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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.32 No.3 pp.193-200
DOI : https://doi.org/10.12750/JET.2017.32.3.193

Chondrogenic Differentiation of Porcine Skin-Derived Stem Cells with Different Characteristics of Spontaneous Adipocyte Formation

Hyo-Kyung Bae1, Bae-Dong Jung1, Seunghyung Lee2, Choon-Keun Park2, Boo-Keun Yang2, Hee-Tae Cheong1
1College of Veterinary Medicine and Institute of Veterinary Science
2College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea
Correspondence: Hee-Tae Cheong +82-33-250-8659htcheong@kangwon.ac.kr
20170804 20170905 20170911

Abstract

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 (P<0.05). Among three chondrogenic markers, collagen type II (Col II) and sex determining region Y-box (Sox9) mRNAs were strongly expressed in pSSCs-IV (P<0.05), but not in aggrecan (Agg), which was significantly higher in pSSCs-II (P<0.05). These results demonstrate that the spontaneous adipocyte generation during chondrogenic differentiation has a positive effect on the chondrogenesis of pSSCs. More research is needed on the correlation between adipocyte generation and cartilage formation.


초록


    National Research Foundation of Korea
    NRF-2013R 1A1A2006182

    INTRODUCTION

    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 (Dick and Scott, 1992, Lermen et al., 2010; Derks et al., 2013). The pSKPs are multipotent stem cells that can differentiate into various mesenchymal cell types (Dyce et al., 2006). Recently, our group also demonstrated the ability of porcine skin-derived stem cells (pSSCs) to differentiate into mesodermal cells (adipocytes, chondrocytes, and osteoblasts) in vitro (Hwang et al., 2015).

    Despite the large number of cells required to regenerate the damaged tissue, the number of cells for cell-based therapies is not sufficient. Many in vitro studies have demonstrated the use of a variety of culture methods to improve the efficiency of cell isolation and expansion and direct differentiation into specific lineage in vitro. These various culture environments, such as three-dimensional (3-D) structure-alginate (Marsich et al., 2008), collagen hydrogel (Zheng et al., 2009), Poly (D,L-lactide-co-glycolide) scaffold (Xin et al., 2007), standard culture medium (Peister et al., 2004; Bekhite et al., 2014), feeder layers (Hattori et al., 2008), and oxygen tension (Bekhite et al., 2014; Lin et al., 2014) can influence the differentiation ability of MSCs into numerous cell types in the humans, mice and other species (Araña et al., 2013).

    Unlike in vitro, the in vivo biochemical environment of cartilage and bone is composed of connective tissue, adipose tissue, nerves and tendons. The induction process for cell-specific differentiation can be influenced by various native environments of cartilage and bone in vivo. The major cell types observed in cartilage are chondrocyte, osteoprogenitor cells, osteocytes and adipocytes, all of which are derived from common multipotential mesenchyme cells. We have recently observed that lipid (such as free fatty acids) droplets are generated during the chondrogenic differentiations of pSSCs (unpublished data). In fact, adipocytes exist within the bones and may around the cartilage in the body. In addition, it was suggested that the addition of leptin, an obesity-associated cytokine-like hormone, during pellet culture of rat growth plate chondrocytes increased terminal differentiation and proliferation by activating the mitogen-Activated Protein Kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (Wang et al., 2012). Although, many of in vitro or in vivo studies have addressed adipose tissue-produced factors such as adiponectin, plasminogen activation inhibitor-1, tumor necrosis factor-α, resistin, and leptin (Friedman and Halaas, 1998; Steppan et al., 2001; Wang et al., 2012), the relationship between the existence of adipocytes and other cells during specific-lineage differentiation remains unclear.

    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.

    MATERIALS and METHODS

    1.Cell isolation

    Porcine skin samples were obtained from ear tissues of pigs (6 month-old females, n = 4). First, ear tissues were collected and transferred to the laboratory in DPBS supplemented with 2% penicillin/streptomycin (P/S, Corning Cellgro, Manassas, VA, USA). To isolate pSSCs, cartilage tissue was completely removed from the ear skin samples and the epidermis and dermis of the skin tissue were washed twice with warm Hank’s balanced salt solution (HBSS, WelGENE, Daegu, Korea). The specimens were then finely chopped with a scalpel blade and digested with 10 mL digestion solution containing 0.25% trypsin-EDTA (Sigma- Aldrich, St. Louis, MO, USA) and 0.1% collagenase type I in DPBS by agitation for 30 min at 37°C. Cells were collected and transferred to 100-mm culture dishes (BD biosciences, Bedford, MA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, N.Y., USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% P/S at 37°C, 5% CO2 in air for 10 days or more. The cells from each pig were cultured separately and passaged one or two times while the culture medium was changed every 2 days. The MSC characteristics of pSSCs were confirmed by fluorescence activated cell sorting (FACS) analysis in our previous report (Hwang et al., 2015).

    2.Induction of chondrogenic differentiation in vitro

    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-β1, ProSpec-Tany TechnoGene, Rehovot, Israe), 1% Insulin-Transferrin-Selenium-A Supplement (Gibco), 50 μM ascorbic acid (Sigma-Aldrich), 0.1 μM dexamethasone (Sigma-Aldrich), 1% antibiotics, and 10% FBS. Cells were further cultured for 24 days to induce chondrogenic differentiation. The induction medium was carefully replaced every 2–3 days. The pSSCs cultured in DMEM supplemented with 10% FBS and 1% P/S were considered as non-induced controls.

    3.Induction of chondrogenic differentiation in 3-D pellet culture

    Three-dimensional pellets were created from pSSCs according to our previous report (Hwang et al., 2015). Briefly, 2.5 × 105 cells/cm2 were transferred to a 15 mL centrifuge tube and centrifuged at 500 X g for 5 minutes to form a micropellet. The pelleted cells were incubated in a chondrogenic induction medium for 26 days. The induction medium was replaced every 2 to 3 days. Histologic specimens of pellets were fixed in formalin, embedded in paraffin, and sectioned transversely into 4-μm-thick slices. Slices were stained with H&E (Sigma-Aldrich) or Alcian blue stain.

    4.Analysis of differentiation potential

    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.

    5.RNA preparation and quantitative real-time polymerase chain reaction (qRT-PCR) analysis

    Total 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 (PPARγ2), adipocyte Protein 2 (aP2), CCAAT/enhancer-binding protein-alpha (C/EBP-α), collagen type II (Col II), aggrecan (Agg), sex determining region Y-box (Sox9), and glyceraldehyde 3- phosphate dehydrogenase (GAPDH) are shown in Table 1.

    6.Statistical analysis

    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 P<0.05 was considered statistically significant.

    RESULTS

    1.Observation of spontaneous adipocyte formation during in vitro differentiation.

    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 in vitro. Lipid droplets were also observed in 3-D pellet of chondrogenic-induced pSSCs (Fig. 1C and D, arrows).

    2.Expressions of adipose and glycosaminoglycan in the pSSC lines

    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, P<0.05, Fig. 3D).

    3.Specific marker gene expression in the pSSCs lines

    The qRT-PCR analysis revealed that adipogenic markers, PPARγ2 and aP2 mRNA expression levels were significantly higher in pSSCs-IV than those of the other pSSC lines (P<0.05, Fig. 4). Agg mRNA, a chondrogenic marker, was not significantly expressed, but the other two chondrogenic markers Col II and Sox9 mRNAs were strongly expressed in the pSSCs-IV (P<0.05).

    DISCUSSION

    The purpose of this study was to investigate the correlation of chondrogenic differentiations with spontaneously generated adipocytes during in vitro chondrogenic differentiation induction of pSSCs. By observing the spontaneous generation of lipid droplets in the pSSC lines during chondrocyte differentiation, we compared the chondrogenic differentiation potentials in the four individual pSSC lines showing varied amount of adipocyte formation during differentiation induction. As shown in Fig. 2, the induction of chondrogenic differentiation was successfully conducted while simultaneously confirming the detection of lipid droplets. Our results indicated that the pSSCs-IV line exhibited the highest levels of ORO and GAG extraction during chondrogenic induction cultures (see Fig. 3B and D). We could not confirm whether the increased GAG accumulation of the pSSCs-IV line is due to the increased adipocytes or the ability of the cell line itself to differentiate. Although questionable points still remain, it is considered that cytokines secreted by adipocyte such as leptin increase the synthesis of GAG and collagen (Dumond et al., 2003; Wang et al., 2012). We tried to examine the effect of adipocyte generation on the chondrogenic differentiation by pre- or co-culture of pSSCs with adipogenic medium or factors at the early stage of chondrogenic differentiation induction. However, there was no significant effect on the number of lipid droplets and the differentiation of pSSCs into chondrocyte (data not shown).

    The qRT-PCR results on gene expressions revealed that the pSSCs-IV line showed high expression of adipocyte-specific markers (PPARγ2 and aP2) and high expression levels of chondrogenic factors (Col II and Sox9), while low Agg expression level simultaneously (See Fig. 4). We suggested that the expression of Agg mRNA was directly regulated by the transcriptional factor PPARγ2, unlike the expression of other genes that may be affected by the differentiation environments. PPARγ activity interferes with the onset of chondrogenic differentiation of MSCs by induction of adipocyte differentiation (Kubota et al., 1999; Zou et al., 2008). The different expression patterns of the three chondrocyte specific markers Agg, Col II and Sox9 may be due to the different operating periods of each marker. PPARγ2 and Agg mRNAs are mainly expressed at the early stages, whereas aP2 and Col II mRNAs are expressed at the late stages of adipogenic and chondrogenic differentiation, respectively (Zou et al., 2008). In chondrogenesis, prominent gene expression is observed during proliferation, extracellular matrix (ECM) synthesis and maturation of multipotential progenitor cells (Zou et al., 2008). However, the expression pattern of representative genes during differentiation of pSSCs into a specific lineage is unclear.

    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.

    ACKNOWLEDGMENTS

    This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R 1A1A2006182) and 2016 Research Grant from Kangwon National University (No. 520160212).

    Figure

    JET-32-193_F1.gif

    Generation of spontaneous lipid droplets during and after chondrogenic induction of pSSCs. A-B) Morphological changes of generated lipid droplets (arrows) during chondrogenic-differentiation in vitro at Day 10 (A) and Day 24 (B). C-D) Cell pellets after induction into chondrocytes in 3-D cell culture for 26 days showing distinguished lipid droplets (arrows). (C) Alcian blue staining, (D) H&E staining. Scale bars = 100 μm.

    JET-32-193_F2.gif

    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.

    JET-32-193_F3.gif

    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 (P<0.05). a-cValues with different letters differ significantly (P<0.05).

    JET-32-193_F4.gif

    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. PPARγ2, Peroxisome proliferator-activated receptor gamma 2; aP2, adipocyte Protein 2; Sox9, sex determining region Y-box; Col II, collagen type II; Agg, aggrecan. a-cValues with different letters differ significantly (P<0.05).

    Table

    Primers sequences used for qRT-PCR.

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