Differentiation of Stromal-Vascular Cells Isolated from Canine Adipose Tissues in Primary Culture.

Abstract

ABSTRACT. A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 µM indomethacin, 10 µg/ml insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental), demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 ± 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development. KEY WORDS: adipocyte differentiation, canine, cell culture, dimethylsulfoxide, indomethacin.J. Vet. Med. Sci. 63(1): 17-23, 2001 In 1974 Green and Kehinde reported that sublines of 3T3 cells underwent adipose differentiation in vitro [19], and since then many other adipogenic cell lines have been established [7,30,34,36]. The development of culture conditions supporting the proliferation and differentiation of adipogenic cell lines or stromal-vascular (S-V) cells from adipose tissues has provided interesting models to study the nature and mechanisms of factors which control the process of adipocyte differentiation. In primary culture, cells carry physiological host factors from the donor animal to the culture [4]. This fact might be an advantage in the study of the physiological regulatory mechanisms when compared with other adipose precursor cells in established cell lines [40]. In recent years, primary culture systems have been established for rat, mouse, sheep, pig, cattle and human preadipocyte differentiation [3,4,6,10,23,37]. However, such culture conditions developed for one species cannot be simply applied to other species, because the specific properties of preadipocytes are different among animal species. In addition, obesity due to excessive formation of adipose tissue is the most common nutritionally related-health problem for dogs and cats [15], and 25 to 44% of pet dogs are obese [16,29]. Therefore, in order to gain a better understanding of factors involved in the regulation of canine adipo...

Newly developed primary culture of rat visceral adipocytes and their in vitro characteristics

“…In a recent study using SVCs isolated from a variety of canine adipose tissue, the rate of differentiation to mature adipocytes was significantly lower from omental SVCs than from perirenal, subcutaneous or inguinal SVCs under the same medial conditions (Wu et al, 2001). Although peroxisome proliferator-activated receptor (PPAR)-g agonists are known to promote differentiation (Shao and Lazar, 1997;Wu et al, 1999) and maturation in adipocytes (Okuno et al, 1998), they increase the weight of only subcutaneous white and brown adipose tissues but not visceral white adipose tissue in rats in vivo (Berthiaume et al, 2004).…”

Adipokine expression and secretion by canine adipocytes: stimulation of inflammatory adipokine production by LPS and TNFα

“…This was followed by two washes before resuspension in erythrocyte lysis buffer for maximally 10 min. Finally, cells were resuspended in inoculation medium [52], counted with a haemocytometer and diluted in inoculation medium to a plating density of 40,000-60,000 cells/cm 2 . The cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 /95% air.…”

“…The cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 /95% air. After 72 h, the preconfluent cells were washed three times with PBS and the inoculation medium replaced by induction medium; after 48 h, the induction medium was replaced by feeding medium which was renewed every 48 h [52]. All culture media contained 100 U/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B (Invitrogen).…”

In Vitro Differentiation of Canine Celiac Adipose Tissue-Derived Stromal Cells into Neuronal Cells

“…Adipose tissue-derived stromal Cells (ATSCs): ATSCs were prepared according to the method of Wu et al [22]. Briefly, celiac adipose tissues were collected aseptically from 1-to 2-years-old beagle dogs under general anesthesia [4].…”

“…Among various adult stem cells, adipose tissue-derived stromal cells (ATSCs) are multipotential adult mesenchymal stem cells and differentiate into mesenchymal lineage cells such as adipocytes, osteocytes, chondrocytes, and myocytes [22,24]. Recently, Ashjian et al [1] reported that ATSCs was able to differentiate into nonmesodermal cells included ectodermal neural cells.…”