INTRODUCTION: Stem cells have become promising treatments for osteoarthritis due to the cells’ ability to regenerate cartilage and availability from bone marrow. Various studies have established the chondrogenic potential of human marrow stromal cells (hMSCs) upon treatment with transforming growth factor β1 (TGF-β1), yet the difference in potential between cells derived from young subjects and those derived from elder subjects has not been confirmed.
OBJECTIVES: This study seeks to establish whether the chondrogenic potential of hMSCs changes with age and sex. This study used a high-density 2D model to measure the acute response of hMSCs to chondrogenic induction over a short time course and various treatment levels. The experiments investigated the expression of chondrogenic genes and expression of TGF-β1 receptors (ALK5) in hMSCs after TGF-β1 treatment to determine whether pediatric hMSCs have more potential for chondrocyte differentiation than adult hMSCs.
METHODS: With IRB approval, nine bone marrow samples were obtained from discarded tissue of adults undergoing total hip replacement and juveniles requiring bone graft for alveolar cleft repair. Subject ages ranged from age 8 to 66. Low-density mononucleated cells were cultured in plastic tissue culture dishes. Adherent hMSCs were expanded in monolayer culture with phenol red-free α-MEM medium with 10% fetal bovine serum. After 48 hours of treatment with TGF-β1, cells were collected for RNA extraction and RT-PCR analysis of chondrogenic genes and TGF-β1 receptor levels. Alcian blue staining in 24-well plates of hMSCs was performed after 10 days to compare the effects of different concentrations of TGF-β1, and the effects of another inducer of chondrogenesis, kartogenin (KGN) on matrix accumulation.
RESULTS: Gel electrophoresis of PCR products revealed no consistent trend in chondrogenic mRNA expression in pediatric cells compared to adult cells, or female cells compared to male cells. The data indicate that the change in chondrogenic potential of hMSCs with age and sex is inconsistent. KGN showed no consistent effect on hMSCs. Cells with high baseline levels of TGF-β1 receptor (ALK5) showed no upregulation of ALK5 after TGF-β1 treatment, while samples with low basal expression of TGF-β1 receptors showed upregulation after TGF-β1 treatment.
CONCLUSIONS: There is still much debate in the literature regarding the potential of adult hMSC chondrogenesis compared to juveniles. This study confirms the irreproducibility of displaying differences between young and adult hMSCs. A larger sample size is needed to establish a correlation between age and chondrogenic potential. Further in vitro studies will consider the optimum time course and concentration of TGF-β1 to observe differences in gene expression of cells, and will identify other clinical determinants of differentiation potential.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/23744 |
| Date | 12 July 2017 |
| Creators | Burke, Elaine |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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