Effect of Substrate Elasticity on In Vitro Aging of Human Mesenchymal Stem Cells
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Effect of Substrate Elasticity on In Vitro Aging of Human Mesenchymal Stem Cells Courtney E. LeBlon,1 Caitlin R. Fodor,2 Tony Zhang,2 Xiaohui Zhang,1 Sabrina S. Jedlicka2,3,4 1
Mechanical Engineering & Mechanics, 2Bioengineering Program, 3Materials Science & Engineering, 4Center for Advanced Materials & Nanotechnology Lehigh University, Bethlehem, PA
ABSTRACT Human mesenchymal stem cells (hMSCs) were routinely cultured on tissue-culture polystyrene (TCPS) to investigate the in vitro aging and cell stiffening. hMSCs were also cultured on thermoplastic polyurethane (TPU), which is a biocompatible polymer with an elastic modulus of approximately 12.9MPa, to investigate the impact of substrate elastic modulus on cell stiffening and differentiation potential. Cells were passaged over several generations on each material. At each passage, cells were subjected to osteogenic and myogenic differentiation. Local cell elastic modulus was measured at every passage using atomic force microscopy (AFM) indentation. Gene and protein expression was examined using qRT-PCR and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Results show that the success of myogenic differentiation is highly reliant on the elastic modulus of the undifferentiated cells. The success of osteogenic differentiations is most likely somewhat dependent on the cell elastic modulus, as differentiations were more successful in earlier passages, when cells were softer. INTRODUCTION hMSCs are adult stem cells that can differentiate into a variety of lineages, including bone, cartilage, fat, tendon, muscle, and marrow stroma.1 hMSCs are able to proliferate in culture while retaining their multilineage potential. Because of their multipotency, hMSCs have great potential for regenerative medicine and tissue engineering. However, MSCs only represent 0.01% to 0.001% of cells in bone marrow.2 Therefore, to produce sufficient cell numbers for therapeutic use, hMSCs must be expanded in vitro. TCPS is the standard growth substrate in cell culture laboratories. It has an elastic modulus of approximately 3 GPa, is several orders of magnitude stiffer than most values for cells, which are typically in the kPA range.3-6 This stiff culture substrate is likely not the ideal material for stem cell maintenance. hMSCs are subject to in vitro aging, which is hallmarked by telomere shortening, slowed proliferation, and decreased differentiation capacity.5, 7-11 For example, Bonab et al. studied the osteogenic and adipogenic differentiation potential of expanded hMSCs. By the 10th passage in vitro, 20% and 60% of the samples lost their osteogenic and adipogenic differentiation potential, respectively.7 Aging in vitro is accompanied by changes in the mechanical properties of hMSCs, most likely due to extremely stiff in vitro culture substrates, as it has been shown that the elasticity of hMSCs can be modulated by substrate.12 To examine the mechanical properties of cells, AFM indentation is commonly used.13-15 Maloney et al. used AFM to indent hMSCs maint
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