We demonstrated that MDW is a practical and versatile system for ECM micropatterning and for the generation of ECM microarrays. The salient features of this technology include the printing of multicomponent ECM compositions in nonrepeating geometries that can span the area of a standard microscope slide; the micropatterning of ECMs for regulating cell attachment, phenotype, and function; and the fabrication of ECM microarrays for assessment of matrix-mediated induction of ESC differentiation. To date, there are limited numbers of groups that have utilized this type of MDW technology. Mei et al.
developed multicomponent ECM microarrays with 6–9 µm feature sizes to assess the attachment and spreading of skeletal myoblasts on varying gap distances and protein compositions [14
]. In contrast, we demonstrated the features of ECM micropatterning at the supracellular scale (~30–300 µm features).
The role of ECMs on the modulation of ESC self-renewal or differentiation has only recently been studied. Using traditional DNA microarray technology, several investigators have assessed the role of ECM composition on ESC differentiation towards hepatic, cardiac, and ectodermal differentiation [15
]. More recently, ECM microarrays have been adapted in a multiwell format for combined assessment of matrix and soluble inducing factors on cardiac differentiation of murine ESCs [22
]. Besides assessing stem cell fate specification, the ECM microarray platform has also been shown to define the ECM and soluble factor microenvironment that promotes human ESC pluripotency and proliferation [23
In comparison to DNA robotic spotting technology, we provide another method to generate ECM microarrays for stem cell fate determination using MDW, which can be further adapted to generate ECM microarrays with varying geometries. Although a simplified ECM microarray containing 4 compositions was demonstrated, this technology can be extended to larger quantities of ECM as well as compositions consisting of multiple ECM components. Furthermore, this system can incorporate repeated time point measurements using fluorescent reporter genes for non-invasive temporal assessment of cell fate specification. Despite the limitations of this simplified ECM microarray, our data provides evidence of ECM-mediated effects on ectodermal differentiation of ESCs.
In addition to the generation of ECM microarrays for stem cell fate specification, the MDW technology can be utilized for a number of other biological applications. For example, to improve cell survival in ischemic tissues in vivo, the ECM microarray can serve as an in vitro platform for identifying the ECM requirements that support cell viability and proliferation under conditions that mimic ischemia, such as hypoxia and reduced serum media. In addition, the MDW technology can be used for tissue engineering constructs with preferential ECM geometries and cellular alignments that mimic physiological tissues. Scaffold designs incorporating MDW can promote efficient homing of cells in predefined patterns. Other applications address questions of basic biology, such as the role of cell–cell, cell–matrix, and cell–soluble factors on cell phenotype and function. These applications are interesting and warrant future research.
Cell–ECM interactions are involved in a wide range of biological processes, from the formation of embryonic organs to pathological remodeling in disease states. In chick embryos, for example, fibronectin is critical for embryonic development and is expressed in the dorsal aorta of embryonic day 6 (E6) [24
], and laminin is prevalent in the aortic vascular wall at E10 [24
]. In the adult vasculature, for example, collagen IV is highly abundant in the basement membrane of blood vessels [25
]. Owing to the importance of cell–ECM interactions during embryonic development and physiological maintenance, ECMs likely play an important role in specifying stem cell fate.
The mechanism by which ECMs mediate the induction of stem cell differentiation is not well characterized. ECM-mediated regulation of cell behavior is largely due to integrins, which are heterodimeric transmembrane adhesion receptors. The extracellular domain of integrins binds to ECMs, whereas the intracellular domain connects to the actin cytoskeleton through the focal adhesion complex. Emerging evidence implicates integrins as mechanotransducers that relay external cues from the ECM into the intracellular space and activate downstream signaling pathways that regulate stem cell renewal and differentiation [26
]. Other mediators include focal adhesion kinase, which have been shown to modulate cardiogenesis in ESCs [28
]. As further research in cell–ECM interactions continue, we anticipate these mechanisms will reveal interesting insights regarding the process by which cells sense and respond to matrix stimuli.
Although MDW is a powerful and versatile platform for studying the role of ECM spatial patterning and chemical composition in directing cellular organization and stem cell fate specification, it is limited by several technical issues. The use of MDW with two-dimensional ECM printing does not mimic the three-dimensional ECM microenvironment, which can be assessed using microwell technology [29
]. Future application of MDW with a microwell platform may enable the generation of ECM spatial geometries and chemical compositions in a three-dimensional microenvironment. Another limitation of MDW is the open cell culture system that enables paracrine factors from neighboring cells of an ECM microarray to influence each other. To overcome this limitation, individual ECM compositions can be isolated from one another using gaskets to create an ECM microarray with multiwell platform [22
]. Despite these limitations, our results highlight the utility of MDW as a reproducible platform for studying cell-ECM interactions
In summary, we have demonstrated a MDW approach for generating micropatterned ECMs and ECM microarrays. This method is amenable to the deposition of varying ECM compositions, geometries, and sizes. MDW shows tremendous potential for micropatterning applications related to cellular patterning and cell-matrix interactions that influence stem cell renewal or differentiation. We anticipate that this technology will have increasing value for the study of cellular function and behaviour.