Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/18661
Title: Applying Macromolecular Crowding to Promote the Expansion and Adipogenic Differentiation of Human Mesenchymal Stem Cells in vitro; an Effect of Matrix Reciprocity
Authors: LOE HUI LI @ FELICIA CELESTE LOE
Keywords: Macromolecular Crowding, Mesenchymal Stem Cells, Adipogenesis, Proliferation, Matrix Reciprocity
Issue Date: 21-Jul-2010
Source: LOE HUI LI @ FELICIA CELESTE LOE (2010-07-21). Applying Macromolecular Crowding to Promote the Expansion and Adipogenic Differentiation of Human Mesenchymal Stem Cells in vitro; an Effect of Matrix Reciprocity. ScholarBank@NUS Repository.
Abstract: Two bottlenecks impede use of human bone marrow-derived mesenchymal stem cells (hMSCs) in tissue engineering and regenerative medicine -- (a) Need for extensive ex vivo expansion (self-renewal) to generate clinically relevant numbers and (b) Attenuation in differentiation potential with prolonged ex vivo propagation. hMSCs reside in a physiologically crowded microenvironment composed of soluble factors, cellular components and dense arrays of solid extracellular matrix (ECM); which is crucial in maintaining their self-renewal, multipotentiality via cell fate determination and preconditioning of progeny daughter cells. Maintenance of their phenotype or differentiation is governed by specific cues within each unique local microenvironment. Macromolecular crowding (MMC) is a novel method that exploits the properties of inert macromolecules to generate an Excluded Volume Effect (EVE). While theoretical papers have described the importance of MMC in recapitulating the physiological environment and the possible consequences, limited literature describe actual experimental application of MMC on biological cellular systems. The aim of this study was to determine if the cocktail of macromolecules containing FicollTM70 and FicollTM400; could emulate the physiological environment in the ¿liquid phase¿ (direct crowding in the media) and the ¿solid phase¿ (matrix remodeling and deposition). This would enable novel applications such as enhanced proliferation while maintaining multipotentiality of hMSCs during ex vivo culturing; as well as enhanced adipogenic differentiation via matrix reciprocity. During long-term ex vivo expansion of hMSCs, MMC resulted in a higher proliferative rate compared to controls and generated a fold increase in cell numbers. hMSCs expanded under MMC retained their multipotentiality and self-renewal capacity as determined by cell surface markers analysis and colony forming units assay, respectively. hMSCs propagated under MMC for 28 days also retained higher adipogenic differentiation potential compared to controls when induced in the absence of crowding. Macromolecular crowding enhanced differentiation response (cytoplasmic lipid droplet accumulation) during adipogenesis at an early (p4) and late passage (p8); and enabled more effective use of inductive ingredients. Immunocytochemistry of the cell layer highlighted extent of matrix remodeling during differentiation and demonstrated that MMC increased both deposition and remodeling of collagen IV, a key matrix protein involved in adipogenesis. This observation was also confirmed by immunoblot. Interestingly, MMC reduced gene expression of collagen IV, indicating feedback from the matrix (matrix reciprocity). Matrices deposited by hMSCs, preadipocytes and mature adipocytes in the absence and presence of crowding were decellularised via detergent lysis -- then reseeded with naïve hMSCs and adipogenically induced. Adipocyte matrix deposited under MMC served as the best substrate for adipogenesis while hMSCs seeded on hMSC matrix and preadipocyte matrices differentiated less than those on TCPS, indicated intrinsic signaling capacity from the matrix. To further elucidate signaling from the matrix, naïve hMSCs were reseeded on adipocytes and preadipocyte matrices deposited ± MMC; then maintained in basal media. The hMSCs differentiated into the same phenotype as the origin of the matrix. 0.5M NaCl treatment of the matrices to remove elution-sensitive factors resulted in a loss of this signaling; indicating that biochemical cues from the matrix were due to sequestered factors in the matrix.
URI: http://scholarbank.nus.edu.sg/handle/10635/18661
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