Phenotypic characterization of equine synovial fluid-derived chondroprogenitor cells

Abstract

Background: Progenitor cells exist in most tissues and body fluids. Synovial fluid chondroprogenitor cells have been described in several species; however, the specific phenotypic characteristics of these cells have not been defined. This study addressed the impacts of joint location and donor age variation on synovial fluid chondroprogenitor numbers, and determined whether synovial fluid chondroprogenitors express hypertrophic characteristics or a non-endochondral phenotype in chondrogenic culture. Methods: Synovial fluid was aspirated from the joints of healthy adult horses, and cells in the aspirates were expanded through two monolayer passages. The number of colony-forming units (CFU) in each aspirate was assessed after 14 days. Population doublings (PD) and PD times were calculated during the subsequent two passages. Passage 3 cells were used to determine osteogenic and adipogenic capacities, or were transferred to pellet cultures in chondrogenic medium containing TGF-β1 or BMP-2. Bone marrowderived MSCs and primary articular chondrocytes were cultured under similar conditions to provide reference values for chondrogenesis. Chondrogenesis was assessed by measuring collagen type II protein and sulfated glycosaminoglycan secretion, expression of chondrogenic mRNAs, and induction of ALP activity. Results: CFU counts varied considerably, but the majority of aspirates contained <50 CFU/ml. PDs were consistently between 2.0 and 3.0 during both passages. PD times varied from 0.2-0.4 days. Proliferation was not significantly influenced by anatomical location or donor age. Equine synovial fluid progenitors expressed osteogenic, adipogenic and chondrogenic phenotypes under appropriate conditions. Under chondrogenic conditions, synovial fluid cells increased collagen and aggrecan mRNA expression to levels comparable to bone marrow MSCs, although collagen type II protein secretion was less than half that of articular chondrocytes. In contrast to bone marrow MSCs, synovial fluid chondroprogenitors did not express collagen type X or increase ALP activity in response to TGF-β1 or BMP-2. Consistent with these observations, Runx2 and Mef2C expression in synovial fluid chondroprogenitors was 20-40 fold lower than in bone marrow MSCs. Conclusions: Synovial fluid chondroprogenitors are capable of robust non-hypertrophic chondrogenesis, whether stimulated by TGF-β1 or by BMP-2. These results indicate that synovial fluid cells are phenotypically suitable for articular cartilage repair/regeneration, although strategies to accelerate cell proliferation and synthesize a functional cartilage matrix in vivo will be required for clinically feasible cellbased therapies.

Bone morphogenetic protein 2 stimulates chondrogenesis of equine synovial membrane-derived progenitor cells

“…Bone morphogenetic protein 2 administration stimulated significantly more sGAG secretion (4.2 ± 0.65 mg/pellet) than TGF-β1 (1.9 ± 0.26 mg/pellet; ▶ Figure 4 D). Collagen type X transcript levels and ALP activities were consistently low in SMPC, comparable to values previously found in equine articular chondrocytes, and markedly lower than expression in bone marrow-derived stem cells that express a hypertrophic chondrogenic phenotype (34). Neither growth factor significantly up-regulated hypertrophic marker expression (▶ Figure 5).…”

“…Three pellets were pooled for each sGAG measurement, and within each donor, the assays were run in triplicate. Representative pellets from the adenoviral experiments were also fixed in 4% paraformaldehyde, processed for histology and stained with toluidine blue to detect sGAG in the pellet matrices (34,35).…”

“…The genespecific primers used for these analyses and the annealing temperatures used for each primer set are provided in ▶ Table 1. To provide some comparative context, transcriptional outcomes from SMPC were referenced to analogous data derived from experiments completed on equine articular chondrocyte pellet cultures (the target phenotype) and bone marrow-derived stem cell pellet cultures, using equivalent culture protocols and analyses (34).…”

“…Collagen type II protein secretion was measured with a commercially available ELISA assay n , as previously described, using the manufacturer's recommended protocol (35). Sulfated glycosaminoglycan (sGAG) secretion into the pellet matrices was measured using the dimethylmethylene blue (DMMB) assays, also as previously described (34,35).…”

“…Third passage SMPC were transferred to pellet cultures (2.5 × 10 5 cells per pellet) by centrifugation at 390 rcf in 1.5 ml microcentrifuge tubes, as previously described (34,35). Pellets were maintained in DMEM, supplemented with insulin/transferrin/selenous acid (ITS), 37.5 μg/ml ascorbic acid, 100 μg/ml sodium pyruvate, 10 -7 M dexamethasone, 1% penicillinstreptomycin and either 10 ng rhTGF-β1 i or 100 ng rhBMP-2/ml i for 20 days.…”