Alteration of gene expression levels during osteogenic induction of human adipose derived stem cells in long-term cultur
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ORIGINAL PAPER
Alteration of gene expression levels during osteogenic induction of human adipose derived stem cells in long-term culture Wan Kamarul Zaman Wan Safwani • Suzana Makpol • Somasundaram Sathapan Kien Hui Chua
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Received: 9 December 2011 / Accepted: 26 March 2012 / Published online: 5 April 2012 Springer Science+Business Media B.V. 2012
Abstract Adipose tissue is a source of multipotent stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic and adipogenic cells. Most studies on human adipose-derived stem cells (ASCs) have been carried out at the early passages. For clinical usage, ASCs need to be expanded in vitro for a period of time to get sufficient cells for transplantation into patients. However, the impact of long-term culture on ASCs molecular characteristics has not been established yet. Several studies have also shown that osteogenic and adipogenic cells have the ability to switch pathways during in vitro culture as they share the same progenitor cells. This data is important to ensure their functionality and efficacy before being used clinically in the treatment of bone diseases. Therefore, we aim to investigate the effect of long-term culture on the adipogenic, stemness and osteogenic genes expression during osteogenic induction of ASCs. In this study, the W. K. Z. W. Safwani K. H. Chua (&) Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia e-mail: [email protected]; [email protected] S. Makpol Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan, Malaysia, Kuala Lumpur, Malaysia S. Sathapan Subang Jaya Medical Centre, Selangor, Malaysia
molecular characteristics of ASCs during osteogenic induction in long-term culture was analysed by observing their morphological changes during induction, analysis of cell mineralization using Alizarin Red staining and gene expression changes using quantitative RT-PCR. Morphologically, cell mineralization at P20 was less compared to P5, P10 and P15. Adipogenesis was not observed as negative lipid droplets formation was recorded during induction. The quantitative PCR data showed that adipogenic genes expression e.g. LPL and AP2 decreased but PPAR-c was increased after osteogenic induction in long-term culture. Most stemness genes decreased at P5 and P10 but showed no significant changes at P15 and P20. While most osteogenic genes increased after osteogenic induction at all passages. When compared among passages after induction, Runx showed a significant increased at P20 while BSP, OSP and ALP decreased at later passage (P15 and P20). During long-term culture, ASCs were only able to differentiate into immature osteogenic cells. Keywords Gene expression Human adipose derived stem cells Long-term culture Osteogenesis Stemness
Introduction Stem cells have generated a lot of interests due to their potential in regenerative medicine. Its therapeutic
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