J Vet Sci.  2017 Jun;18(2):141-148. 10.4142/jvs.2017.18.2.141.

Effect of donor age on the proliferation and multipotency of canine adipose-derived mesenchymal stem cells

• Lee J
• Lee KS
• Kim CL
• Byeon JS
• Gu NY
• Cho IS
• Cha SH

• KMID: 2412566
• DOI: http://doi.org/10.4142/jvs.2017.18.2.141

Abstract

Research into adipose tissue-derived mesenchymal stem cells (AD-MSCs) has demonstrated the feasibility of their use in clinical applications due to their ease of isolation and abundance in adipose tissue. We isolated AD-MSCs from young and old dogs, and the cells were subjected to sequential sub-passaging from passage 1 (P1) to P7. Canine AD-MSCs (cAD-MSCs) were examined for proliferation kinetics, expression of molecules associated with self-renewal, expression of cell surface markers, and differentiation potentials at P3. Cumulative population doubling level was significantly higher in cAD-MSCs of young donors than in those of old donors. In addition, expressions of CD73, CD80, Oct3/4, Nanog, cell survival genes and differentiation potentials were significantly higher in young donors than in old donors. The present study suggests that donor age should be considered when developing cell-based therapies for clinical application of cAD-MSCs.

Keyword

adipose mesenchymal stem cells; age; canine; differentiation; multipotency

MeSH Terms

Adipose Tissue/*cytology/physiology
Age Factors
Animals
Cell Differentiation/physiology
Cell Proliferation/*physiology
Dogs
Gene Expression Profiling/veterinary
Mesenchymal Stromal Cells/*physiology
Real-Time Polymerase Chain Reaction/veterinary
Reverse Transcriptase Polymerase Chain Reaction/veterinary

Figure

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  Fig. 1 Morphology and proliferation of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). (A) Morphology of cells passage 1–6 (P1–P6) derived from adipose tissues typically appeared as fibroblast-like. (B) Cumulative population doubling level (CPDL) of cAD-MSCs during continuous passages. CPDL of the cAD-MSCs increased at each passage until P3. Cells were grown in two age groups, YAD-MSCs and OAD-MSCs. GAPDH was used as a housekeeping control gene. The results are shown as the mean ± standard error of the mean (n = 5) obtained by three determinations. YAD, 7-month-old dogs; OAD, 10- to 11-year-old dogs. 100× (A).

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  Fig. 2 Expression pluripotency markers (Oct3/4, Sox2, and Nanog) of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). Using reverse transcriptase polymerase chain reaction (RT-PCR; A) and real-time quantitative RT-PCR (qRT-PCR; B), the expressions of stemness genes of cAD-MSCs were examined in two age groups (young and old) at passage 3. GAPDH was used as a housekeeping control gene. All mRNA data were normalized to the GAPDH levels, and the relative fold change in expression level is shown as a mean ± SEM (n = 3). *p < 0.05. YAD, 7-month-old dogs; OAD, 10- to 11-year-old dogs.

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  Fig. 3 Surface markers of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). MSC-specific cell surface markers (positive: CD44, CD54, CD61, CD73, CD80, CD90, and CD105; not detectable: CD29, CD34, CD117, and MHC-class II) of cAD-MSCs were observed by real-time quantitative reverse transcriptase polymerase chain reaction in two age groups (young and old) at passage 3. GAPDH was used as a housekeeping control gene. All mRNA data were normalized to the levels of GAPDH, and relative fold changes in expression levels are shown as the mean ± SEM (n = 3). *p < 0.05. YAD, 7-month-old dogs; OAD, 10- to 11-year-old dogs.

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  Fig. 4 Differentiation potentials of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). Adipocytes (Oil Red O; A), osteocytes (Alizarin Red; B), and chondrocytes (Alcian Blue; C) were positively stained in two age groups (young and old) at passage 3. (D) The mRNA expressions of adipocyte, osteocyte, and chondrocyte-related genes were detected by real-time quantitative reverse transcriptase polymerase chain reaction and compared between undifferentiated and differentiated cells in the two groups at passage 3. All mRNA data were normalized to the levels of GAPDH, and relative fold changes in expression levels are shown as the mean ± SEM (n = 3). *p < 0.05. 100× (A–C). YAD, 7-month-old dogs; OAD, 10- to 11-year-old dogs; LPL, lipoprotein lipase; OP, osteopontin; OC, osteocalcin; COL2A, type II-collagen.

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  Fig. 5 Cell survival-related genes of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). The mRNA expressions of cell survival-related genes such as TERT, DKC1, HDAC1, DNMT1, Bcl-2, LDHA, and SLC2A1 were examined by real-time quantitative reverse transcriptase polymerase chain reaction in two groups (young and old) at passage 3. GAPDH was used as a housekeeping control gene. All mRNA data were normalized to the levels of GAPDH, and relative fold changes in expression levels are shown as the mean ± SEM (n = 3). *p < 0.05. YAD, 7-month-old dogs; OAD, 10- to 11-year-old dogs; TERT, telomerase reverse transcriptase; DKC1, dyskerin pseudouridine synthase 1; HDAC1, histone deacetylase 1; DNMT1, DNA (cytosine-5)-methyltransferase 1; LDHA, lactate dehydrogenase A; SLC2A1, glucose transporter member 1.

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