Cell Transplantation, Vol. 25, pp. 449–461, 2016 Printed in the USA. All rights reserved. Copyright Ó 2016 Cognizant, LLC.
0963-6897/16 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368915X688641 E-ISSN 1555-3892 www.cognizantcommunication.com
Fetal Cartilage-Derived Cells Have Stem Cell Properties and Are a Highly Potent Cell Source for Cartilage Regeneration Woo Hee Choi,* Hwal Ran Kim,* Su Jeong Lee,* Nayoung Jeong,* So Ra Park,† Byung Hyune Choi,‡ and Byoung-Hyun Min*§¶ *Department of Molecular Science and Technology, Ajou University, Suwon, Korea †Department of Physiology, Inha University College of Medicine, Incheon, Korea ‡Department of Biomedical Sciences, Inha University College of Medicine, Incheon, Korea §Cell Therapy Center, Ajou University Medical Center, Suwon, Korea ¶Department of Orthopedic Surgery, Ajou University School of Medicine, Suwon, Korea
Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. However, the characteristics and the potential of progenitor cells from fetal tissue remain poorly defined. In this study, we examined cells from human fetal cartilage at 12 weeks after gestation in comparison with bone marrow-derived MSCs or cartilage chondrocytes from young donors (8–25 years old). The fetal cartilage-derived progenitor cells (FCPCs) showed higher yields by approximately 24 times than that of chondrocytes from young cartilage. The morphology of the FCPCs was polygonal at passage 0, being similar to that of the young chondrocytes, but it changed later at passage 5, assuming a fibroblastic shape more akin to that of MSCs. As the passages advanced, the FCPCs showed a much greater proliferation ability than the young chondrocytes and MSCs, with the doubling times ranging from 2~4 days until passage 15. The surface marker profile of the FCPCs at passage 2 was quite similar to that of the MSCs, showing high expressions of CD29, CD90, CD105, and Stro-1. When compared to the young chondrocytes, the FCPCs showed much less staining of SA-b-gal, a senescence indicator, at passage 10 and no decrease in SOX9 expression until passage 5. They also showed a much greater chondrogenic potential than the young chondrocytes and the MSCs in a three-dimensional pellet culture in vitro and in polyglycolic acid (PGA) scaffolds in vivo. In addition, they could differentiate into adipogenic and osteogenic lineages as efficiently as MSCs in vitro. These results suggest that FCPCs have stem cell properties to some extent and that they are more active in terms of proliferation and chondrogenic differentiation than young chondrocytes or MSCs. Key words: Fetal cartilage-derived progenitor cells (FCPCs); Mesenchymal stem cells (MSCs); Chondrocytes; Cartilage regeneration
INTRODUCTION The self-repair of articular cartilage is difficult when it becomes injured due to its low regenerative capacity caused by the lack of blood supply, low cellularity, and a limited number of progenitor cells (32). To replenish cells in areas with cartilage defects, surgical techniques such as the abrasion and microfracture arthroplasties have been clinically applied, but both are associated with the formation of fibrocartilage, which is weaker and less durable. Autologous chondrocyte transplantation (ACT) has also been used clinically for cartilage regeneration,
showing reasonable results as a cartilage repair strategy (5,42); however, it also has shortcomings in that only a small piece of cartilage tissue can be taken from a patient, and the number of chondrocytes is very limited. In addition, the chondrogenic phenotype and redifferentiation potential can vary depending on the patient and is easily lost, particularly in aged chondrocytes during expansion in a monolayer culture (3,15,21,39). To overcome the limitations of autologous chondrocytes, many studies have utilized mesenchymal stem cells (MSCs) isolated from various tissues, such as bone marrow,
Received January 18, 2014; final acceptance July 28, 2015. Online prepub date: July 13, 2015. Address correspondence to Professor Byoung-Hyun Min, Department of Orthopedic Surgery, Ajou University School of Medicine, Wonchon-dong, Youngtong-gu, Suwon, Gyeonggi-do, Korea. Tel: +82-31-219-4444; Fax: +82-31-219-4193; E-mail: [email protected] or Professor Byung Hyune Choi, Department of Biomedical Sciences, Inha University College of Medicine, 100 Inharo, Nam-gu, Incheon, 22212, Korea. Tel: +82-32-860-9881; Fax: +82-32-885-8302; E-mail: [email protected]
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synovial membrane, fat, or cord blood (10,29,30,35). MSCs are regarded as an attractive cell source for cartilage regeneration because they have high self-renewal ability and the multipotent ability to differentiate into various cell types, including chondrocytes in vitro (43). For cartilage regeneration, MSCs are simply injected into the defect area with or without cell carriers, or they are differentiated into chondrocytes to form cartilage tissue in vitro before implantation (7,12,25,33). However, MSCs have also been shown to have limited capacity to differentiate fully into chondrocytes and form intact cartilage tissues both in vitro and in vivo. In particular, cartilage tissues formed using MSCs were mostly subject to hypertrophic changes and lost their chondrogenic properties in vivo (8,11,13,41). Stem cells or progenitors isolated from fetal tissues such as liver, bone marrow, blood, lung, kidney, pancreas, placenta, brain, and spinal cord are another potent cell source for cell therapy and tissue engineering efforts (40). They have often been shown to have high multipotentiality and proliferation capacity, better sometimes than adult stem cells isolated from aged donors (23,28,36). Moreover, MSCs from fetal liver have been shown to elicit no alloreactive T-cell response, which suggests that fetal stem cells do not display an allogenic immune response (19,20). Previously, several studies attempted to use fetal cartilage-derived cells in cartilage tissue engineering. Fuchs et al. reported that ovine fetal cartilage cells formed better cartilage tissue than adult chondrocytes by producing more matrix molecules in the pellet culture (17). Quintin et al. showed that human fetal femoral head cells differentiated well into chondrogenic, adipogenic, and osteogenic lineages but with less efficiency than bone marrow MSCs (44). These studies imply that fetal cartilage-derived cells could be a potential cell source for cartilage regeneration, but it is necessary to identify the characteristics and therapeutic utility of fetal cartilage-derived cells further for additional clinical applications. In this study, we examined the characteristics of fetal cartilage-derived progenitor cells (FCPCs) in comparison with bone marrow MSCs and young chondrocytes in terms of their proliferation ability over passages, their plasticity, and their chondrogenic potential in vitro and in vivo. MATERIALS AND METHODS Cell Isolation and Culture The study was approved by the institutional review board (IRB) of the Ajou University Medical Center (AJIRB-CRO-07-139) and was carried out with the written consent of all donors. Human fetal cartilage tissues (n = 4, M12w-a, M12w-b, F12w-c, M11w) were obtained from patients following elective termination at 12 weeks after gestation, and cells were isolated from the femoral
Choi ET AL.
head of the cartilage tissue. Young chondrocytes were isolated from the knee cartilage tissue of surgery patients after trauma at 11~25 years old (n = 3, M11y, M18y, M25y). Aged chondrocytes were isolated from the knee cartilage tissue of three donors undergoing total knee replacement surgery (n = 3, F45y, M54y, F56y). Cartilage tissues were minced into small pieces and treated with 0.1% collagenase type 2 (Worthington Biochemical Corp., Freehold, NJ, USA) in high-glucose Dulbecco’s modified Eagle medium (DMEM; HyClone, Logan, UT, USA) containing 1% fetal bovine serum (FBS; HyClone) at 37°C under 5% CO2. After 12 h, isolated cells were cultured in DMEM supplemented with 10% FBS, 100 U/ ml penicillin G, and 100 μg/ml streptomycin (Pen-Strep; HyClone) at a density of 8 × 103 cells/cm2. MSCs were isolated from the bone marrow of fractured femurs of surgery patients who were 8~25 years old (n = 3, F8y, M11y, M25y). Briefly, mononuclear cells (MNCs) were collected by Ficoll (Ficoll-Paque PLUS; Amersham Biosciences, Piscataway, NJ, USA) gradient centrifugation, suspended in a-modified Eagle’s medium (a-MEM; HyClone) supplemented with 10% FBS, and plated at a density of 8 × 104 cells/cm2. After 6 days, nonadherent cells were removed, and the adherent MSCs were replenished with fresh medium. Cells were passaged at 80% confluence, where the plating density was approximately 8 × 103 cells/cm2. Flow Cytometry Analysis Cells at passages 2~3 were analyzed for the expression of stem cells markers on the cell surface. Cells in the suspension were incubated with anti-CD34-FITC, antiCD29-PE, anti-CD90-FITC (BD Biosciences, San Jose, CA, USA), anti-CD105-FITC (Ancell, Bayport, MN, USA), and anti-Stro-1 (R&D Systems, Minneapolis, MN, USA) antibodies for 40 min at 4°C. For the anti-Stro-1 antibody, cells were washed with phosphate-buffered saline (PBS; Welgene, Daegu, Korea) and then incubated with goat anti-mouse IgM-FITC (R&D Systems) for 30 min at 4°C. The dilution factors of each antibody were determined according to the manufacturers’ instructions. Stained cells were analyzed by flow cytometry (Becton Dickinson FACSvantage). Analysis of mRNA Levels The total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of RNA was used to synthesize cDNA by reverse transcription. PCR was performed using 0.5 mg of synthesized cDNA and specific primers for collagen II, SOX9, aggrecan, collagen I, and GAPDH. The sequences of the primers and the reaction conditions are listed in Table 1. Quantitative real-time PCR was performed with a CFX ConnectTM PCR machine (Bio-Rad, Hercules, CA, USA)
STEM CELL CHARACTERISTICS OF FETAL CARTILAGE CELLS
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Table 1. The Primers Used for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Primer
Sequence
Annealing Temp./ Time/Cycle
Product Size
GAPDH
Forward Reverse
5¢-GGTCATGAGTCCTTCCACGAT-3¢ 5¢-GGTGAAGGTCGGAGTCAACGG-3¢
59°C/30 s/27 cycles
520 bp
COL2A1
Forward Reverse
5¢-CTCCTGGAGCATCTGGAGAC-3¢ 5¢-ACCACGATCACCCTTGACTC-3¢
59°C/30 s/35 cycles
152 bp
ACAN
Forward Reverse
5¢-CCCCACTGGCCCCAAGAATCAAG-3¢ 5¢-CGCTGCGCCCTGTCAAAGTCG-3¢
65°C/30 s/27 cycles
319 bp
SOX9
Forward Reverse
5¢-CACACAGCTCACTCGACCTTG-3¢ 5¢-TTCGGTTATTTTTAGGATCATCTCG-3¢
60°C/30 s/30 cycles
76 bp
COL1A2
Forward Reverse
5¢-CTTCTGGCCCTGCTGGAAAAGATG-3¢ 5¢-CCCGGATACAGGTTTCGCCAGTAG-3¢
57°C/30 s/27 cycles
444 bp
using SYBR Premix Ex TaqTM (Takara Bio Inc., Otsu, Shiga, Japan). All reactions were duplicated, and each amplified signal of the (sex-determining region Y)-box 9 (SOX9) and collagen II a 1 (COL2A1) were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cellular Senescence Assay Senescent cells were determined by senescence-associated b-galactosidase (SA-b-gal) staining using a senescence cell histochemical staining kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were first fixed for 6 min at room temperature and then incubated with a staining mixture for 8 h at 37°C without CO2. Senescent cells were observed in blue color under a microscope (TE-2000; Nikon, Tokyo, Japan). Multilineage Differentiation For osteogenic and adipogenic differentiation, cells were plated in six-well plates (TPP, Trasadingen, Switz erland) at densities of 2 × 103 cells/cm2 and 2 × 104 cells/ cm2, respectively. After 24 h, the cells were incubated in a differentiation medium for each lineage. The osteogenic medium consisted of a-MEM supplemented with 10% FBS, 10 mM b-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), and 50 μg/ml ascorbate-2 phosphate (Sigma-Aldrich). The adipogenic medium consisted of a-MEM supplemented with 10% FBS, 1 µM dexamethasone, 10 μg/ml insulin (SigmaAldrich), 0.5 mM isobutyl-methylxanthine (IBMX; SigmaAldrich), and 0.1 mM indomethacin (Sigma-Aldrich). The control cells were incubated in a-MEM supplemented with 10% FBS for 3 weeks. After 3 weeks of differentiation, cells were stained with Alizarin red S (SigmaAldrich) and Oil red O (Sigma-Aldrich) to observe the degree of mineralization and the lipid droplets, respectively. For quantitative analysis, the adsorbed dyes were eluted with 3 ml of 10% acetic acid (Duksan Chemical,
Seoul, Korea) and 1.5 ml of 10% ammonium hydroxide (Duksan Chemical) for Alizarin red S and with 1 ml of isopropyl alcohol (Duksan Chemical) for Oil red O. The absorbance was measured at 405 nm and 500 nm, respectively. To present the data, values from the differentiated samples were normalized by the control values of undifferentiated cells. For chondrogenic differentiation, 3 × 105 cells were centrifuged at 500 × g for 5 min, and the cell pellet was cultured in a chondrogenic medium. The chondrogenic medium consisted of DMEM supplemented with 100 nM dexamethasone, 50 μg/ml ascorbate-2 phosphate, ITS supplement (Sigma-Aldrich), 40 μg/ml proline (SigmaAldrich), 1.25 mg/ml bovine serum albumin (BSA; Sigma-Aldrich), 100 μg/ml sodium pyruvate (SigmaAldrich), and 10 ng/ml TGF-b3 (R&D Systems). After 3 weeks of induction, the samples were fixed with 4% formaldehyde (Duksan Chemical) and embedded in paraffin wax. Sections with a thickness of 4 μm were prepared and stained with Safranin O (Sigma-Aldrich) to observe the sulfated glycosaminoglycans (sGAG). Cartilage Tissue Formation In Vivo To examine the potential for cartilage tissue formation, cells were seeded in a scaffold and implanted subcutaneously into the backs of BALB/c nude mice (male, 6 weeks of age; Orient Bio, Seongnam, Korea). Briefly, 5 × 106 cells were seeded in a PGA scaffold (Biofelt®; Concordia Medical, Warwick, RI, USA) with a diameter of 5 mm and a height of 2 mm. The cell–PGA constructs were cultured in the chondrogenic media mentioned above for 1 week before implantation into the nude mice. To minimize individual variations in the animals, a set of constructs from all of the different groups was implanted in a nude mouse (n ³ 3 per group). The tissues formed were sampled at 1, 2, and 4 weeks after implantation. The experimental protocol for animal use was reviewed and approved by the Institutional Animal Experiment
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Committee of the Gyeonggi Institute of Science and Technology Promotion. Histology and Immunohistochemistry The samples were fixed with 4% formaldehyde (Duksan Chemical) and embedded in paraffin wax (Merck, Darmstadt, Germany). Sections 4 mm thick were stained with Safranin O and von Kossa staining (SigmaAldrich) to identify the degree of sGAG and calcium deposition, respectively. For an immunohistochemical analysis of collagen II, sections were treated with 3% hydrogen peroxide (Duksan Chemical) in methanol for 10 min and reacted with a pepsin solution (Golden Bridge International, Inc., Mukilteo, WA, USA) for 10 min. After
Choi ET AL.
blocking the sections with 1% BSA in PBS, they were incubated with an anti-collagen II antibody (1:100; Calbiochem, Darmstadt, Germany) or an anti-collagen X antibody (1:100; Abcam, Cambridge, MA, USA) for 1.5 h at room temperature. The sections were then incubated with a biotinylated secondary antibody against mouse IgG (SPlink HRP Detection Kit; Golden Bridge International) for 30 min and with a peroxidase-conjugated streptavidin solution (SPlink HRP Detection Kit; Golden Bridge International) for 30 min. Finally, the sections were reacted with a 3,3¢-diaminobenzidine (DAB) solution (Golden Bridge International) and counterstained with Mayer’s hematoxylin (YD Diagnostics, Seoul, Korea) before mounting.
Figure 1. Isolation of FCPCs from human fetal cartilage tissue. (A) Human fetal cartilage tissue 12 weeks after gestation (a, b) and young cartilage tissue (c, d) were stained with Safranin O/fast green staining for sulfated glycosaminoglycans and were observed under a microscope. (B) Human fetal cartilage tissue was immunostained for collagen II (upper panel) and collagen I (lower panel). (C) The yields of cells isolated from 1 g of tissue were compared between FCPCs (n = 4) and young chondrocytes (n = 3) isolated from each of the tissues. (D) The morphologies of the FCPCs and chondrocytes at 24 h and MSCs at 6 days after initial plating were observed. Y-Chon, young chondrocytes.
STEM CELL CHARACTERISTICS OF FETAL CARTILAGE CELLS
Statistical Analysis The quantitative data from the multilineage differentiation experiments were analyzed by a one-way analysis of variance (ANOVA) with Tukey’s post hoc test using SPSS 12.0.1 (IBM, Armonk, NY, USA). Values of p
0963-6897/16 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368915X688641 E-ISSN 1555-3892 www.cognizantcommunication.com
Fetal Cartilage-Derived Cells Have Stem Cell Properties and Are a Highly Potent Cell Source for Cartilage Regeneration Woo Hee Choi,* Hwal Ran Kim,* Su Jeong Lee,* Nayoung Jeong,* So Ra Park,† Byung Hyune Choi,‡ and Byoung-Hyun Min*§¶ *Department of Molecular Science and Technology, Ajou University, Suwon, Korea †Department of Physiology, Inha University College of Medicine, Incheon, Korea ‡Department of Biomedical Sciences, Inha University College of Medicine, Incheon, Korea §Cell Therapy Center, Ajou University Medical Center, Suwon, Korea ¶Department of Orthopedic Surgery, Ajou University School of Medicine, Suwon, Korea
Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. However, the characteristics and the potential of progenitor cells from fetal tissue remain poorly defined. In this study, we examined cells from human fetal cartilage at 12 weeks after gestation in comparison with bone marrow-derived MSCs or cartilage chondrocytes from young donors (8–25 years old). The fetal cartilage-derived progenitor cells (FCPCs) showed higher yields by approximately 24 times than that of chondrocytes from young cartilage. The morphology of the FCPCs was polygonal at passage 0, being similar to that of the young chondrocytes, but it changed later at passage 5, assuming a fibroblastic shape more akin to that of MSCs. As the passages advanced, the FCPCs showed a much greater proliferation ability than the young chondrocytes and MSCs, with the doubling times ranging from 2~4 days until passage 15. The surface marker profile of the FCPCs at passage 2 was quite similar to that of the MSCs, showing high expressions of CD29, CD90, CD105, and Stro-1. When compared to the young chondrocytes, the FCPCs showed much less staining of SA-b-gal, a senescence indicator, at passage 10 and no decrease in SOX9 expression until passage 5. They also showed a much greater chondrogenic potential than the young chondrocytes and the MSCs in a three-dimensional pellet culture in vitro and in polyglycolic acid (PGA) scaffolds in vivo. In addition, they could differentiate into adipogenic and osteogenic lineages as efficiently as MSCs in vitro. These results suggest that FCPCs have stem cell properties to some extent and that they are more active in terms of proliferation and chondrogenic differentiation than young chondrocytes or MSCs. Key words: Fetal cartilage-derived progenitor cells (FCPCs); Mesenchymal stem cells (MSCs); Chondrocytes; Cartilage regeneration
INTRODUCTION The self-repair of articular cartilage is difficult when it becomes injured due to its low regenerative capacity caused by the lack of blood supply, low cellularity, and a limited number of progenitor cells (32). To replenish cells in areas with cartilage defects, surgical techniques such as the abrasion and microfracture arthroplasties have been clinically applied, but both are associated with the formation of fibrocartilage, which is weaker and less durable. Autologous chondrocyte transplantation (ACT) has also been used clinically for cartilage regeneration,
showing reasonable results as a cartilage repair strategy (5,42); however, it also has shortcomings in that only a small piece of cartilage tissue can be taken from a patient, and the number of chondrocytes is very limited. In addition, the chondrogenic phenotype and redifferentiation potential can vary depending on the patient and is easily lost, particularly in aged chondrocytes during expansion in a monolayer culture (3,15,21,39). To overcome the limitations of autologous chondrocytes, many studies have utilized mesenchymal stem cells (MSCs) isolated from various tissues, such as bone marrow,
Received January 18, 2014; final acceptance July 28, 2015. Online prepub date: July 13, 2015. Address correspondence to Professor Byoung-Hyun Min, Department of Orthopedic Surgery, Ajou University School of Medicine, Wonchon-dong, Youngtong-gu, Suwon, Gyeonggi-do, Korea. Tel: +82-31-219-4444; Fax: +82-31-219-4193; E-mail: [email protected] or Professor Byung Hyune Choi, Department of Biomedical Sciences, Inha University College of Medicine, 100 Inharo, Nam-gu, Incheon, 22212, Korea. Tel: +82-32-860-9881; Fax: +82-32-885-8302; E-mail: [email protected]
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synovial membrane, fat, or cord blood (10,29,30,35). MSCs are regarded as an attractive cell source for cartilage regeneration because they have high self-renewal ability and the multipotent ability to differentiate into various cell types, including chondrocytes in vitro (43). For cartilage regeneration, MSCs are simply injected into the defect area with or without cell carriers, or they are differentiated into chondrocytes to form cartilage tissue in vitro before implantation (7,12,25,33). However, MSCs have also been shown to have limited capacity to differentiate fully into chondrocytes and form intact cartilage tissues both in vitro and in vivo. In particular, cartilage tissues formed using MSCs were mostly subject to hypertrophic changes and lost their chondrogenic properties in vivo (8,11,13,41). Stem cells or progenitors isolated from fetal tissues such as liver, bone marrow, blood, lung, kidney, pancreas, placenta, brain, and spinal cord are another potent cell source for cell therapy and tissue engineering efforts (40). They have often been shown to have high multipotentiality and proliferation capacity, better sometimes than adult stem cells isolated from aged donors (23,28,36). Moreover, MSCs from fetal liver have been shown to elicit no alloreactive T-cell response, which suggests that fetal stem cells do not display an allogenic immune response (19,20). Previously, several studies attempted to use fetal cartilage-derived cells in cartilage tissue engineering. Fuchs et al. reported that ovine fetal cartilage cells formed better cartilage tissue than adult chondrocytes by producing more matrix molecules in the pellet culture (17). Quintin et al. showed that human fetal femoral head cells differentiated well into chondrogenic, adipogenic, and osteogenic lineages but with less efficiency than bone marrow MSCs (44). These studies imply that fetal cartilage-derived cells could be a potential cell source for cartilage regeneration, but it is necessary to identify the characteristics and therapeutic utility of fetal cartilage-derived cells further for additional clinical applications. In this study, we examined the characteristics of fetal cartilage-derived progenitor cells (FCPCs) in comparison with bone marrow MSCs and young chondrocytes in terms of their proliferation ability over passages, their plasticity, and their chondrogenic potential in vitro and in vivo. MATERIALS AND METHODS Cell Isolation and Culture The study was approved by the institutional review board (IRB) of the Ajou University Medical Center (AJIRB-CRO-07-139) and was carried out with the written consent of all donors. Human fetal cartilage tissues (n = 4, M12w-a, M12w-b, F12w-c, M11w) were obtained from patients following elective termination at 12 weeks after gestation, and cells were isolated from the femoral
Choi ET AL.
head of the cartilage tissue. Young chondrocytes were isolated from the knee cartilage tissue of surgery patients after trauma at 11~25 years old (n = 3, M11y, M18y, M25y). Aged chondrocytes were isolated from the knee cartilage tissue of three donors undergoing total knee replacement surgery (n = 3, F45y, M54y, F56y). Cartilage tissues were minced into small pieces and treated with 0.1% collagenase type 2 (Worthington Biochemical Corp., Freehold, NJ, USA) in high-glucose Dulbecco’s modified Eagle medium (DMEM; HyClone, Logan, UT, USA) containing 1% fetal bovine serum (FBS; HyClone) at 37°C under 5% CO2. After 12 h, isolated cells were cultured in DMEM supplemented with 10% FBS, 100 U/ ml penicillin G, and 100 μg/ml streptomycin (Pen-Strep; HyClone) at a density of 8 × 103 cells/cm2. MSCs were isolated from the bone marrow of fractured femurs of surgery patients who were 8~25 years old (n = 3, F8y, M11y, M25y). Briefly, mononuclear cells (MNCs) were collected by Ficoll (Ficoll-Paque PLUS; Amersham Biosciences, Piscataway, NJ, USA) gradient centrifugation, suspended in a-modified Eagle’s medium (a-MEM; HyClone) supplemented with 10% FBS, and plated at a density of 8 × 104 cells/cm2. After 6 days, nonadherent cells were removed, and the adherent MSCs were replenished with fresh medium. Cells were passaged at 80% confluence, where the plating density was approximately 8 × 103 cells/cm2. Flow Cytometry Analysis Cells at passages 2~3 were analyzed for the expression of stem cells markers on the cell surface. Cells in the suspension were incubated with anti-CD34-FITC, antiCD29-PE, anti-CD90-FITC (BD Biosciences, San Jose, CA, USA), anti-CD105-FITC (Ancell, Bayport, MN, USA), and anti-Stro-1 (R&D Systems, Minneapolis, MN, USA) antibodies for 40 min at 4°C. For the anti-Stro-1 antibody, cells were washed with phosphate-buffered saline (PBS; Welgene, Daegu, Korea) and then incubated with goat anti-mouse IgM-FITC (R&D Systems) for 30 min at 4°C. The dilution factors of each antibody were determined according to the manufacturers’ instructions. Stained cells were analyzed by flow cytometry (Becton Dickinson FACSvantage). Analysis of mRNA Levels The total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of RNA was used to synthesize cDNA by reverse transcription. PCR was performed using 0.5 mg of synthesized cDNA and specific primers for collagen II, SOX9, aggrecan, collagen I, and GAPDH. The sequences of the primers and the reaction conditions are listed in Table 1. Quantitative real-time PCR was performed with a CFX ConnectTM PCR machine (Bio-Rad, Hercules, CA, USA)
STEM CELL CHARACTERISTICS OF FETAL CARTILAGE CELLS
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Table 1. The Primers Used for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Primer
Sequence
Annealing Temp./ Time/Cycle
Product Size
GAPDH
Forward Reverse
5¢-GGTCATGAGTCCTTCCACGAT-3¢ 5¢-GGTGAAGGTCGGAGTCAACGG-3¢
59°C/30 s/27 cycles
520 bp
COL2A1
Forward Reverse
5¢-CTCCTGGAGCATCTGGAGAC-3¢ 5¢-ACCACGATCACCCTTGACTC-3¢
59°C/30 s/35 cycles
152 bp
ACAN
Forward Reverse
5¢-CCCCACTGGCCCCAAGAATCAAG-3¢ 5¢-CGCTGCGCCCTGTCAAAGTCG-3¢
65°C/30 s/27 cycles
319 bp
SOX9
Forward Reverse
5¢-CACACAGCTCACTCGACCTTG-3¢ 5¢-TTCGGTTATTTTTAGGATCATCTCG-3¢
60°C/30 s/30 cycles
76 bp
COL1A2
Forward Reverse
5¢-CTTCTGGCCCTGCTGGAAAAGATG-3¢ 5¢-CCCGGATACAGGTTTCGCCAGTAG-3¢
57°C/30 s/27 cycles
444 bp
using SYBR Premix Ex TaqTM (Takara Bio Inc., Otsu, Shiga, Japan). All reactions were duplicated, and each amplified signal of the (sex-determining region Y)-box 9 (SOX9) and collagen II a 1 (COL2A1) were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cellular Senescence Assay Senescent cells were determined by senescence-associated b-galactosidase (SA-b-gal) staining using a senescence cell histochemical staining kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were first fixed for 6 min at room temperature and then incubated with a staining mixture for 8 h at 37°C without CO2. Senescent cells were observed in blue color under a microscope (TE-2000; Nikon, Tokyo, Japan). Multilineage Differentiation For osteogenic and adipogenic differentiation, cells were plated in six-well plates (TPP, Trasadingen, Switz erland) at densities of 2 × 103 cells/cm2 and 2 × 104 cells/ cm2, respectively. After 24 h, the cells were incubated in a differentiation medium for each lineage. The osteogenic medium consisted of a-MEM supplemented with 10% FBS, 10 mM b-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), and 50 μg/ml ascorbate-2 phosphate (Sigma-Aldrich). The adipogenic medium consisted of a-MEM supplemented with 10% FBS, 1 µM dexamethasone, 10 μg/ml insulin (SigmaAldrich), 0.5 mM isobutyl-methylxanthine (IBMX; SigmaAldrich), and 0.1 mM indomethacin (Sigma-Aldrich). The control cells were incubated in a-MEM supplemented with 10% FBS for 3 weeks. After 3 weeks of differentiation, cells were stained with Alizarin red S (SigmaAldrich) and Oil red O (Sigma-Aldrich) to observe the degree of mineralization and the lipid droplets, respectively. For quantitative analysis, the adsorbed dyes were eluted with 3 ml of 10% acetic acid (Duksan Chemical,
Seoul, Korea) and 1.5 ml of 10% ammonium hydroxide (Duksan Chemical) for Alizarin red S and with 1 ml of isopropyl alcohol (Duksan Chemical) for Oil red O. The absorbance was measured at 405 nm and 500 nm, respectively. To present the data, values from the differentiated samples were normalized by the control values of undifferentiated cells. For chondrogenic differentiation, 3 × 105 cells were centrifuged at 500 × g for 5 min, and the cell pellet was cultured in a chondrogenic medium. The chondrogenic medium consisted of DMEM supplemented with 100 nM dexamethasone, 50 μg/ml ascorbate-2 phosphate, ITS supplement (Sigma-Aldrich), 40 μg/ml proline (SigmaAldrich), 1.25 mg/ml bovine serum albumin (BSA; Sigma-Aldrich), 100 μg/ml sodium pyruvate (SigmaAldrich), and 10 ng/ml TGF-b3 (R&D Systems). After 3 weeks of induction, the samples were fixed with 4% formaldehyde (Duksan Chemical) and embedded in paraffin wax. Sections with a thickness of 4 μm were prepared and stained with Safranin O (Sigma-Aldrich) to observe the sulfated glycosaminoglycans (sGAG). Cartilage Tissue Formation In Vivo To examine the potential for cartilage tissue formation, cells were seeded in a scaffold and implanted subcutaneously into the backs of BALB/c nude mice (male, 6 weeks of age; Orient Bio, Seongnam, Korea). Briefly, 5 × 106 cells were seeded in a PGA scaffold (Biofelt®; Concordia Medical, Warwick, RI, USA) with a diameter of 5 mm and a height of 2 mm. The cell–PGA constructs were cultured in the chondrogenic media mentioned above for 1 week before implantation into the nude mice. To minimize individual variations in the animals, a set of constructs from all of the different groups was implanted in a nude mouse (n ³ 3 per group). The tissues formed were sampled at 1, 2, and 4 weeks after implantation. The experimental protocol for animal use was reviewed and approved by the Institutional Animal Experiment
452
Committee of the Gyeonggi Institute of Science and Technology Promotion. Histology and Immunohistochemistry The samples were fixed with 4% formaldehyde (Duksan Chemical) and embedded in paraffin wax (Merck, Darmstadt, Germany). Sections 4 mm thick were stained with Safranin O and von Kossa staining (SigmaAldrich) to identify the degree of sGAG and calcium deposition, respectively. For an immunohistochemical analysis of collagen II, sections were treated with 3% hydrogen peroxide (Duksan Chemical) in methanol for 10 min and reacted with a pepsin solution (Golden Bridge International, Inc., Mukilteo, WA, USA) for 10 min. After
Choi ET AL.
blocking the sections with 1% BSA in PBS, they were incubated with an anti-collagen II antibody (1:100; Calbiochem, Darmstadt, Germany) or an anti-collagen X antibody (1:100; Abcam, Cambridge, MA, USA) for 1.5 h at room temperature. The sections were then incubated with a biotinylated secondary antibody against mouse IgG (SPlink HRP Detection Kit; Golden Bridge International) for 30 min and with a peroxidase-conjugated streptavidin solution (SPlink HRP Detection Kit; Golden Bridge International) for 30 min. Finally, the sections were reacted with a 3,3¢-diaminobenzidine (DAB) solution (Golden Bridge International) and counterstained with Mayer’s hematoxylin (YD Diagnostics, Seoul, Korea) before mounting.
Figure 1. Isolation of FCPCs from human fetal cartilage tissue. (A) Human fetal cartilage tissue 12 weeks after gestation (a, b) and young cartilage tissue (c, d) were stained with Safranin O/fast green staining for sulfated glycosaminoglycans and were observed under a microscope. (B) Human fetal cartilage tissue was immunostained for collagen II (upper panel) and collagen I (lower panel). (C) The yields of cells isolated from 1 g of tissue were compared between FCPCs (n = 4) and young chondrocytes (n = 3) isolated from each of the tissues. (D) The morphologies of the FCPCs and chondrocytes at 24 h and MSCs at 6 days after initial plating were observed. Y-Chon, young chondrocytes.
STEM CELL CHARACTERISTICS OF FETAL CARTILAGE CELLS
Statistical Analysis The quantitative data from the multilineage differentiation experiments were analyzed by a one-way analysis of variance (ANOVA) with Tukey’s post hoc test using SPSS 12.0.1 (IBM, Armonk, NY, USA). Values of p
