Extracellular matrix microarrays to study inductive signaling for endoderm specification.

During tissue development, stem and progenitor cells are faced with fate decisions coordinated by microenvironmental cues. Although insights have been gained from in vitro and in vivo studies, the role of the microenvironment remains poorly understood due to the inability to systematically explore combinations of stimuli at a large scale. To overcome such restrictions, we implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high-throughput manner. Using embryonic stem cells as a model system, we derived definitive endoderm progenitors and applied them to the array platform to study the influence of ECM, including the interactions of ECM with growth factor signaling, on the specification of definitive endoderm cells towards 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 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. STATEMENT OF SIGNIFICANCE: Cellular microarrays can provide the capability to perform high-throughput investigations into the role of microenvironmental signals in a variety of cell functions. This study demonstrates the utility of a high-throughput cellular microarray approach for analyzing the effects of extracellular matrix (ECM) in liver and pancreas differentiation of endoderm progenitor cells. Despite an appreciation that ECM is likely involved in these processes, the influence of ECM, particularly combinations of matrix proteins, had not been systematically explored. In addition to the identification of relevant ECM compositions, this study illustrates the capability of the cellular microarray platform to be integrated with a diverse range of cell fate measurements, which could be broadly applied towards the investigation of cell fate regulation in other tissue development and disease contexts.

Cutting edge: differential requirements for Stat4 in expression of glycosyltransferases responsible for selectin ligand formation in Th1 cells.

A role for Stat4 in IL-12-induced up-regulation of selectin ligands on Th1 cells was explored. Th1 cells generated from Stat4(-/-) mice exhibited no IL-12-inducible P-selectin ligands, no up-regulation of core 2 beta1,6-glucosaminyltransferase I (C2GlcNAcT-I), and low levels of the Th1 transcription factor T-bet. In contrast, Stat4(-/-) Th1 cells exhibited only a partial defect in expression of IL-12-inducible E-selectin ligands and expressed equivalently high levels of alpha1,3-fucosyltransferase VII (FucT-VII) as wild-type Th1 cells. FucT-VII expression was induced by T cell activation, and was enhanced by IL-12 independently of Stat4, whereas C2GlcNAcT-I up-regulation was mediated exclusively by IL-12, acting through Stat4. These data show that FucT-VII and C2GlcNAcT-I are controlled through distinct pathways and imply the existence of at least one other IL-12-inducible glycosyltransferase required for E-selectin and possibly P-selectin ligand formation in Th1 cells.