Activating Synthetic Notch Receptors by a Micropatterned Surface for Spatial Control of Differentiation

Introduction:: Spatial organization of multiple cell types is intrinsic to tissue function in vivo. Recapitulating this cellular organization in vitro is essential to creating readily available and accurate tissues for drug screening, disease modeling, and regenerative medicine. However, recreating the organized complexity of native tissues with cellular scale precision remains a major technological hurdle. The synthetic Notch receptor (synNotch) is a modular receptor that can be genetically engineered into mammalian cells to induce expression of user-prescribed transgenes upon binding of user-prescribed ligands. To date, these ligands have been presented by expressing them in another cell or uniformly coating them on a surface. In this study, our goal was to spatially activate the synNotch receptor with cellular scale precision by culturing cells on a surface microcontact printed with the cognate ligand. As a case study, we used this approach to spatially control the transdifferentiation of fibroblasts into skeletal muscle precursor cells by activating myoD, a master myogenic transcription factor. We also created a dual-fate cell line and orthogonally activated two synNotch pathways with spatial control, laying the foundation for more accurate, multi-cell tissues in vitro.


Materials and Methods:: C3H mouse embryonic fibroblasts were transduced with an anti-GFP-synNotch receptor with myoD-mCherry target gene. Upon synNotch binding to GFP, the mCherry reporter and the transcription factor myoD are expressed (Fig. 1A). To print GFP on a surface: (1) biopsy punches were used to cut out cylindrical stamps (8mm diameter) from PDMS, or (2) a Digital Light Processing 3D printer was used to create replica molds of finer patterns, such as 500μm thick concentric circles, aligned rows, and letters. To maximize cell adhesion and GFP adsorption, PDMS coated coverslips were treated with APTES and glutaraldehyde before fibronectin coating and microcontact printing with GFP. Cells were then cultured on the micro patterned surface (Fig. 1B, C). To measure differentiation of multi-nucleated muscle fibers, cells were fixed and immunostained for DAPI and sarcomeric α-actinin, a marker for myotubes (FIG 1C). Another line of C3H mouse embryonic fibroblasts were transduced with anti-GFP-synNotch receptor activating miRFP reporter gene and anti-mCherry-synNotch activating tBFP reporter gene (Fig 1D). These cells were then cultured on 500µm rows of GFP and mCherry that were perpendicularly microcontact printed (Fig 1E). ImageJ was used to quantify the plot profiles of both activation pathways (Fig 1E).


Results, Conclusions, and Discussions:: We observed localized myotube formation primarily in C3H synNotch cells in contact with the patterned GFP (Fig. 1B). By spatially activating myoD, we obtained myotube-like cells that express alpha-actinin, a myotube marker. Alpha-actinin immunostaining closely recaptures the geometry of the GFP patterns (Fig 1C). Dual-fate cells spatially and orthogonally activate miRFP on GFP pattern and tBFP on mCherry pattern, validated by our microscopy and plot profiles (Fig 1E).

By combining synthetic biology and microfabricated surfaces, we can drive spatial transgene activation and cell differentiation towards the myogenic lineage with microscale precision. By using multiple synNotch receptors in the same cells, we can spatially control activation of multiple differentiation pathways, as demonstrated here by spatially controlling the dual-activation of two fluorescent reporters. These technologies lay the foundation for the generation of multi-cell tissue constructs using cells engineered with multiple synNotch receptors and transgene modules in combination with multi-ligand patterning, establishing a new paradigm for the field of tissue engineering.


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