Journal of Embryo Transfer 2017; 32(3): 193-200
Published online September 29, 2017
https://doi.org/10.12750/JET.2017.32.3.193
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Hyo-Kyung Bae1, Bae-Dong Jung1, Seunghyung Lee2, Choon-Keun Park2, Boo-Keun Yang2, and Hee-Tae Cheong1,†
Correspondence to: Correspondence: Hee-Tae Cheong
The purpose of this study is to confirm whether spontaneous adipocyte generation during chondrogenic induction culture affects the chondrogenic differentiation of porcine skin-derived stem cells (pSSCs). For this purpose, chondrogenic differentiation characteristics and specific marker gene expression were analyzed using cell lines showing different characteristics of spontaneous adipocyte formation. Of the four different lines of pSSCs, the pSSCs-IV line showed higher Oil red O (ORO) and glycosaminoglycan (GAG) extraction levels. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the levels of adipogenic markers peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and adipocyte Protein 2 (aP2) mRNAs were significantly higher in pSSCs-IV than those of the other pSSC lines (
Keywords: Porcine skin-derived stem cells, Chondrogenesis, Spontaneous adipocyte generation
Since pig skin has similar histological and physiological characteristics to human skin, porcine skin-derived progenitors (pSKPs) are a precursor source for scientific researches and a variety of human biological studies (
Despite the large number of cells required to regenerate the damaged tissue, the number of cells for cell-based therapies is not sufficient. Many
Unlike
The purpose of this study is to investigate whether spontaneous adipocyte generation during chondrocyte induction cultures affects the chondrogenic differentiation of pSSCs. For this purpose, it was analyzed the chondrogenic differentiation potential and the expression of specific marker gene using cell lines showing different characteristics of spontaneous adipocyte formation.
Porcine skin samples were obtained from ear tissues of pigs (6 month-old females,
Prior to differentiation induction, the frozen pSSCs were cultured with high glucose (4.5 g/L)-DMEM supplemented with 10% (v/v) FBS and 1% P/S in 100-mm petri dishes (2.5 × 105 cells per dish). After 5 days, cells were harvested by treatment with 0.05% trypsin-EDTA for induction of specific differentiation. To induce chondrogenic differentiation, pSSCs were seeded in 24-well plates (BD biosciences) at a density of 1 × 104 cells/cm2 and grown in basic high glucose-DMEM containing 10% FBS for 2-3 days. At 70–80% confluence of the cells, the medium in test wells was replaced with chondrogenic induction medium consisting of 10 ng/mL transforming growth factor-beta 1 (TGF-
Three-dimensional pellets were created from pSSCs according to our previous report (
At the end of induction culture, each line of chondrogenicinduced pSSCs was stained with 0.6% oil Red O (ORO) solution (w/v, Sigma-Aldrich) for 1 hour or 0.5% Alcian blue (Sigma-Aldrich) in hydrochloric acid (pH, 1.0) for 30 minutes at room temperature to confirm the presence of adipose cells or acidic mucosubstances suggestive of chondrogenic differentiation, respectively. For quantification, ORO was eluted with isopropanol and quantified by measuring the optical density (OD) at 510 nm with an ELISA plate reader (VersaMax, Molecular Device, Sunnyvale, CA, USA). Alcian blue-stained cultures were extracted with 200 μL of 6 M guanidine-HCl (Daejung, Siheung, KOREA) for 2 h at room temperature. The OD of the extracted dye was read at 650 nm using an ELISA plate reader. We compared chondrogenic differentiation capacity between the four individual pSSCs lines (pSSCs-I, -II, -III, and –IV) showing the variety in the amount of adipocyte formation.
RNA preparation and quantitative real-time polymerase chain reaction (qRT-PCR) analysisTotal RNA was extracted from the induced cells of four individual pSSCs lines (pSSCs-I, -II, -III, and –IV) using Trizol (Invitrogen, Karlsruhe, Germany) to evaluate gene expression of chondrocyte and adipocyte markers. For analysis total RNA was diluted to 500 ng/μL using RNase free water and reverse transcribed into cDNA using RT-PreMix (Bioneer, Daejeon, Korea). The reaction was carried out using a Veriti® 96-well Thermal cycler (Applied Biosystems, Foster City, CA, USA) at 37°C for 15 sec, 50°C for 4 min, and 60°C for 30 sec. Before qRT-PCR, cDNA was diluted 1:5 (v/v) with RNase free water. The qRT-PCR was performed using 1 μL of diluted cDNA in combination with power SYBR Green PCR master Mix (TOPrealTM qPCR 2X PreMIX; SYBR Green with high ROX, Enzynomics, Daejeon, KOREA). Amplification reactions were conducted in a stepOne Plus instrument (Applied Biosystems) as follows: 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extraction at 72°C for 30 sec. All quantitative data were figured out by the comparative ΔΔCT method as the normalization against the corresponding housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and non-induction groups to obtain relative multiples. Experiments on each sample were performed three times. The primers used in this study peroxisome proliferator-activated receptor gamma 2 (PPAR
Table 1 . Primers sequences used for qRT-PCR.
Gene | Primer sequences (5’-3’) | Size (bp) | GeneBank accession NO. | Annealing temperature (°C, 40 Cycle) |
---|---|---|---|---|
PPARγ2 | F-GCGCCCTGGCAAAGCACT | 238 | AF103946 | 62 |
R-TCCACGGAGCGAAACTGACAC | ||||
aP2 | F-GGCCAAACCCAACCTGA | 167 | AF102872 | 62 |
R-GGGCGCCTCCATCTAAG | ||||
Agg | F-TTCCCTGAGGCCGAGAAC | 194 | AF201722 | 64 |
R-GGGCGGTAATGGAACACAAC | ||||
Col II | F-CTGGAGCTCCTGGCCTCGTG | 138 | AF201724 | 62 |
R-CAGATGCGCCTTTGGGACCAT | ||||
Sox9 | F- TGGCAAGGCTGACCTGAAG | 78 | AF029696 | 60 |
R- GCTCAGCTCGCCGATGT | ||||
GAPDH | F-GGGCATGAACCATGAGAAGT | 230 | AF017079 | 60 |
R-AAGCAGGGATGATGTTCTGG |
Results are representative of triplicates and are expressed as mean ± S.E. Cells from each group were evaluated separately. Statistical significance was determined by analysis of variance (ANOVA) and Duncan’s multiple range test. Differences between the two groups were measured by the Student’s t-tests using the Statistical Analysis System software package (SAS Institute, Cary, NC, USA). A value of
Small lipid droplets appeared on day 10 of chondrogenic induction cultures (Fig. 1A, arrow). Lipid droplets are used as an adipocyte indicator. As shown in Fig. 1B, the number of mature lipid droplets at day 24 of differentiation induction was increased on the monolayers of chondrogenic-inducted pSSCs
Generation of spontaneous lipid droplets during and after chondrogenic induction of pSSCs. A-B) Morphological changes of generated lipid droplets (arrows) during chondrogenic-differentiation
All four pSSCs lines (pSSCs-I, -II, -III, and -IV) showed adipocyte formation as well as accumulation of proteoglycans after chondrogenic induction culture, unlike non-induced control pSSCs. However, the concentrations of lipid droplets varied by individual cell lines, among them, pSSCs-IV showed a high lipid droplet concentration, whereas other cell lines showed few lipid droplets (Fig. 2). Quantitative analysis of ORO and Alcian blue extracts revealed that the levels of lipid droplet and glycosaminoglycan (GAG) extraction were significantly higher in the induced pSSC lines compared with corresponding non-induced controls (Fig. 3A, C). Comparing the levels of ORO extracts among chondrogenic-induced cell lines, pSSCs-IV (OD 18.99±2.35) was higher than other cell lines (pSSCs-I; 3.32±0.23, II; 4.22±0.13, and III; 2.10±0.07, OD, respectively, Fig. 3B). Similar pattern was observed in GAG extraction, the GAG extraction level of pSSCs-IV (OD 5.38±0.41) was higher than those of others (pSSCs-I: 3.13±0.14, II: 3.86±0.24, and III: 2.96±0.06, OD, respectively,
Morphological images of chondrogenic-induced pSSCs from four individual cell lines (pSSCs-I, II, III, and IV). After the induction for 24 days, the lipid droplets generated during chondrogenic differentiation of pSSCs were stained by Oil red O (red positive cells). Chondrogenesis was assessed by Alcian blue staining for the synthesis of glycosaminoglycans (GAGs) in induced cells. Control cells (non-induction) were cultured in DMEM+10% FBS for the same days. Non-indu, non-induction. Scale bars = 100 μm.
Quantitative data of lipid droplet formation and chondrogenic differentiation potentials from four individual cell lines (pSSCs-I, -II, -III, and –IV). The levels of lipid droplets and sulfated glycosaminoglycans (GAGs) in chondrogenic-induced pSSCs were determined by Oil red O and Alcian blue staining extraction after 24 days of chondrogenic differentiation. A and B) Quantitative data were based on Oil red O staining. Data in B represented as fold-change of non-induced control cells. C and D) Quantitative data of GAG accumulation. Data in D were represented as fold-change of 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. Non-indu, non-induction; Chondro, chondrogenic induction. *Significantly higher than non-induction control (
The qRT-PCR analysis revealed that adipogenic markers, PPAR
Gene expression of chondrocyte and adipocyte markers in four chondrogenic-induced pSSC lines. Gene expression levels were analyzed by qRT-PCR, normalized to GAPDH and represented as fold-change of 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. PPAR
The purpose of this study was to investigate the correlation of chondrogenic differentiations with spontaneously generated adipocytes during
The qRT-PCR results on gene expressions revealed that the pSSCs-IV line showed high expression of adipocyte-specific markers (PPAR
These results demonstrate that spontaneous adipocyte generation during chondrogenic differentiation affects chondrogenic differentiation potential. Spontaneous adipocyte generation has a positive effect on the chondrogenic differentiation of pSSCs. However, despite the difference in adipocyte generation, additional studies are needed on the correlation between adipocyte generation and chondrogenic differentiation, as the four pSSCs lines are very well differentiated into chondrocytes.
Journal of Embryo Transfer 2017; 32(3): 193-200
Published online September 29, 2017 https://doi.org/10.12750/JET.2017.32.3.193
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Hyo-Kyung Bae1, Bae-Dong Jung1, Seunghyung Lee2, Choon-Keun Park2, Boo-Keun Yang2, and Hee-Tae Cheong1,†
Correspondence to:Correspondence: Hee-Tae Cheong
The purpose of this study is to confirm whether spontaneous adipocyte generation during chondrogenic induction culture affects the chondrogenic differentiation of porcine skin-derived stem cells (pSSCs). For this purpose, chondrogenic differentiation characteristics and specific marker gene expression were analyzed using cell lines showing different characteristics of spontaneous adipocyte formation. Of the four different lines of pSSCs, the pSSCs-IV line showed higher Oil red O (ORO) and glycosaminoglycan (GAG) extraction levels. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the levels of adipogenic markers peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and adipocyte Protein 2 (aP2) mRNAs were significantly higher in pSSCs-IV than those of the other pSSC lines (
Keywords: Porcine skin-derived stem cells, Chondrogenesis, Spontaneous adipocyte generation
Since pig skin has similar histological and physiological characteristics to human skin, porcine skin-derived progenitors (pSKPs) are a precursor source for scientific researches and a variety of human biological studies (
Despite the large number of cells required to regenerate the damaged tissue, the number of cells for cell-based therapies is not sufficient. Many
Unlike
The purpose of this study is to investigate whether spontaneous adipocyte generation during chondrocyte induction cultures affects the chondrogenic differentiation of pSSCs. For this purpose, it was analyzed the chondrogenic differentiation potential and the expression of specific marker gene using cell lines showing different characteristics of spontaneous adipocyte formation.
Porcine skin samples were obtained from ear tissues of pigs (6 month-old females,
Prior to differentiation induction, the frozen pSSCs were cultured with high glucose (4.5 g/L)-DMEM supplemented with 10% (v/v) FBS and 1% P/S in 100-mm petri dishes (2.5 × 105 cells per dish). After 5 days, cells were harvested by treatment with 0.05% trypsin-EDTA for induction of specific differentiation. To induce chondrogenic differentiation, pSSCs were seeded in 24-well plates (BD biosciences) at a density of 1 × 104 cells/cm2 and grown in basic high glucose-DMEM containing 10% FBS for 2-3 days. At 70–80% confluence of the cells, the medium in test wells was replaced with chondrogenic induction medium consisting of 10 ng/mL transforming growth factor-beta 1 (TGF-
Three-dimensional pellets were created from pSSCs according to our previous report (
At the end of induction culture, each line of chondrogenicinduced pSSCs was stained with 0.6% oil Red O (ORO) solution (w/v, Sigma-Aldrich) for 1 hour or 0.5% Alcian blue (Sigma-Aldrich) in hydrochloric acid (pH, 1.0) for 30 minutes at room temperature to confirm the presence of adipose cells or acidic mucosubstances suggestive of chondrogenic differentiation, respectively. For quantification, ORO was eluted with isopropanol and quantified by measuring the optical density (OD) at 510 nm with an ELISA plate reader (VersaMax, Molecular Device, Sunnyvale, CA, USA). Alcian blue-stained cultures were extracted with 200 μL of 6 M guanidine-HCl (Daejung, Siheung, KOREA) for 2 h at room temperature. The OD of the extracted dye was read at 650 nm using an ELISA plate reader. We compared chondrogenic differentiation capacity between the four individual pSSCs lines (pSSCs-I, -II, -III, and –IV) showing the variety in the amount of adipocyte formation.
RNA preparation and quantitative real-time polymerase chain reaction (qRT-PCR) analysisTotal RNA was extracted from the induced cells of four individual pSSCs lines (pSSCs-I, -II, -III, and –IV) using Trizol (Invitrogen, Karlsruhe, Germany) to evaluate gene expression of chondrocyte and adipocyte markers. For analysis total RNA was diluted to 500 ng/μL using RNase free water and reverse transcribed into cDNA using RT-PreMix (Bioneer, Daejeon, Korea). The reaction was carried out using a Veriti® 96-well Thermal cycler (Applied Biosystems, Foster City, CA, USA) at 37°C for 15 sec, 50°C for 4 min, and 60°C for 30 sec. Before qRT-PCR, cDNA was diluted 1:5 (v/v) with RNase free water. The qRT-PCR was performed using 1 μL of diluted cDNA in combination with power SYBR Green PCR master Mix (TOPrealTM qPCR 2X PreMIX; SYBR Green with high ROX, Enzynomics, Daejeon, KOREA). Amplification reactions were conducted in a stepOne Plus instrument (Applied Biosystems) as follows: 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extraction at 72°C for 30 sec. All quantitative data were figured out by the comparative ΔΔCT method as the normalization against the corresponding housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and non-induction groups to obtain relative multiples. Experiments on each sample were performed three times. The primers used in this study peroxisome proliferator-activated receptor gamma 2 (PPAR
Table 1. Primers sequences used for qRT-PCR..
Gene | Primer sequences (5’-3’) | Size (bp) | GeneBank accession NO. | Annealing temperature (°C, 40 Cycle) |
---|---|---|---|---|
PPARγ2 | F-GCGCCCTGGCAAAGCACT | 238 | AF103946 | 62 |
R-TCCACGGAGCGAAACTGACAC | ||||
aP2 | F-GGCCAAACCCAACCTGA | 167 | AF102872 | 62 |
R-GGGCGCCTCCATCTAAG | ||||
Agg | F-TTCCCTGAGGCCGAGAAC | 194 | AF201722 | 64 |
R-GGGCGGTAATGGAACACAAC | ||||
Col II | F-CTGGAGCTCCTGGCCTCGTG | 138 | AF201724 | 62 |
R-CAGATGCGCCTTTGGGACCAT | ||||
Sox9 | F- TGGCAAGGCTGACCTGAAG | 78 | AF029696 | 60 |
R- GCTCAGCTCGCCGATGT | ||||
GAPDH | F-GGGCATGAACCATGAGAAGT | 230 | AF017079 | 60 |
R-AAGCAGGGATGATGTTCTGG |
Results are representative of triplicates and are expressed as mean ± S.E. Cells from each group were evaluated separately. Statistical significance was determined by analysis of variance (ANOVA) and Duncan’s multiple range test. Differences between the two groups were measured by the Student’s t-tests using the Statistical Analysis System software package (SAS Institute, Cary, NC, USA). A value of
Small lipid droplets appeared on day 10 of chondrogenic induction cultures (Fig. 1A, arrow). Lipid droplets are used as an adipocyte indicator. As shown in Fig. 1B, the number of mature lipid droplets at day 24 of differentiation induction was increased on the monolayers of chondrogenic-inducted pSSCs
Generation of spontaneous lipid droplets during and after chondrogenic induction of pSSCs. A-B) Morphological changes of generated lipid droplets (arrows) during chondrogenic-differentiation
All four pSSCs lines (pSSCs-I, -II, -III, and -IV) showed adipocyte formation as well as accumulation of proteoglycans after chondrogenic induction culture, unlike non-induced control pSSCs. However, the concentrations of lipid droplets varied by individual cell lines, among them, pSSCs-IV showed a high lipid droplet concentration, whereas other cell lines showed few lipid droplets (Fig. 2). Quantitative analysis of ORO and Alcian blue extracts revealed that the levels of lipid droplet and glycosaminoglycan (GAG) extraction were significantly higher in the induced pSSC lines compared with corresponding non-induced controls (Fig. 3A, C). Comparing the levels of ORO extracts among chondrogenic-induced cell lines, pSSCs-IV (OD 18.99±2.35) was higher than other cell lines (pSSCs-I; 3.32±0.23, II; 4.22±0.13, and III; 2.10±0.07, OD, respectively, Fig. 3B). Similar pattern was observed in GAG extraction, the GAG extraction level of pSSCs-IV (OD 5.38±0.41) was higher than those of others (pSSCs-I: 3.13±0.14, II: 3.86±0.24, and III: 2.96±0.06, OD, respectively,
Morphological images of chondrogenic-induced pSSCs from four individual cell lines (pSSCs-I, II, III, and IV). After the induction for 24 days, the lipid droplets generated during chondrogenic differentiation of pSSCs were stained by Oil red O (red positive cells). Chondrogenesis was assessed by Alcian blue staining for the synthesis of glycosaminoglycans (GAGs) in induced cells. Control cells (non-induction) were cultured in DMEM+10% FBS for the same days. Non-indu, non-induction. Scale bars = 100 μm.
Quantitative data of lipid droplet formation and chondrogenic differentiation potentials from four individual cell lines (pSSCs-I, -II, -III, and –IV). The levels of lipid droplets and sulfated glycosaminoglycans (GAGs) in chondrogenic-induced pSSCs were determined by Oil red O and Alcian blue staining extraction after 24 days of chondrogenic differentiation. A and B) Quantitative data were based on Oil red O staining. Data in B represented as fold-change of non-induced control cells. C and D) Quantitative data of GAG accumulation. Data in D were represented as fold-change of 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. Non-indu, non-induction; Chondro, chondrogenic induction. *Significantly higher than non-induction control (
The qRT-PCR analysis revealed that adipogenic markers, PPAR
Gene expression of chondrocyte and adipocyte markers in four chondrogenic-induced pSSC lines. Gene expression levels were analyzed by qRT-PCR, normalized to GAPDH and represented as fold-change of 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. PPAR
The purpose of this study was to investigate the correlation of chondrogenic differentiations with spontaneously generated adipocytes during
The qRT-PCR results on gene expressions revealed that the pSSCs-IV line showed high expression of adipocyte-specific markers (PPAR
These results demonstrate that spontaneous adipocyte generation during chondrogenic differentiation affects chondrogenic differentiation potential. Spontaneous adipocyte generation has a positive effect on the chondrogenic differentiation of pSSCs. However, despite the difference in adipocyte generation, additional studies are needed on the correlation between adipocyte generation and chondrogenic differentiation, as the four pSSCs lines are very well differentiated into chondrocytes.
Generation of spontaneous lipid droplets during and after chondrogenic induction of pSSCs. A-B) Morphological changes of generated lipid droplets (arrows) during chondrogenic-differentiation
Morphological images of chondrogenic-induced pSSCs from four individual cell lines (pSSCs-I, II, III, and IV). After the induction for 24 days, the lipid droplets generated during chondrogenic differentiation of pSSCs were stained by Oil red O (red positive cells). Chondrogenesis was assessed by Alcian blue staining for the synthesis of glycosaminoglycans (GAGs) in induced cells. Control cells (non-induction) were cultured in DMEM+10% FBS for the same days. Non-indu, non-induction. Scale bars = 100 μm.
Quantitative data of lipid droplet formation and chondrogenic differentiation potentials from four individual cell lines (pSSCs-I, -II, -III, and –IV). The levels of lipid droplets and sulfated glycosaminoglycans (GAGs) in chondrogenic-induced pSSCs were determined by Oil red O and Alcian blue staining extraction after 24 days of chondrogenic differentiation. A and B) Quantitative data were based on Oil red O staining. Data in B represented as fold-change of non-induced control cells. C and D) Quantitative data of GAG accumulation. Data in D were represented as fold-change of 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. Non-indu, non-induction; Chondro, chondrogenic induction. *Significantly higher than non-induction control (
Gene expression of chondrocyte and adipocyte markers in four chondrogenic-induced pSSC lines. Gene expression levels were analyzed by qRT-PCR, normalized to GAPDH and represented as fold-change of 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. PPAR
Table 1 . Primers sequences used for qRT-PCR..
Gene | Primer sequences (5’-3’) | Size (bp) | GeneBank accession NO. | Annealing temperature (°C, 40 Cycle) |
---|---|---|---|---|
PPARγ2 | F-GCGCCCTGGCAAAGCACT | 238 | AF103946 | 62 |
R-TCCACGGAGCGAAACTGACAC | ||||
aP2 | F-GGCCAAACCCAACCTGA | 167 | AF102872 | 62 |
R-GGGCGCCTCCATCTAAG | ||||
Agg | F-TTCCCTGAGGCCGAGAAC | 194 | AF201722 | 64 |
R-GGGCGGTAATGGAACACAAC | ||||
Col II | F-CTGGAGCTCCTGGCCTCGTG | 138 | AF201724 | 62 |
R-CAGATGCGCCTTTGGGACCAT | ||||
Sox9 | F- TGGCAAGGCTGACCTGAAG | 78 | AF029696 | 60 |
R- GCTCAGCTCGCCGATGT | ||||
GAPDH | F-GGGCATGAACCATGAGAAGT | 230 | AF017079 | 60 |
R-AAGCAGGGATGATGTTCTGG |
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