Quantitative real-time pcr analysis for chondrogenic differentiation of human mesenchymal stem cell in alginate scaffolds

T. Kamarul, Pan-Pan Chong, L. Selvaratnam, Cheh-Chin Tai, Azlina A. Abbas

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INTRODUCTION: Despite the manyadvances seen in cartilage repair techniques,cartilage damage remains a difficult conditionto treat [1]. Mesenchymal stem cells (MSCs)being capable of self-renewal and undergomultilineage differentiation was thought to bethe answer to this problem [2]. It has beenproposed that in order to achieve this, MSCextracted from tissues undergo cell proliferationin vitro prior to reimplantation to the defect site.However, the question remains as to whetherMSCs are able retain their multilineage andphenotypic potential in prolonged in vitroculture environment. A study was thereforeconducted to assess the ability of MSCs tomaintain its differentiated phenotype bychemically inducing chondrogenicdifferentiation and using quantitative real-timepolymerase chain reaction (qRT-PCR) todetermine their gene expression over a periodof time. METHODS: Human bone marrow samples (2ml) were collected from patients undergoingarthroplasty surgery whom were otherwisehealthy. The mononuclear cells extracted wereseparated using Ficoll–Paque PLUS viacentrifugation. Subsequently, suspended cellswere removed after 5 days of culture, andadherent cells left to grow. Cells were detachedupon reaching 80-90% confluence and subcultured up to 3 passages prior to furtherexperiments. MSC antigens were recognizedusing monoclonal antibodies CD29, CD105 andCD166. To distinguish MSCs from residenthematopoietic stem cells, CD34 surfacemarkers were used as negative controls. Thecharacterised MSCs were then cultured inalginate scaffolds using chondrogenic medium.To assess chondrogenesis, gene expressionanalyses using RT-PCR were conducted onchondrogenic-MSCs using cartilage-specificphenotypic markers at pre-determined timepoints. RESULTS: Flow cytometry analyses confirmthe presence of MSCs and absence ofhaematopetic stem cells. The gene expressionlevel of COMP for chondrogenic-MSCs wassignificantly higher than that of control (MSCcultured in basal growth medium) and upregulated in long-term alginate 3-dimensionalscaffolds culture environment. However, thegene expression levels of aggrecan appear todecrease in long-term culture conditions. DISCUSSION & CONCLUSIONS: Flowcytometry and RT-PCR analysis revealed thatMSCs and chondrogenic-MSCs in culture retainthe appropriate phenotypes comparable to thatof in vivo MSCs. However, certain genesappear to be down regulated when maintainedin prolonged cell culture conditions and mayaffect therapeutic outcomes when introduced inin vivo conditions.
Original languageEnglish
Pages (from-to)44
Number of pages1
JournalEuropean Cells and Materials
Issue numberSuppl 1
Publication statusPublished - Aug 2009

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