Cell Tissue Res (2007) 327:449–462 DOI 10.1007/s00441-006-0308-z
REGULAR ARTICLE
Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle Hideya Yoshimura & Takeshi Muneta & Akimoto Nimura & Akiko Yokoyama & Hideyuki Koga & Ichiro Sekiya
Received: 17 June 2006 / Accepted: 17 July 2006 / Published online: 13 October 2006 # Springer-Verlag 2006
Abstract Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (j), CD45 (j), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential,
This study was supported in part by grants from the Japan Latest Osteoarthritis Society and from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University (to T.M.), and by the Japan Society for the Promotion of Science (grant no. 18591657 to I.S.). Recombinant human bone morphogenetic protein-2 was kindly provided by Astellas Pharma. H. Yoshimura : T. Muneta : A. Nimura : A. Yokoyama : H. Koga Section of Orthopaedic Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan T. Muneta Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan I. Sekiya (*) Section of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan e-mail:
[email protected]
synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colonyrate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model. Keywords Mesenchymal stem cells . Expansion . Chondrogenesis . Adipogenesis . Calcification . Rat (Sprague Dawley, male)
Introduction Mesenchymal stem cells (MSCs) have attracted much interest because of their self-renewing potential and multipotentiality for possible clinical use (Prockop 1997). Friedenstein et al. (1976) initially isolated bone-marrowderived MSCs by their adherence to plastic dishes and demonstrated that the MSCs grew as foci with a fibroblastlike morphology, termed colony-forming unit-fibroblasts. Several recent reports have shown that human MSCs are also present in other tissues, such as the synovium (De Bari et al. 2001a), periosteum (De Bari et al. 2001b), subcuta-
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neous adipose tissues (Zuk et al. 2002), and muscle (Asakura et al. 2001). Bone-marrow-derived MSCs have been isolated from a variety of species, including mouse (Peister et al. 2004), rat (Javazon et al. 2001), rabbit (Johnstone et al. 1998), and human subjects (Colter et al. 2001). Although MSCs from the different species have similar characteristics in part, some data suggest that variations occur among species. MSCs from human bone marrow are relatively easy to harvest and to expand in culture (Sekiya et al. 2002a), whereas rodent MSCs have proven more difficult (Friedenstein et al. 1976; Simmons et al. 1991; Aubin 1999), although this is not without controversy (Javazon et al. 2001). The technical difficulties in preparing MSCs from rodent bone marrow have limited the number of possible experiments, because animal transplantation models are required for preclinical studies. The selection of suitable cell populations is apparently crucial for the outcome of in vivo experiments with MSCs. We have hypothesized that mesenchymal tissues other than bone marrow might be good sources for MSCs from rodents. Here, we have isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality.
Materials and methods
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trypsinized and harvested to count the cell numbers. The remaining three dishes were stained with 0.5% crystal violet to count cell colony numbers. Colonies less than 2 mm in diameter and faintly stained colonies were ignored. For further analyses, the optimal initial cell density was chosen based on the following criteria: (1) that the colony size was not affected by colony-to-colony contact inhibition, and (2) that a large number of colonies was obtained. Similarly, nucleated cells from bone marrow were plated at 104, 105, or 106 cells/60-cm2 dish in six dishes each and cultured for 7 days as Passage 0. To evaluate colony number per adherent cells, nucleated cells from bone marrow were plated at 106 cells/60-cm2 dish, those from synovium, periosteum, and adipose at 104 cells/60-cm2 dish, and those from muscle at 105 cells/ 60-cm2 dish. The number of adherent cells was counted in 50 fields, at a magnification of 100 under a microscope, 1 day after initial plating. Colony number was counted in three dishes for each plating density at 7 days after initial plating. Three rats were used for the experiments. All of the cells were cultured in complete medium consisting of αMEM (Invitrogen), 20% fetal bovine serum (FBS; lot-selected for rapid growth of human MSCs, Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (Invitrogen) and incubated at 37-C with 5% CO2. At 24 h after initial plating, the cells were washed twice with phosphate-buffered saline (PBS) to remove nonadherent cells.
Isolation and culture of rat MSCs Culture density and proliferation Sprague-Dawley rats (12-week-old males) were used. All experiments were conducted in accordance with the institutional guidelines for the care and use of experimental animals. Bone marrow was collected from femurs and tibias. Bone marrow was extruded by inserting a 22-gauge needle into the shaft of the bone and flushed out with 1 ml Hanks_ balanced salt solution (Invitrogen, Carlsbad, Calif., USA). The cells were then size-fractionated by being layered onto a Ficoll-Paque (Pharmacia, Piscataway, N.J., USA) gradient and centrifuged for 20 min at 1,000g at room temperature. The synovial membranes of bilateral knee joints, periosteum of femurs and tibias, subcutaneous adipose tissue, and hindlimb skeletal muscles were excised from the same rats. The tissues were minced, digested for 3 h at 37-C with type II collagenase (0.2%; Sigma, Lakewood, N.J., USA), and passed through a 40-μm filter (Becton Dickinson, Franklin Lakes, N.J., USA) to yield single-cell suspensions. Cell numbers were counted with a hemocytometer. Nucleated cells from the synovium, periosteum, adipose tissue, and skeletal muscle were plated at 103, 104, or 105 cells/60-cm2 dish in six dishes each and cultured for 7 days as Passage 0. Three dishes from each tissue type were
Passage 1 cells derived from various mesenchymal tissues were cultured at 100, 500, and 1,000 cells/cm2/60-cm2 dish. Every 2 days, cells from three plates from each culture density were harvested and counted with a hemocytometer. To elucidate the kinetics of continuous growth, the cells at Passage 2 were replated at 100 cells/cm2 in complete medium every 4 days. Colony-forming assays For these assays, 100 cells at Passage 1 were plated and cultured for 7 days in 60-cm2 dishes. The cells were subsequently fixed with 4% paraformaldehyde, stained with 0.5% crystal violet in 4% paraformaldehyde for 5 min, and washed twice with distilled water. The number of colonies was then counted. Colonies of less than 2 mm in diameter and faintly stained colonies were ignored. Flow cytometry Passage 2 cells (1106) were suspended in 500 μl PBS containing 20 ng/ml fluorescein isothiocyanate (FITC)-
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coupled antibodies against CD11b, CD45, or CD90 (Becton Dickinson, Franklin Lakes, N.J., USA) or, as an isotype control, FITC-coupled nonspecific IgG (Becton Dickinson). After incubation for 30 min at 4-C, the cells were washed with PBS and resuspended in 1 ml PBS for analysis. Cell fluorescence was evaluated by flow cytometry in a FACSCalibur instrument (Becton Dickenson); data were analyzed by using CellQuest software (Becton Dickinson).
water and filtering through a 0.2-μm filter. After being stained, the cultures were washed three times, and the number of colonies positive with Oil Red-O was counted. Colonies less than 2 mm in diameter or faint colonies were ignored. The same adipogenic cultures were subsequently stained with crystal violet, and the number of total cell colonies was counted (Sekiya et al. 2004).
Chondrogenesis
Passage 1 cells were plated at the same densities as indicated for the adipogenesis assay and cultured in complete medium for 7 days. The medium was then switched to a calcification medium in the presence of 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid (Sigma) and incubated for 21 days. These dishes were stained with fresh 0.5% alizarin red solution.
For chondrocyte differentiation, a pellet culture system was used. Approximately 8105 cells were placed in a 15-ml polypropylene tube (Falcon, Bedford, Mass., USA) and pelleted into micromasses by centrifugation at 450g for 10 min. The pellet was cultured for 21 days in chondrogenic media, which contained 500 ng/ml bone morphogenetic protein-2 (Astellas Pharma, Tokyo, Japan), in addition to high-glucose Dulbecco_s modified Eagle_s medium (Invitrogen) supplemented with 10 ng/ml transforming growth factor-β3, 10j7M dexamethasone, 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate, and 50 mg/ml ITS+ Premix (Becton Dickinson). For microscopy, the pellets were embedded in paraffin, cut into 5-μm-thick sections, and stained with 1% toluidine blue (Sekiya et al. 2001, 2005; Ichinose et al. 2005). Analysis of glycosaminoglycans and hyaluronan The amount of chondroitin sulfate and hyaluronan were quantified according to a previously described method (Shinmei et al. 1992). Each pellet (n=5) was digested with 3 mg/ml collagenase in 0.25 ml DMEM for 3 h at 37-C, and the cell fraction was removed. The levels of unsaturated disaccharides derived from chondroitin sulfates and hyaluronan were evaluated (Yoshida et al. 1989; Yokoyama et al. 2005). Adipogenesis in a colony-forming assay Passage 1 cells were plated at 100 cells for synovium, periosteum, adipose, and muscle-derived cells, and at 1,000 cells for bone-marrow-derived cells per 60-cm2 dish, cultured in complete medium for 7 days, and then switched to adipogenic medium consisting of complete medium supplemented with 0.5 μM dexamethasone (Sigma), 0.5 mM isobutylmethylxanthine (Sigma), and 50 μM indomethacin (Sigma). After 4 days, the adipogenic cultures were fixed in 4% paraformaldehyde for at least 1 h and stained with fresh Oil Red-O solution for 2 h. The Oil Red-O solution was prepared by mixing three parts of stock solution (0.5% in isopropanol; Sigma) with two parts of
Calcification
RNA collection and reverse transcription/polymerase chain reaction Total RNA was prepared from the cultured cells and the cultured pellets by using TRIzol reagent (Invitrogen) according to the manufacturer_s protocol. cDNA synthesis was performed for 1 h at 42-C in a reaction mixture containing 5 μg total RNA, 100 U ReverTra Ace (TOYOBO), 4 μl 5RT buffer, 2 μl 10 mM dNTPs, and 1 μl 5 pM oligo (dT) adjusted with RNase-free water to a total volume of 20 μl. Primer sequences were taken from the literature (Neubauer et al. 2004; Nishida et al. 2004; Rodriguez et al. 2004; Santa Maria et al. 2004). Polymerase chain reaction (PCR) amplification of the resulting cDNAs was performed under the following conditions: 35 cycles at 94-C for 30 s, 60-C for 30 s, and 72-C for 30 s for type 2 collagen, Myo D, and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or 94-C for 30 s, 56-C for 30 s, and 72-C for 30 s for PPARγ2, C/EBPα, and CD44. After PCR, 10-μl aliquots were electrophoresed on 1.5% Agarose H14 (Takara Bio, Japan) in TRIS-acetate plus EDTA buffer, stained with SYBR Gold, and photographed. PCR primers were as follows: – – –
C O L 2 A 1 : 5 ¶- AT G A C A AT C T G G C T C C C A A CACTGC-3¶ (forward) and 5¶-GACCGGCCC TATGTCCACACCGAAT-3¶ (reverse), 364 bp; PPARγ2: 5¶-GAGCATGGTGCCTTCGCTGA-3¶ (forward) and 5¶-AGCAAGGCACTTCTGAAACCGA-3¶ (reverse), 564 bp; C/EBPα: 5¶-AAGGCCAAGAAGTCGGTGGA-3¶ (forward) and 5¶-CAGTTCGCGGCTCAGCTGTT-3¶ (reverse), 189 bp;
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– – –
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Myo D: 5¶-CACTACAGCGGCGACTCCGACGCG-3¶ (forward) and 5¶-CGCTCCACTATGCTGGACAGG-3¶ (reverse), 200 bp; CD44: 5¶-CAGACCTGCCCAATGCCTTTGATG GACC-3¶ (forward) and 5¶-CAAAGCCAAGGCCAA GAGGGATGCC-3¶ (reverse), 420 bp; GAPDH: 5¶-TGAACGGGAAGCTCACTGG-3¶ (forward) and 5¶-TCCACCACCCTGTTGCTGTA-3¶ (reverse), 360 bp.
Alkaline phosphatase (ALP) activity Passage 2 cells were plated at 1105 cells in 6-well culture dishes and incubated in calcification medium. ALP activity was measured after 14 days of differentiation. The cells were harvested with lysis buffer (0.1 M TRIS-HCl, 5 mM MgCl2, 2% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride) and sonicated. Total protein concentrations of supernatant were determined by the Bradford method (BioRad, Hercules, Calif.). An aliquot (10 μl) of supernatant was added to 100 μl 50 mM p-nitrophenylphosphatase hexahydrate containing 1 mM MgCl2, and the mixture was incubated at 37-C for 30 min. Absorption at 405 nm was measured with a spectrophotometer. The ALP activity per total protein (μg) represented the millimoles of p-nitrophenol released after a 30-min incubation at 37-C.
Results High yield and proliferation potential in synovium-derived cells Colony number increased together with nucleated cell number (Fig. 1a,b). When plated at the same cell density, colony number per nucleated cell was much lower from bone marrow (Fig. 1b). According to observations of dishes stained with crystal violet, cell colony size was smaller in synovium-, periosteum-, and adipose-derived cells plated at 105 cells/60 cm2 dish (Fig. 1a). Based on our criteria, we determined that 106 cells/60-cm2 dish for bone marrow, 104 cells/60-cm2 dish for synovium, periosteum, and adipose, and 105 cells/60-cm2 dish for muscle were the optimal initial cell density for further analyses. Under these conditions, colony number per nucleated cell (Fig. 1c), colony number per adherent cell (Fig. 1d), and cell number per colony (Fig. 1e) were the highest for synovium and the lowest for bone marrow (P
