ABSTRACT
The stem cell niche is crucial to the control of stem cell fate determination in vitro as well as in vivo, and an understanding of these niches is required for the progression of stem cell and tissue engineering. The goal of our study was to commit human mesenchymal stem cells (hMSCs) to the epithelial lineage. To do this, we cultured bone marrow-derived mesenchymal stem cells (MSCs) on plates coated with type I collagen gel with or without 10 µM all-trans retinoic acid (ATRA).We found depth-dependent differentiation of hMSCs to the epithelial lineage, with the thick collagen gel (1900 µm) generating more than 80% cytokeratin-18 (CK-18)-positive cells, whereas the thin collagen gel (100 µm) generated significantly fewer CK-18-positive cells. In addition, we found that supplementation of 10 µM ATRA enhanced CK-18 expression and induced cluster-formation in cells grown on the thick collagen gel. The effect of gel depth on hMSC differentiation appears to be caused by partial cytoskeletal disruption.These results suggest that ATRA and a collagen extracellular matrix may have a synergistic effect on differentiation of human mesenchymal stem cells to epithelial lineage.
Subject(s)
Cell Differentiation/drug effects , Collagen Type I/pharmacology , Epithelial Cells/cytology , Gels/chemistry , Mesenchymal Stem Cells/cytology , Tretinoin/pharmacology , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Drug Synergism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Extracellular Matrix/metabolism , Fluorescent Antibody Technique , Humans , Keratin-18/analysis , Tissue Engineering/methodsABSTRACT
It is crucial to understand how gravitational force affects the osteogenic differentiation of mesenchymal stem cells (MSCs), and these fundamental aspects hold promise for the development of a novel model of MSC regulation for cell proliferation and differentiation. The objective of this study was to investigate how significantly gravitational dispersion affects the spontaneously induced osteogenic differentiation of MSCs. Expression of surface antigen was measured by flow cytometry prior to two axial rotational cultures. About 12,500 hMSC cells were spread on culture wells of 1.8 cm(2) surface area and incubated for 7 days at 5% CO(2). The culture medium, 10% FCS/DMEM containing 3 ng/ml bFGF, was replaced every 3 days. Four wells then were placed in a 50-ml centrifugal tube filled with 10% FCS/DMEM without bFGF. The centrifugal tube was attached to the center of the rotor, and two axial rotational cultures were started at 10 rpm each of both rotational speeds. It was confirmed that the hMSCs used in this study expressed typical surface antigens as well as a multipotent differentiation ability for either osteogenic or adipogenic differentiation. Spontaneous expression of alkaline phosphatase (Alp) mRNA following the conventional static culture (1G condition) was suppressed by two axial rotational cultures for 7 days (p < 0.05). A separate study indicated that the cell count number eventually increased from 24,700 ± 6,400 to 78,400 ± 18,700 (p < 0.05). In addition, suppressed Alp mRNA was recovered after an additional 7-day culture under static conditions. This result indicated that dispersion of gravity is a promising modality to regulate osteogenic differentiation of hMSCs.
