Morphometric Analysis to Characterize the Differentiation of Mesenchymal Stem Cells into Smooth Muscle Cells in Response to Biochemical and Mechanical Stimulation

Abstract

The morphology and biochemical phenotype of cells are closely linked. This relationship is important in progenitor cell bioengineering, which generates functional, tissue-specific cells from uncommitted precursors. Advances in biofabrication have demonstrated that cell shape can regulate cell behavior and alter phenotype-specific functions. Establishing accessible and rigorous techniques for quantifying cell shape will therefore facilitate assessment of cellular responses to environmental stimuli, and will enable more comprehensive understanding of developmental, pathological, and regenerative processes. For progenitor cells being induced into specific lineages, this ability becomes a pertinent means for validating their degree of differentiation and may lead to novel strategies for controlling cell phenotype. In our approach, we used the differentiation of adult human mesenchymal stem cells (MSCs) into smooth muscle cells (SMCs) as a model system to investigate the relationship between cell shape and phenotype. These cell types are responsive to mechanical and biochemical stimuli and the shape of SMCs is a recognized marker of differentiated state, providing a system in which morphological and biochemical phenotype are both understood and inducible. By applying exogenous stimuli, we changed cell shape and examined the corresponding cellular phenotype. In the first Aim, we applied stretch to MSCs on 2D collagen sheets to promote differentiation. Using mathematical shape factors, we quantified the morphological changes in response to defined stretch parameters. In the second Aim, we investigated the use of input energy as a means of controlling cell shape and corresponding differentiation. We examined how combinations of stretch parameters that produce equal energy input impacted morphology, and postulated that cell shape is a function of energy input. In the third Aim, we translated our method of quantifying shape factors into 3D culture, and validated the method by investigating the differentiation of MSCs into SMCs by mechanical and growth factor stimulation. We used the shape factors to quantify morphological differences and compared these changes to biochemical markers. Our results demonstrate that mechanical stretch influences multiple aspects of MSC phenotype, including cell morphology. Shape factors described these changes objectively and quantitatively, and enabled the identification of relationships between SMC shape and differentiated state. Similar morphological responses could be induced using different combinations of stretch parameters that resulted in equal energy input. Cell shape followed a linear relationship with energy input despite the variance introduced by using MSCs from different patients. Only one SMC gene marker directly exhibited this relationship; however, partial least squares regression analysis revealed that other genes were also associated with shape factors. Translation of the shape quantification method into 3D systems revealed that while the additional dimensionality hindered comparison of morphology between 2D and 3D samples, these shape factors were still applicable within 3D systems. Differences in cell morphology caused by growth factors and mechanical stretch in 3D constructs were elucidated by shape analysis, and these phenotypic changes were corroborated through biochemical assays. Taken together, these results validate the use of cell shape as means of characterizing phenotype and the process of progenitor cell differentiation. The automated method we developed generates a robust set of morphological parameters that provide a way to characterize the differentiation of MSCs into SMCs. This work has implications in our understanding of the relationship between cell morphology and phenotype, and may lead to new ways to control and improve differentiation efficiency in a variety of cell and tissue systems.