Chondrogenic Potential of CD90+ Progenitors Figure 1. Micro-mass differentiation of sfMPCs. Normal (human N = 5 A–D & canine N = 2 H–K) and OA (human N = 5 E–G & canine N = 2 L–N) derived sfMPCs where aggregated using the micro-mass technique and induced to differentiate over a 14 day period with media supplements. qRT- PCR results demonstrated that Sox9, Collagen 2 and Aggrecan are significantly elevated at days 3, 5, 8 and 14 of differentiation (A,E,H,L), furthermore, at day 14 Collagen 2 protein is expressed in the cell cultures (B,F,I,M). Secondary controls (C,J) demonstrate minimal non-specific staining. Scale bars represent 50 mM. * = p.0.05. doi:10.1371/journal.pone.0043616.g001 tissue-like structures were highly cellular (Figure 5 E). TEM analysis demonstrated that the cells were in close association with each other (Figure 5 F), and were also in proximity to collagen fibres (Figure 5 G). The cells in these self-assembled structures were attached to the surrounding collagen fibre network of the extracellular matrix (Figure 5 E and F). Discussion OA is a degenerative disorder of synovial joints characterized by articular cartilage degradation and meager evidence of repair or regeneration. Although evidence of cartilage stem/progenitor cells has been difficult to obtain, which may explain this response, recent animal studies have raised the possibility that MPCs present in the synovial membrane or joint fluid may play a role in repair cartilage in vivo [9,10]. MPCs are of particular interest to regenerative medicine as they have the ability to generate bone, cartilage, and muscle under certain conditions. Indeed, properly harnessed, these MPCs could be utilized in-vivo to promote the repair or replacement of tissue damaged by trauma or disease. MPCs for skeletal regeneration are most commonly sourced from the bone marrow or fat, and it is increasing recognized that many adult tissues have resident stem cell populations. Although seemingly the ideal source of MPCs for cartilage regeneration, it has been claimed that adult articular cartilage uniquely lacks ‘true’ MPCs [21]. Interestingly, MPC populations have been isolated from the synovial membrane and synovial fluid bathing the joint [14]. Moreover, when compared to MPC populations isolated from bone marrow or fat [12], sfMPCs demonstrate an increased chondrogenic capacity compared to MPC populations isolated from bone marrow or fat [12]. This current study demonstrates that normal and OA (human and canine) derived sfMPCs undergo effective chondrogenesis when a micro-mass step is included in the differentiation protocol, which corroborates previously published findings [2]. Further- more, when sfMPCs (normal/OA) are enriched for the CD90- positive fraction, enhanced chondrogenesis is observed compared to the CD90-depleted fraction. This is of particular interest as a recent study by Nagase et al. reports that the chondrogenic potential of adult stem cells decrease with the subsequent loss of CD90 [19], while another study seems to contradict this observation [22]. The results of the present study supports those of Nagase et al. suggesting that CD90 is a cell surface marker that correlates positively with chondrogenic potential [19]. It is also noteworthy that patients with even advanced OA have sfMPCs, and in some cases with numbers far exceeding normal individuals [2], which could be interpreted as a cellular repair response. A particularly noteworthy finding in the present study is that CD90-positive sfMPCs (normal/OA) can differentiate into chon- drocytes without a micro-mass step. However, whereas whole populations of sfMPCs from normal individuals aggregate and express high levels of chondrogenic markers, whole populations of sfMPCs from OA joints do not aggregate spontaneously, grow only as a monolayer, and express low levels of chondrogenic markers during differentiation. The exact functional significance of micromass aggregation is unclear, yet micro-mass-induced aggre- gation clearly increases the efficiency of chondrogenesis [23,24], possibly by mimicking embryonic tissue condensation. Although the promotion of cell differentiation by aggregation is not limited to sfMPCs and chondrogenesis, (having been demonstrated with pancreatic, hepatic, fibroblastic, bone marrow, and cord blood MPCs [25–30]), it seems curious that all normal and OA sfMPCs behave differently in non-aggregating culture even though they PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43616