br Results br Discussion WNT proteins in vivo

Results
Discussion WNT proteins in vivo are often secreted locally and presented to responsive LY2228820 in a spatially controlled manner (Alexandre et al., 2014; Clevers et al., 2014; Farin et al., 2016; Goldstein et al., 2006; van den Heuvel et al., 1989). In this study, we present a covalently immobilized WNT platform that can act as a basal signal to support stem cell maintenance and tissue engineering. The aldehyde-amine chemistry does not require the continued presence of detergent and is easier than the multistep immobilization onto the recently published microbeads (Habib et al., 2013). In addition, the dynamics of the culture media makes it challenging to spatially control the WNT microbeads. The WNT platform can be stored and maintain its signaling activity for a prolonged period. We demonstrate the ability of the basal WNT surfaces to induce WNT/β-catenin signaling as well as enrich and maintain adult stem cells and ESCs in 2D cultures. Unlike soluble WNT3A proteins where ESC passaging increases the proportion of ALP+ pluripotent colonies, the WNT platform maintains the heterogeneity of ALP colonies in serum-free medium. This system may provide a way to investigate fluctuations between pluripotency/differentiation states of ESCs under defined conditions. We speculate that the non-uniform distribution of immobilized WNT proteins on the surface can be a potential reason for this observation. Alternatively, the division of ESCs on the WNT platform can yield a cell in close proximity to the WNT source, while the daughter cell has less access to the immobilized WNT. Importantly, the WNT platform can be adapted to 3D tissue culture. To demonstrate this, we used hMSCs, which require WNT signaling for their maintenance and differentiation in 2D culture (Boland et al., 2004; de Boer et al., 2004; Ling et al., 2009; Narcisi et al., 2015). By combining the WNT platform with a 3D culturing system of primary hMSCs, we recapitulated layers of a maturing cell environment of the periosteal bone niche (Bonewald, 2011). Unlike WNT DTT control surfaces, cells in close proximity to the basal WNT platform maintained high expression of the stem cell marker STRO1. In addition, the basal WNT directed migration and differentiation of cells toward an osteogenic phenotype within 7 days of culture. Importantly, adding soluble WNT3A to the 3D system maintained cell proliferation and expression of the stem cell marker STRO1, but migration and differentiation processes were significantly reduced. This implicates the role of spatially confined WNT signals in the maintenance of stem cells and directed cell differentiation. Therefore, spatial presentation of WNT signals to cells in a 3D context can be used for tissue engineering purposes. Our ultimate goal is to mimic basic cellular, signaling, and mechanical (Bonewald and Johnson, 2008; Foster et al., 2015; Robinson et al., 2006; Tu et al., 2012) elements of the bone environment by generating a controlled microsystem of stem cells and a directed differentiation into osteogenic cells in 3D culture. As many types of stem cells are WNT3A responsive, we anticipate that this platform can be adapted to generate WNT-mediated tissue formation in vitro. Developmental studies show the involvement of other WNTs in tissue patterning (van Amerongen and Nusse, 2009). Fortunately, the predicted protein sequence for the known WNTs shows extensive similarity (Thrasivoulou et al., 2013), suggesting they are likely to be amenable for immobilization using this aldehyde chemistry. With the advances in single-cell analysis and cell population profiling, the proposed WNT platform provides a unique opportunity to further investigate many aspects of localized signaling including the transcriptome and proteome of WNT-responsive cells. Such analysis in vivo is challenging, as it is difficult to visualize WNT proteins in mammalian systems and correlate the timing of the cellular response. This basic understanding is crucial for studying development and improving cell-based therapy.