Bone Marrow-Derived Mesenchymal Stem Cells Express the Pericyte Marker 3G5 in Culture and Show Enhanced Chondrogenesis in Hypoxic Conditions Wasim S. Khan, Adetola B. Adesida, Simon R. Tew, Emma T. Lowe, and Timothy E. Hardingham United Kingdom Centre for Tissue Engineering and Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom Received 20 January 2009; accepted 24 September 2009 Published online 7 January 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.21043 ABSTRACT: Bone marrow-derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow-derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony-forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow-derived stem cells. ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:834 – 840, 2010 Keywords: bone marrow-derived mesenchymal stem cells; pericytes; chondrogenesis; hypoxia; 3G5 Articular cartilage has very limited intrinsic capacity for repair after injury. Most focal cartilage lesions, left untreated, progress to more extensive lesions and, in the long term, these require joint arthroplasty. Autol- ogous chondrocytes harvested from low weight-bearing areas of articular cartilage are being used for the repair of focal hyaline cartilage defects. 1 Although short-term clinical results have been good, evidence suggests some incidence of progressive degenerative changes in the joint. 2 This procedure is also accompanied by donor site morbidity, and the limited amount of tissue available necessitates prolonged cell expansion. There is, therefore, interest in alternative sources of adult stem cells for cell-based tissue engineering approaches for cartilage repair. Cells with stem cell characteristics have been reported in many tissues including the bone marrow. 3–6 Bone marrow-derived mesenchymal stem cells (BMSCs) rep- resent a small portion of the cells in the stromal compartment, and conditions for the differentiation of these cells into chondrocytes, osteoblasts, and adipocytes have been used to show that they are multipotent. 7 Because of their multipotency and practical access, cells from the bone marrow are of interest as a potential source of cells for the repair of focal cartilage defects in the knee. 8 There have been limited reports of human autologous bone marrow stromal cell implantation for cartilage repair. 6,9 The procedure involved the harvest of bone marrow stroma from the iliac crest and required culture expansion of cells. A better understanding of the nature of these mesenchymal stem cells is essential to harness their optimal potential. In previous studies, a minor popula- tion of BMSCs has also been found to be positive for the pericyte marker 3G5. 10 Pericytes have been shown to have multidifferentiation potential 11,12 and it has been suggested that, if distributed widely with vascular capillaries, pericytes may account for stem cells in other tissues. 13 This theory is supported by the observation that many of the tissues from which stem cells have been isolated have good vascularization. In further support of this theory, a subendothelial network of pericyte-like cells has been identified using 3G5 in the vascular bed in many human tissues. 14 Mammalian cells are normally cultured in air (con- taining 20% oxygen) with added 5% carbon dioxide, but some cells, including human bone marrow-derived hematopoetic stem cells, have been reported to prolifer- ate more rapidly in lower oxygen concentrations. 15–17 This is not surprising as the bone marrow oxygen tension in vivo is in the range of 4% – 7%. 18 Grayson et al. 19 have shown that hypoxia in human BMSCs results in the ability to maintain a significantly higher number of stem cells, higher levels of stem cell genes, and higher levels of osteoblastic and adipocytic differentiation markers on differentiation. They did not look at the effects of hypoxia on the chondrogenic differentiation of human BMSCs. In chondrocyte culture systems, it has been shown that hypoxia results in increased synthesis of extracellular matrix by chondrocytes, 20,21 and this has been extended to stem cells from adipose tissue 22 and infrapatellar fat 834 JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2010 Correspondence to: Wasim S. Khan (T: þ44 (0) 7971 190720; F: þ44 (0) 161 275 5752; E-mail: wasimkhan@doctors.org.uk) ß 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.