(615d) Correlating Effects of Gel Microstructural Features with Specific Differentiation Patterning of Mouse Embryonic Stem Cells

Embryonic stem cells (ESC) have the potential to be used in many applications due to their ability to differentiate into cells of any of the three germ layers (ectoderm, endoderm, and mesoderm). There are several different routes by which ESC can be guided to differentiate into specific germ layers, including genetic manipulation, chemical cues, and mechanical stimuli. With respect to the latter, it has been observed that substrate stiffness plays a significant role in governing specific phenotype differentiation.

In our previous study we investigated the effect of mechanical stiffness of fibrin on ESC differentiation. Our results indicated that while ectoderm and mesoderm germ layers displayed weak response to the change in fibrin substrate stiffness in the chosen range (2 – 250KPa), endoderm markers were strongly responsive, with softer substrates up-regulating endoderm specific markers. Although cells respond differently to substrates of varying stiffness, it was also observed that gels with the same stiffness but fabricated under different conditions lead to different differentiation patterning. Therefore, this macroscopic property is perhaps not what the cell truly experiences; modification of fabrication conditions changes the gel microstructure, which in turn affects the stiffness, and these micro-characteristics directly interact with the cells. However, it is not clear what specific microstructural features are the most influential in inducing cellular differentiation, and correspondingly, how they affect ESC behavior.

In the current work, we investigate the effect of microstructural features of fibrin gels on the differentiation of mouse ESC. Twelve different fibrin gels were fabricated by varying the fibrinogen concentration and fibrinogen to thrombin crosslinking ratio. The fibrin gels were used to induce ESC differentiation employing both 2D and 3D cultures. After the differentiation protocol the ESC were analyzed for phenotypic commitment by performing qRT-PCR for the specific germ layer markers. Each of the 12 different fibrin gels was imaged with scanning electron microscopy. Microstructural features of each of these gels were quantified using an image analysis tool for the characterization of fibrous scaffolds [1]. Specific features which were characterized include fiber diameter, node density, fiber length, and pore size.  A comparison of these attributes along with principal component analysis led to a subset of features which vary most across gel conditions. Gel microstructural features were correlated with the ESC differentiation patterning using regression analysis. The gels are heterogeneous in nature, and therefore a bootstrapping technique was used to account for the variability of the system. Interestingly, only a subset of genes shows a significant relationship with microstructural features, and that only a few of these features significantly affect differentiation, including fibrin pore size distribution and node density. These results are also compared to the correlation of gel stiffness, determined by both by rheological measurements and atomic force microscopy, to phenotype commitment. This analysis reveals the sensitivity of cellular phenotype commitment and differentiation patterning to each of the microstructural features. Moreover, such information can be used to help guide the design of scaffolds with specific properties for tissue engineering applications. Results of these studies will be presented and discussed.

 [1] A. D’Amore et al. Biomaterials, 2010, Vol. 31, 5345-5354.