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. 2016 Jul 1;78(6):987-95.
doi: 10.1292/jvms.15-0596. Epub 2016 Feb 26.

Evaluation of phenotypic, functional and molecular characteristics of porcine mesenchymal stromal/stem cells depending on donor age, gender and tissue source

Sun-A Ock  1 , Yeon-Mi Lee, Ji-Sung Park, Sharath Belame Shivakumar, Seon-Woung Moon, Nak-Ju Sung, Won-Jae Lee, Si-Jung Jang, Ju-Mi Park, Seung-Chan Lee, Sung-Lim Lee, Gyu-Jin Rho
Affiliations

Evaluation of phenotypic, functional and molecular characteristics of porcine mesenchymal stromal/stem cells depending on donor age, gender and tissue source

Sun-A Ock et al. J Vet Med Sci. .

Abstract

The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells (MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, in vitro differentiation ability, senescence-associated β-galactosidase (SA-β-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFβ, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA-β-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under in vitro culture conditions.

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Figures

Fig. 1.
Fig. 1.
Analysis of alkaline phosphatase (AP) activity and CD90 expression of MSCs harvested at 1 week and 8 months after birth. (A) AP activity. Scale bars=500 µm. (B) CD90 expression determined by flow cytometry. Ba, open and filled blue histograms display isotype-matched controls and positive reaction, respectively. Bb, CD90 expression levels of MSCs. Significant differences were considered when P<0.05.
Fig. 2.
Fig. 2.
In vitro differentiation of MSCs. (A) MSCs harvested at 1 week after birth. (B) MSCs harvested at 8 months after birth. Scale bars=200 µm.
Fig. 3.
Fig. 3.
Senescence-associated (SA)-β-galactosidase activity of MSCs obtained at 1 week and 8 months after birth. Scale bars=200 µm. The blue color indicates positivity for cell senescence.
Fig. 4.
Fig. 4.
Analysis of telomere length, telomere activity-related TERT protein expression and Oct 3/4 protein expression in MSCs harvested at 1 week and 8 months after birth. (A) Telomere restriction fragment lengths determined by Southern blotting using a telomere length assay. Significant differences were considered when P<0.05. (B) TERT and Oct3/4 protein expression determined by western blot analysis.
Fig. 5.
Fig. 5.
Cell cycle analysis of MSCs harvested at 1 week (A) and 8 months after birth (B). The first and second black filled peaks indicate the G0/G1 and G2/M phases, respectively, and the black oblique line-filled peak in the middle indicates the S phase.
Fig. 6.
Fig. 6.
Senescence-related proteins expression in MSCs harvested at 1 week and 8 months after birth. (A) IL6 as an immunomodulator, c-myc as a cell proliferation factor and oncoprotein, TGF β as a differentiation and cell proliferation factor, p53 as an external stress and antitumor factor, and p21 as a cell cycle arrest protein were assayed. (B) Bak as a proapoptotic protein and Bcl2 as an antiapoptotic protein were analyzed.
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