Z24 - Engineering Aligned Multi-Lineage Musculoskeletal Tissues by Spatially Controlling the Activation of SynNotch Receptors

Introduction: : New approaches for precisely controlling the spatial organization of cells are critically needed to recapitulate the complex architecture and function of human tissues. For example, one existing approach to engineer vascularized tissues is to rely on the spontaneous self-organization of endothelial cells within a mixed co-culture of cells or an organoid. However, this stochastic process of self-assembly leads to irregular vascular architectures that typically do not fully perfuse the tissue. Thus, there is a need for new technologies to spatially control both the differentiation and architecture of cellular structures, such as vasculature, to engineer advanced tissues that are reproducible and physiologically relevant.
Recent advances in synthetic biology have led to new technologies for engineering mammalian cells that can complement existing technologies for engineering the extracellular matrix, opening up new opportunities for tissue engineering. For example, the synthetic Notch (synNotch) receptor is a synthetic transmembrane receptor modified to recognize a user-defined ligand and induce expression of user-defined transcription factors. Our team previously engineered SynNotch receptors that recognize GFP and mCherry and induce MyoD and ETV2 expression, respectively. We also used microcontact and microfluidic patterning to dictate the location of these two synNotch ligands on a synthetic material surface such that we could spatially control myogenic and endothelial cell fates, towards engineering vascularized muscle tissues. Here, our objective is to spatially pattern synNotch ligands on the surface of a natural extracellular matrix-derived hydrogel (gelatin) to improve cell adhesion and biocompatibility.

Materials and

Methods: : Gelatin and GFP were functionalized with methacrylate anhydride (MA) to form gelatin methacroyl (Gel-MA) and GFP-MA, respectively. A solution of Gel-MA and Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate (LAP) was added to a glass coverslip and a flat or micromolded PDMS stamp with 10x10 µm ridges was placed on top before the surface was exposed to UV light for initial crosslinking. A GFP-MA solution was added, and the coverslip was crosslinked a second time either as-is or covered with a photomask comprising various squares or lines of different dimensions. Atomic Force Microscopy (AFM) was conducted to determine the average elastic modulus and surface topography within a 200 µm scan of a hydrogel surface that had been uniformly exposed to UV light once, twice, or twice with a photomask of 100 µm squares with 100 µm spacing. Lentivirus transfection was used to insert an anti-GFP synNotch receptor and either a mCherry reporter gene or a MyoD transgene and mCherry reporter gene into L929 mouse fibroblasts (referred to as synNotch cells). Anti-GFP mCherry synNotch cells were seeded on Gel-MA hydrogels that had either been photopatterned with various squares or capillary shaped geometry (Fig. 1A). Live cell fluorescence imaging was performed to determine the colocalization between the mCherry reporter and photopatterned GFP signal. Anti-GFP mCherry-MyoD synNotch cells were seeded on hydrogels with micromolded or flat surface topography with no GFP, a uniform coating of GFP, or photopatterned regions of GFP (Fig. 2A). The orientation and myogenic index of the immunostained myotubes were analyzed via imageJ.

Results, Conclusions, and Discussions:: Photopatterning enabled spatial control over the location of GFP on Gel-MA hydrogels. For example, by using a photomask with 100 μm squares with 100 μm spacing, Gel-MA hydrogels were patterned with GFP squares visible with both fluorescence and brightfield microscopy (Fig. 1B,C). AFM mapping of surface topography and elastic modulus demonstrated a 30 µm difference in height and an approximately 20 kPa difference in elastic modulus between photopatterned and non-photopatterned regions; there was an approximately 10 kPa difference in the elastic modulus of the bulk hydrogels that had been crosslinked twice for photopatterning versus once for the initial crosslinking (Fig. 1D-F). Anti-GFP mCherry synNotch cells demonstrated visible colocalization of mCherry and GFP fluorescence, as quantified by the Spearman colocalization coefficient (Fig. 1G-I). For smaller squares (50 μm), colocalization decreased as spacing between squares increased; for larger squares (250 μm), increased spacing between squares improved colocalization (Fig. 1H). Photomasks of complex geometries, such as capillary-like structures, were also generated to guide cell activation into more physiologically relevant structures (Fig. 1I).
We next evaluated the spatial control over myogenic synNotch transgene fate in anti-GFP mCherry-MyoD synNotch cells. Myotubes differentiated on a 250x250 µm photopatterned hydrogel demonstrated a slightly lower alignment compared to myotubes cultured on a micromolded Gel-MA hydrogel with 10x10 µm ridges and a uniform surface coating of GFP, but both photopatterning and micromolding resulted in higher myotube alignment compared to myotubes grown on a flat surface with uniform GFP coating (Fig. 2A-C). However, spatial control over myotube activation needs further optimization (Fig. 2D). We expect that more precise colocalization can be accomplished through photomasks with additional features to enhance surface topography.
In summary, photopatterning gelatin hydrogels with synNotch ligands is a promising approach for activating synNotch receptors with spatial control on an extracellular matrix-derived biomaterial and holds potential for spatially patterning multiple synNotch ligands, allowing for cellular scale control over multiple synNotch pathways. In the future, this technology can be expanded to generate more sophisticated tissue models that incorporate multiple cell types with precise spatial control, such as vascularized muscle, to better recapitulate human tissue structures and functions.

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