Supplementary Materialssupplemental. the liver and pancreas fates. We identified ECM combinations that influence endoderm fate decisions towards these lineages, and demonstrated the utility of this platform for studying ECM-mediated modifications to signal activation during liver specification. In particular, defined combinations of fibronectin and laminin isoforms, as well as combinations of distinct collagen subtypes, were shown to influence SMAD pathway activation and the degree of hepatic differentiation. Overall, our systematic high-throughput approach suggests that ECM components of the microenvironment have modulatory effects on endoderm differentiation, including effects on lineage fate choice and cell adhesion and survival during the SAG inhibitor differentiation process. This platform represents a robust tool for analyzing effects of ECM composition towards the continued improvement of stem cell differentiation protocols and further elucidation of tissue development processes. microenvironments in order to study cell differentiation and tissue development. These approaches have emphasized the reduction of multicomponent cellular microenvironments into distinct individual signals that can be tightly controlled in engineering environments, and have provided insights into regulatory mechanisms. However, the understanding of how combinations of microenvironmental cues act together to regulate stem cell fates has been restricted by the iterative nature of these methods. Cellular microarrays can facilitate the combination of distinct biochemical cues in a high-throughput manner, and the quantitative assessment of how such combinatorial microenvironments regulate cell fate decisions. Cellular microarray platforms have been employed to study neural stem cell fate , and towards the clarification of the role of the microenvironment in the mammary gland . Anderson and co-workers used this approach to identify synthetic materials that maintain human embryonic stem cell pluripotency . We have previously developed an extracellular matrix (ECM) microarray, which has been applied towards the examination of hepatocyte survival and stem cell differentiation . Recently, we expanded the throughput of this platform towards the study of lung adenocarcinoma cell adhesion and potential mechanisms underlying metastasis . Here, we have employed an ECM microarray-based approach towards the systematic analysis of liver and pancreas differentiation of endoderm progenitor cells within distinct SAG inhibitor ECM microenvironments. [13, 14]. Together with growth factor signaling, epigenetic changes have additionally been demonstrated to be critical for directing the fate of endoderm cells, for example, increased methylation of distinct promoter regions has been identified to be necessary for hepatic Rabbit Polyclonal to OR10A7 (liver)-lineage commitment . Notably, limited studies have examined the expression of ECM components during liver [16, 17] and pancreas  development, or during the differentiation of stem and progenitor cells [19C21]. Thus, the role of ECM in the SAG inhibitor differentiation of hepatic and pancreatic lineages remains primarily unclear. In this report, we demonstrate an unbiased high-throughput approach for identifying ECM combinations that modulate liver and pancreas differentiation and for investigating the signaling events underlying cell lineage commitment. In particular, we illustrate the scope of this approach by employing multiple iterations of an ECM microarray platform consisting of 741 unique pairwise combinations of 38 ECM molecules, as well as subsequent arrays formulated from focused subsets of ECM combinations for defined mechanistic studies. Overall, our studies highlight the capabilities of a high-throughput cell microarray platform for deconstructing the complex signals regulating endoderm differentiation. 2. Materials and methods 2.1. ECM array fabrication and cell seeding Vantage acrylic slides (CEL-1 Associates VACR-25C) were coated with polyacrylamide gel pads (6022mm) as described previously . ECM domains were arrayed using a DNA Microarray spotter (Cartesian Technologies Pixsys Microarray Spotter and ArrayIt 946 Pins) from 384-well V-bottom source plates containing the ECM combinations previously prepared using a Tecan EVO 150 liquid handler. Specifically, 10 l volumes of ECM combinations were prepared in the 384-well source plate using the liquid handler, and these ECM combinations were prepared to a final concentration of 200 g/ml in a previously described buffer , consisting of 100 mM acetate, 5mM EDTA, 20% glycerol, 0.25% Triton X-100, and pH adjusted to 5.0. 741 combinations were spotted in replicates of five and rhodamine dextran (Invitrogen) was arrayed to dually serve as.