and CD1) have the potential to serve as early biomarkers of inflammation as well as indicators of NSAID efficacy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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