The authors have declared that no competing interests exist.
Osteoarthritis (OA), a common chronic joint disorder in both humans and canines, is characterized by a progressive loss of articular cartilage. Canines can serve as an animal model of OA for human medicine, and this research can simultaneously establish effective veterinary treatments for canine OA. One attractive treatment that can lead to cartilage regeneration is the use of mesenchymal stem cells (MSCs). However, for canine OA, little information is available regarding the best source of MSCs. The purpose of this study was to identify a promising MSC source for canine cartilage regeneration. We collected synovial, infrapatellar fat pad, inguinal adipose, and bone marrow tissues from six canines and then conducted a donor-matched comparison of the properties of MSCs derived from these four tissues. We examined the surface epitope expression, proliferation capacity, and trilineage differentiation potential of all four populations. Adherent cells derived from all four tissue sources exhibited positivity for CD90 and CD44 and negativity for CD45 and CD11b. The positive rate for CD90 was higher for synovium-derived than for adipose-derived and bone marrow-derived MSCs. Synovium-derived and infrapatellar fat pad-derived MSCs displayed substantial proliferation ability, and all four populations underwent trilineage differentiation. During chondrogenesis, the wet weight was heavier for cartilage pellets derived from synovium MSCs than from the other three sources. The synovium is therefore a promising source for MSCs for canine cartilage regeneration. Our findings provide useful information about canine MSCs that may be applicable to regenerative medicine for treatment of OA.
Osteoarthritis (OA), the most common chronic disorder of synovial joints, is characterized by the progressive loss of articular cartilage, which leads to pain and functional impairment. Current treatment options are limited to analgesia and to prosthetic joint replacement for end-stage disease. An unmet need still exists for the development of regenerative medicine that can restore lost cartilage and thereby provide a long-term solution for OA symptoms. [
OA is not specifically a human disease, as canines can spontaneously develop OA, and this is becoming a significant veterinary problem in aging companion dogs [
One attractive regenerative approach for cartilage regeneration is the use of mesenchymal stem cells (MSCs), which can be isolated from various mesenchymal tissues of both dogs and humans. Most canine studies use MSCs derived from bone marrow or subcutaneous adipose tissue [
In the present study, we expanded MSCs from different canine tissues using strictly controlled and similar processes, and we performed a donor-matched quantitative comparison of the MSC properties. Our results show that canine MSCs isolated from synovium, infrapatellar fat pad, adipose, and bone marrow tissues exhibit similarities and differences, and the data provide useful information on canine MSCs that may be applicable to regenerative medicine for OA.
Six healthy, skeletally mature beagle dogs (12–18 months old, 10–17 kg, 5 males and 1 female) were used in the study. All experiments were conducted in accordance with our institutional guidelines. The protocol was approved by the Animal Committee of Tokyo Medical and Dental University (Protocol number: 0170403A) and the Animal Committee of the Graduate School of Agricultural and Life Sciences at the University of Tokyo (Protocol number: P16-279). Dogs were euthanized with an overdose intravenous injection of thiopental (150 mg/kg) or deep anesthesia with isoflurane followed by an intravenous KCL injection (1 ml/kg) for reasons unrelated to this study, including an odontological study (approved by the Animal Committee of Tokyo Medical and Dental University; approval number: 0170333A) and ophthalmological studies (the Animal Care Committee of the Graduate School of Agricultural and Life Sciences at the University of Tokyo; approval number: P15-13, PH15-81 and PH17-116). After euthanasia, synovium with subsynovial tissue was harvested from each dog from the bony side of the suprapatellar pouch in the knee. The infrapatellar fat pad was harvested from the knee joint. Subcutaneous adipose tissue was harvested from the inguinal region. Bone marrow was aspirated from the proximal humerus using bone marrow puncture needles (
Synovium and the infrapatellar fat pad were harvested from the knee joint, adipose tissue from the inguinal region, and bone marrow from the humerus.
Synovium, infrapatellar fat pad, and adipose tissues were minced and digested at 37°C for 3 hours in a 3 mg/ml collagenase D solution (Roche Diagnostics, Mannheim, Germany) in α-minimal essential medium (αMEM; Invitrogen, Carlsbad, CA, USA). The cells released by digestion were filtered through a 70-μm nylon filter (Becton Dickinson, Franklin Lakes, N.J., USA), and the remaining tissues were discarded. The obtained nucleated cell numbers were counted for all tissues using a Luna-FL Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA, USA). The cells were plated at 1 × 103, 1 × 104, or 1 × 105 cells/dish in 6 dishes for each density (60 cm2 culture dish; Nalge Nunc International, Rochester, N.Y., USA) and cultured in complete culture medium: αMEM containing 10% fetal bovine serum (FBS; Invitrogen), 100 units/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 250 ng/ml amphotericin B (Invitrogen). Bone marrow was diluted with αMEM and the number of nucleated cells was counted with a Luna-FL Dual Fluorescence Cell Counter. Then bone marrow-derived cells were plated at 1 × 104, 1 × 105, or 1 × 106 cells/60 cm2 in complete culture medium. The plated cells were incubated at 37°C in a 5% humidified CO2 atmosphere as passage-0. The medium was changed every 3–4 days.
After 14 days, three dishes for each cell concentration were fixed with 10% formalin solution (Wako, Osaka, Japan), stained with 0.5% crystal violet for 5 min, and washed with distilled water. The optimal initial cell density was determined based on the following observations: 1) the colony size was not affected by contact inhibition, and 2) the greatest number of colonies was obtained. We harvested the cells from the remaining 3 dishes and counted them to determine the cell number at passage-0. The data were analyzed with the Friedman test, followed by Dunn’s multiple comparisons test. The passage-0 cells were then replated at 3000 cells/dish (60 cm2 dishes) as passage-1 cells and cultured for 14 days. Surface epitopes were then analyzed in 1 × 106 passage-1 cells, while the remaining cells were cryopreserved at a concentration of 1 × 106 cells/ml in αMEM containing 5% dimethylsulfoxide (Wako, Osaka, Japan) and 10% FBS. Aliquots of 1 ml were slowly frozen and cryopreserved at -80°C in a BICELL biofreezing vessel (Nihon Freezer, Tokyo, Japan). The cells were later expanded by thawing a frozen vial of cells, plating them at 3 × 105 cells/dish (150-cm2 dishes), and incubating for 7 days in the recovery plate. These passage-2 cells were used in subsequent analyses.
The colony-forming abilities of the mesenchymal tissue-derived cell types from 6 canine donors were examined by replating passage-2 cells at 1 × 103 cells/dish (60 cm2 dishes) in 6 dishes and incubating them for 14 days in complete medium. Three dishes were then fixed with 10% formalin solution, stained with 0.5% crystal violet (CV), and washed with distilled water for counting of colony numbers in the dishes. Colonies less than 2 mm in diameter or colonies with only faint staining were ignored. The cells from the remaining three dishes were harvested and counted with a hemocytometer to calculate the fold increase and the cell number per colony for each population. The data were analyzed with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
Proliferation potentials were compared by plating cells derived from the four canine mesenchymal tissues at passage-2. Cells were plated at 1 × 103 cells/10 cm2 (3 canine donors, n = 3 in each donor). After 4, 8, and 12 days of culture, the cells were harvested and the cell numbers were counted. The data were analyzed with the Friedman test, followed by Dunn’s multiple comparisons test.
Passage-1 cells (1 × 106 cells) were suspended in 100 μl phosphate buffered saline (PBS) containing 10 μg/ml antibodies for APC-conjugated CD90 (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated CD44 (eBioscience, San Diego, CA, USA), FITC-conjugated CD45 (eBioscience), and PerCP-Cyanine5.5-conjugated CD11b (Biolegend, San Diego, CA, USA). As an isotype control, nonspecific anti-mouse IgG coupled with APC, PE, FITC, or PerCP-Cyanine5.5 (Becton Dickinson) was substituted for the primary antibody. After incubation for 30 minutes at 4°C, the cells were washed with PBS and then resuspended in 500 μl of PBS for analysis. Cell fluorescence was evaluated by flow cytometry using a FACSVerse instrument (Becton Dickinson); the data were analyzed using Flowjo software (Tree Star, Ashland, OR, USA). All analyses were performed on samples from all 6 donors. The data were analyzed with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
Passage-2 cells (2.5 × 105 cells) for chondrocyte differentiation were placed in a 15-ml polypropylene tube (Falcon, Bedford, MA, USA) and centrifuged at 490
For histological analysis, the pellets were embedded in paraffin, cut into 5-μm sections, and stained with toluidine blue. Collagen type II expression was visualized by immunostaining using a rabbit anti-collagen type II antibody (Cosmo Bio Co., LTD., Tokyo, Japan).
After 21 days of culture, pellets from each tissue were digested for 18 hours at 65°C in papain buffer (200 mM sodium phosphate buffer, 100 mM sodium acetate, 10 mM ethylenediaminetetraacetic acid (EDTA), 5 mM L-cysteine HCl, pH 6.4) containing 5 μg/ml of papain and then centrifuged at 10000
The amounts of DNA in the pellets at day 0 were determined by pelleting 2.5 × 105 passage-2 cells from 3 canine donors, 3 pellets each, as already described mentioned. DNA was immediately extracted from the pellets using the NucleoSpin Extract kit (Machery‐Nagel, Düren, Germany), and the amount of DNA in each pellet was determined. The data were analyzed with the Friedman test, followed by Dunn’s multiple comparisons test.
Passage-2 cells from each tissue were plated at 1 × 103 cells per well in 6-well plates (Falcon) and cultured in complete medium for 7 days (3 canine donors, 3 wells each). The medium was then switched to a calcification medium consisting of complete medium supplemented with 10 mM β-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), and 50 μM ascorbic acid (Sigma-Aldrich). Control cells were cultured in complete medium without any of these three supplements. After 21 days, the calcification and the control cultures were fixed in 10% formalin solution for 5 minutes, stained with 0.5% alizarin red solution for 5 minutes, and washed with distilled water.
For quantification, 1 × 103 cells were plated in 6-well plates and cultured for 7 days. The medium was then switched to the calcification medium for an additional 21 days. After staining with alizarin red, the areas positive for alizarin red were measured with Image J software [
Passage-2 cells from each tissue were plated at 2 × 104 cells/well in 12-well plates (Falcon) (3 canine donors, 3 wells each) and cultured in complete medium for 7 days. The culture medium was then switched to an adipogenic medium consisting of complete medium supplemented with 1 μM dexamethasone (Sigma-Aldrich), 5 μM rosiglitazone (Wako), 5 μg/ml insulin (Wako), and 3.5 g/L glucose (Sigma-Aldrich) [
For quantification, nine areas surround by a square 548 μm in height and 730 μm in width were randomly selected from each well, and the ratio of oil-red-o positive areas per total area was measured with Image J software. The data were analyzed with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
All data were statistically evaluated with GraphPad Prism 6 (Graphpad Software, La Jolla, CA, USA). The results are presented as means ± standard deviation (SD). P values of < 0.05 were considered to be statistically significant.
Maximum yields of nucleated cells were obtained by plating nucleated cells derived from the four tissue types of the six canine donors at 3 different cell densities (
(A) Experimental design. Nucleated cells derived from solid tissues were plated in six dishes at high, middle, and low density (1 ×103, 1 ×104, or 1 ×105 cells per dish; 60-cm2 dish), while cells derived from bone marrow were plated at 1 ×104, 1 ×105, or 1 ×106 cells per dish (60-cm2 dish). After culturing for 14 days, three dishes from each condition were stained with crystal violet and the optimal initial cell density was determined based on the following criteria: 1) the colony size was not affected by colony-to-colony contact inhibition and 2) the greatest number of colonies was obtained. The cells from the remaining three dishes were used for further analyses. (B) Cell colonies stained with crystal violet. The optimal initial cell density is indicated by a red square.
(A) The numbers of nucleated cells tissue weight and per bone marrow volume. Synovium, infrapatellar fat pad, and adipose tissues were digested with collagenase D solution, and nucleated cell numbers were counted for these three tissues as well as for bone marrow. (B) Harvested cell number after 14 days of culture. The nucleated cells were plated at the determined optimal plating density and after 14 days of culture, the numbers of harvested cells were counted. Values are the mean ± standard deviation (n = 6). Statistical analyses were carried out only for the three solid tissues. **, p < 0.01 with the Friedman test, followed by Dunn’s multiple comparisons test. Sy, synovium; IFP, infrapatellar fat pad; AT, adipose tissue; BM, bone marrow. (C) Cell morphology of plastic-adherent cells derived from four canine mesenchymal tissues.
Cells derived from all four tissue types were positive for CD90 and CD44 and negative for CD45 and CD11b (
Colony forming abilities were compared by plating passage-2 cells derived from the four tissues at 1 × 103 cells in 60 cm2 dishes and culturing for 14 days (
(A) Experimental design. Passage-2 cells were plated at 1 × 103 cells/dish (60 cm2 dishes) in 6 dishes. After 14 days of culture, three dishes were stained with crystal violet for colony counting and the other three dishes were used for cell counting and further analyses. (B) Images of colonies stained with crystal violet. (C) Colony number (1 × 105)/dish (60 cm2 dishes). (E) Cell number/colony. The means ± SD are shown (n = 18). *, p < 0.05. **, p < 0.01 with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
Proliferation potential was compared by plating passage-2 cells derived from the four tissues at 1 × 103 cells/10 cm2. After 8 and 12 days of culture, the numbers of proliferated cells were significantly greater for synovium and infrapatellar fat pad tissues than for bone marrow. At 12 days of culture, the numbers of proliferated cells were greater for synovium than for adipose tissue (
Cells were plated at 1 × 103 cells/10 cm2 (3 canine donors, n = 3 for each donor) and cell numbers were counted after 4, 8, and 12 days of culture. The means ± SD are shown. *, p < 0.05. **, p < 0.01 with the Friedman test, followed by Dunn’s multiple comparisons test.
After chondrogenic differentiation, each cartilage pellet was translucent, but the size and wet weight varied depending on the donor and the type of mesenchymal tissue harvested (
An aliquot containing 2.5 × 105 passage-2 cells was pelleted and cultured in chondrogenic differentiation medium for 21 days. (A) Macroscopic photographs of cartilage pellets (6 pellets per canine; 6 canine donors). (B) Diameter of cartilage pellets (n = 36). (C) Wet weight of cartilage pellets (n = 36). (D) Histology of sections stained with toluidine blue. (E) Histology of sections immunostained for collagen type II. (F) Amount of sulfated glycosaminoglycan (sGAG) per pellet (n = 36). (G) DNA amount per pellet (n = 36). (H) Ratio of sGAG amount/DNA amount (n = 36). (I) DNA residual rate during chondrogenesis (= DNA amount at 21 days/DNA amount at 0 day; 3 pellets per canine; 3 canine donors). The means ± SD are shown. *, p < 0.05. **, p < 0.01 with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
After calcification, the cells from each mesenchymal tissue were stained with alizarin red (
(A) Cells cultured with and without calcification medium. Passage-3 cells were plated at 1 × 103 cells/10 cm2 well, precultured for 7 days, and then cultured with or without calcification induction medium for 21 days. (B) Alizarin red positive colonies and total colonies. The wells were first stained with alizarin red, and then the same wells were stained with crystal violet. (C) Ratio (%) of alizarin red positive/crystal violet positive areas. The means ± SD are shown. *, p < 0.05. **, p < 0.01 with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
Control cultures showed no droplets that stained with oil red-o. After adipogenesis with adipogenic differentiation medium, the cells derived from four canine tissue sources produced lipid droplets that stained with oil red-o (
(A) Cells cultured with and without adipogenic medium. Passage-3 cells were plated at 2 × 104 cells/5 cm2 well, precultured for 7 days, then cultured with or without adipogenic differentiation medium for 21 days. (B) Relative values of the ratios of the oil red-o positive areas against the ratios for synovium-derived cells. The means ± SD are shown. *, p < 0.05. **, p < 0.01 with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
A precise definition for the term MSC remains confusing, but the minimal criteria for human MSCs, as advocated by the International Society for Cellular Therapy (ISCT), are that the cells (1) are plastic-adherent under standard culture conditions; (2) express CD105, CD73, and CD90, but lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR surface molecules; and (3) differentiate into osteoblasts, adipocytes, and chondrocytes [
In this study, we used donor-matched samples for comparisons because of the known occurrence of variations in the properties of MSCs among individuals [
The canine MSCs from each tissue were positive for CD90 and CD44 and negative for CD45 and CD11b (
We quantitatively evaluated the chondrogenic differentiation potential of MSCs from each of the four tissue sources (
For calcification, we used a differentiation medium similar to that used for human MSCs. Unlike human MSCs, we found that canine MSCs were easily detached during calcification, irrespective of their sources, as previously reported for canine adipose tissue-derived stem cells [
We were unable to differentiate canine MSCs from each source into adipocytes using an adipogenesis differentiation medium designed for human MSCs [
In the present study, the synovium and infrapatellar fat pad were harvested from the knee, adipose tissue was harvested subcutaneously from the inguinal region, and bone marrow was aspirated from the proximal humerus (
The currently available reports of canine MSC production typically have utilized adipose tissue and bone marrow [
We expanded canine MSCs by conducting a similar and strictly controlled process, and then performed a donor-matched quantitative and qualitative comparison to clarify the properties of MSCs derived from synovium, infrapatellar fat pad, adipose tissue, and bone marrow. The colony formation rates of nucleated cells, positive rate for CD90, proliferation potential, and differentiation capacity of the MSCs depended on the source of mesenchymal tissue. The MSCs derived from synovium had superior chondrogenic ability and high proliferation potential, making the synovium suitable as a source for MSCs for cartilage regeneration in canines.
Macroscopic images of control pellets and pellets cultured in chondrogenic differentiation medium.
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We thank Mr. Keiichiro Komori and Ms. Shizuka Fujii for their technical assistance with the experiments; Ms. Mika Watanabe and Ms. Kimiko Takanashi for the management of our laboratory; and Ms. Ellen Roider for proofreading the paper.