| Abstrakt: |
Human cell line HS-27A represents an immortalized subpopulation of human stromal cells derived from bone marrow. HS-27A cells meet the criteria set by the International Society of Cell Therapy (ISCT) for the classification as mesenchymal stem cells (MSCs) by expression of surface molecules CD73, CD90, CD105 and HLA-ABC with no expression of CD14, CD31, CD34, CD45 and HLA-DR. We hypothesized that these cells may undergo osteogenesis similar to human bone marrow-derived stromal cells (BMSCs) and serve as a model of osteogenic cell responses under normal and inflammatory conditions. HS-27A cells were treated with: standard osteogenic factors, i.e., ascorbic acid (Asc), dexamethasone (Dex) and b-glycerophosphate (BGP); recombinant human bone morphogenetic protein 2 (rhBMP-2), MEK1/2 kinase inhibitor (PD98059) and/or lipopolysaccharide (LPS). Cells were examined for alkaline phosphate (ALP) activity, mRNA expression of osteoblastic markers (qRT-PCR) and mineralization of extracellular matrix. Besides, we analyzed mRNA expression of proinflammatory cytokines IL-1β and TNF-α in LPS-treated cells and evaluated transfection efficiency of these cells with Lipofectamine 3000 for potential future genetic manipulations. We determined that HS-27A cell line increase alkaline phosphatase activity (P<0.05), while mineralization of extracellular matrix remains low. Cells treatment with rhBMP-2 and PD98059 resulted in increased mRNA levels for osteogenesis-related transcription factor MSX-2, bone sialoprotein and osteocalcin (all P<0.05). In osteogenic cultures with and without rhBMP-2, addition of LPS led to increased ALP activity, mRNA levels for collagen type I and osteocalcin as well for IL-1β and TNF-α (all P<0.05). We also show that HS-27A cells can be transfected with phMGFP plasmid using Lipofectamine 3000 with low efficiency that may be sufficient for some genetic manipulations. Thus, HS-27A cell line appear as a useful in vitro cell culture model to study short-term osteogenic and inflammatory-related responses of human bone marrow stromal cells. [ABSTRACT FROM AUTHOR] |
