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

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.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/145833/1/brandanw_1.pd

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffnessâ defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverseâ transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretchâ induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterialâ associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/1/term2263.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/2/term2263_am.pd

Shaping the Cell and the Future: Recent Advancements in Biophysical Aspects Relevant to Regenerative Medicine

In a worldwide effort to generate clinically useful therapeutic or preventive interventions, harnessing biophysical stimuli for directing cell fate is a powerful strategy. With the vision to control cell function through engineering cell shape, better understanding, measuring, and controlling cell shape for ultimately utilizing cell shape-instructive materials is an emerging “hot” topic in regenerative medicine. This review highlights how quantitation of cellular morphology is useful not only for understanding the effects of different microenvironmental or biophysical stimuli on cells, but also how it could be used as a predictive marker of biological responses, e.g., by predicting future mesenchymal stromal cell differentiation. We introduce how high throughput image analysis, combined with computational tools, are increasingly being used to efficiently and accurately recognize cells. Moreover, we discuss how a panel of quantitative shape descriptors may be useful for measuring specific aspects of cellular and nuclear morphology in cell culture and tissues. This review focuses on the mechano-biological principle(s) through which biophysical cues can affect cellular shape, and recent insights on how specific cellular “baseline shapes” can intentionally be engineered, using biophysical cues. Hence, this review hopes to reveal how measuring and controlling cellular shape may aid in future regenerative medicine applications