Microarrays were prepared at a stiffness of 1 kPa or 25 kPa, as measured via nanoindentation (Optics11 Life Piuma Nanoindenter). Furthermore, an OmniGrid Micro automated microspotter (Digilab, MA) created array grids of 10 ECM proteins in one- and two-way combinations, for a total of 55 combinations (Figure 1A). The ECM proteins included human collagen I (C1; Millipore, Burlington, MA), collagen III (C3; Millipore), collagen IV (C4; Abcam, Cambridge, MA), collagen V (C5; Abcam), decorin (DC; R&D Systems, Minneapolis, MN), fibronectin (FN, Millipore-Sigma, Burlington, MA), hyaluronic acid (HA; Lifecore Biomedical, Chaska, MN), laminin (LN, Millipore), lumican (LU, Acro Biosystems, Newark, DE), and tenascin C (TC; R&D Systems); these ECM proteins were selected based on our previous work with liver progenitors[19 (link)] and also since these are major ECM proteins present in the liver.[24 (link)] Each microspot or ‘island’ was spotted with a pin diameter of 450 µm and 1 mm center-to-center spacing. Single proteins were spotted at 250 ng µL−1 while 125 ng µL−1 per protein was utilized for two-way combinations. The retention of ECM proteins on PA microarrays was previously characterized using fluorescently labeled C1.[11 (link), 45 ] Lastly, rhodamine-labeled dextran spots were spotted for microarray alignment.