J Vet Sci.  2019 Jan;20(1):16-26. 10.4142/jvs.2019.20.1.16.

Correlation of spontaneous adipocyte generation with osteogenic differentiation of porcine skin-derived stem cells

• Bae HK
• Jung BD
• Lee S
• Park CK
• Yang BK
• Cheong HT

• KMID: 2434773
• DOI: http://doi.org/10.4142/jvs.2019.20.1.16

Abstract

The objective of this study was to examine effects of spontaneous adipocyte generation on osteogenic differentiation of porcine skin-derived stem cells (pSSCs). Correlation between osteogenic differentiation and adipocyte differentiation induced by osteocyte induction culture was determined using different cell lines. Osteogenic differentiation efficiency of pSSCs was then analyzed by controlling the expression of adipocyte-specific transcription factors during osteogenic induction culture. Among four cell lines, pSSCs-II had the lowest lipid droplet level but the highest calcium content (p < 0.05). It also expressed significantly low levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and adipocyte protein 2 (aP2) mRNAs but very high levels of runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) mRNAs as osteogenic makers (p < 0.05). Oil red O extraction was increased by 0.1 µM troglitazone (TGZ) treatment but decreased by 50 µM bisphenol A diglycidyl ether (BADGE) (p < 0.05). Calcium content was drastically increased after BADGE treatment compared to that in osteogenic induction control and TGZ-treated pSSCs (p < 0.05). Relative expression levels of PPARγ2 and aP2 mRNAs were increased by TGZ but decreased by BADGE. Expression levels of Rucx2 and ALP mRNAs, osteoblast-specific marker genes, were significantly increased by BADGE treatment (p < 0.05). The expression level of BCL2 like 1 was significantly higher in BADGE-treated pSSCs than that in TGZ-treated ones (p < 0.05). The results demonstrate that spontaneous adipocyte generation does not adversely affect osteogenic differentiation. However, reducing spontaneous adipocyte generation by inhibiting PPARγ2 mRNA expression can enhance in vitro osteogenic differentiation of pSSCs.

Keyword

Porcine skin-derived stem cells; Osteogenesis; Spontaneous adipocyte generation; Peroxisome proliferator-activated receptor gamma 2

MeSH Terms

Adipocytes*
Alkaline Phosphatase
Calcium
Cell Line
Ether
In Vitro Techniques
Lipid Droplets
Osteocytes
Osteogenesis
PPAR gamma
RNA, Messenger
Stem Cells*
Transcription Factors
Alkaline Phosphatase
Calcium
Ether
PPAR gamma
RNA, Messenger
Transcription Factors

Figure

• 

  Fig. 1 Analysis of osteogenic differentiation in porcine skin-derived stem cells (pSSCs). (A) Morphological images of osteogenic-induced pSSCs from four individual cell lines (pSSCs-I, -II, -III, and -IV). After induction for 24 days, spontaneous lipid droplets generated during osteogenic differentiation of pSSCs were stained by Oil red O (ORO; red, positive cells). Osteogenesis was assessed by von Kossa staining for mineralization in induced cells. Control cells (Non-indu) were cultured in Dulbecco's modified Eagle's medium + 10% fetal bovine serum for the same duration. Scale bars = 100 µm (A). (B–E) Quantitative data of lipid droplet formation and osteogenic differentiation potentials from pSSCs-I, -II, -III, and -IV. Levels of lipid droplets and calcium contents in osteogenic-induced pSSCs after 24 days of osteogenic differentiation were determined by ORO and calcium deposit results, respectively. (B and C) Quantitative data based on ORO staining. Data in panel C in Fig. 1 are represented as fold-change from non-induced control cells. (D and E) Quantitative data of calcium contents. Data in panel E in Fig. 1 are represented as fold-change from non-induced control cells. OD, optical density; Non-indu, non-induction; Osteo, osteogenic induction. *Significantly higher than non-induction control (p < 0.05). a–cValues with different letters differ significantly (p < 0.05).

• 

  Fig. 2 Gene expression levels of osteoblast and adipocyte markers in four osteogenic-induced porcine skin-derived stem cell (pSSC) lines. Gene expression levels were analyzed by quantitative real-time polymerase chain reaction, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and represented as fold-change from non-induced control cells. Data (mean ± SE) of pSSCs-I, -II, -III, and -IV were obtained from four donor biological samples with at least three trials. PPARγ2, peroxisome proliferator-activated receptor gamma 2; aP2, adipocyte protein 2; C/EBP-α, CCAAT/enhancer-binding protein-alpha; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; Col I, collagen type I; SPP1, osteopontin. a–dValues with different letters differ significantly (p < 0.05).

• 

  Fig. 3 Osteogenic differentiation potential changes of porcine skin-derived stem cells (pSSCs) treated by peroxisome proliferator-activated receptor gamma (PPARγ) regulators. Detection of lipid droplets and calcium deposition in osteogenic induction culture of pSSCs following treatment with troglitazone (TGZ) or bisphenol A diglycidyl ether (BADGE). (A and B) PPARγ2 (PPARγ2) mRNA expression level following treatment with various concentrations of TGZ and BADGE during osteogenic differentiation. (C) Oil red O (ORO) and von Kossa stained images. Scale bars = 100 µm (C). (D) Relative ORO extraction level of osteogenic-induced pSSCs. (E) Relative calcium contents of osteogenic-induced pSSCs. These pSSCs were cultured in osteogenic medium with 0.1 µM TGZ or 50 µM BADGE for 24 days. Data are expressed as relative activity compared to osteogenic-induced control (mean ± SE) from three replicates. Non-indu, non-induction. a–dValues with different letters differ significantly (p < 0.05).

• 

  Fig. 4 Gene expression of osteoblast and adipocyte markers in osteogenic-induced porcine skin-derived stem cells treated with troglitazone (TGZ) or bisphenol A diglycidyl ether (BADGE). Levels of adipogenic and osteogenic-related genes were assayed by quantitative real-time polymerase chain reaction. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as fold-change from osteogenic-induced control. All data are expressed as relative activity compared to osteogenic-induced control (mean ± SE) from three replicates. Non-indu, non-induction; Control, induced control; PPARγ2, peroxisome proliferator-activated receptor gamma 2; aP2, adipocyte protein 2; C/EBP-α, CCAAT/enhancer-binding protein-alpha; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; SPP1, osteopontin; Col I, collagen type I. a–dValues with different letters differ significantly (p < 0.05).

• 

  Fig. 5 Expression of apoptotic- and key signaling pathways-related genes controlling both adipogenesis and osteogenesis in porcine skin-derived stem cells (pSSCs) treated with troglitazone (TGZ) or bisphenol A diglycidyl ether (BADGE). Apoptosis and key signaling pathways-related gene levels were assayed by quantitative real-time polymerase chain reaction. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as fold-change from non-induced control. All data are expressed as relative activity compared to non-induced control (mean ± SE) from three replicates. These pSSCs were cultured in osteogenic medium with 0.1 µM TGZ or 50 µM BADGE for 24 days. Non-indu, non-induction; Osteo, osteogenic induction; BAX, BCL2-associated X protein; Casp-3, Caspase-3; Bcl2L1, BCL2 like 1; IGF1, insulin-like growth factor 1; IGF1-R, IGF1-receptor. a–cValues with different letters differ significantly (p < 0.05).

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