(I-353) Engineering Gelatin Hydrogels for Activation of Synthetic Notch Receptors

Introduction:: New approaches for controlling the spatial organization of cells are critically needed to recapitulate the complex architecture and function of human tissues. The synthetic Notch (synNotch) receptor is a synthetic transmembrane receptor that is modified to recognize a user-defined ligand and induce expression of user-defined transcription factors. For example, synNotch receptors have been engineered to recognize GFP and then induce MyoD expression. Fibroblasts expressing this receptor will thus transdifferentiate into myoblasts in the presence of GFP. However, myoblasts generated in this way have limited maturity and limited long-term adhesion on conventional culture surfaces. In other studies, our group has shown that muscle differentiation and culture lifetime can be enhanced by culturing cells on gelatin hydrogels cross-linked with microbial transglutaminase (MTG). MTG can also be used to enzymatically cross-link other peptides or proteins to gelatin. Gelatin hydrogels can also be micro-molded to promote myotube alignment, fusion, and differentiation. Thus, our objective is to conjugate GFP to the surface of micromolded gelatin hydrogels and then evaluate if these substrates can activate synNotch to induce transdifferentiation of fibroblasts to aligned myotubes.

Materials and Methods:: Gelatin coverslips were made by laser cutting polystyrene dishes masked with tape. Laser cut tape was removed to expose an adherent surface for a MTG and gelatin solution; isotropic or micromolded 10x10 PDMS stamps were applied immediately to shape surface topography.
GFP-LACE (Addgene plasmid 133913) containing an additional C-terminus lysine tag (GFP-Lys) was extracted to increase reactivity with MTG, which cross-links lysine and glutamine. GFP-Lys was crosslinked to a gelatin surface through incubating a GFP and MTG solution on the gelatin surface at 37 C for an hour.
L929 mouse fibroblasts were transfected to contain an anti-GFP synNotch receptor and miRFP transgene. L929 cells were seeded on isotropic gelatin hydrogels at 0, 10, 100, or 250 ug/ml of GFP-Lys, or 100 ug/ml of unmodified GFP. 3 days post seeding, the gelatin was minced, digested with 4mg/ml collagenase IV, and strained before flow cytometry analysis to used to quantify the percentage of activated cells. C3H mouse fibroblasts were transfected for a anti-GFP synnotch receptor and mCherry and MyoD transgene. C3H cells were seeded on gelatin coverslips with 100 ug/ml of GFP-Lys. Cells were fixed on days 4, 7, and 14, and immunostained for the muscle cell marker alpha-actinin. Myotube count was performed in ImageJ through size and intensity thresholding of the alpha-actinin signal. Total number of cells per image was calculated by size and intensity thresholding of the DAPI signal. Myogenic index was determined by dividing the number of co-localized nuclei within the alpha-actinin by the total number of nuclei.



Results, Conclusions, and Discussions:: Enzymatic conjugation of GFP modified with an additional lysine tag to gelatin hydrogels successfully activated the synNotch response (Figure 1A-G). Quantification of miRFP positive cells demonstrates a dose-dependent response to GFP (Fig A-B) in the L929 reporter cells. Additionally, we show successful myogenic differentiation on our engineered gelatin hydrogel substrates. The micromolded hydrogel surfaces also promoted the alignment of myotubes. We also observed an increase in myotube count on the micromolded substrate compared to a flat isotropic gelatin surface on day 7 (Fig C-D). Additionally, the micromolded gelatin surface increased the myogenic index of the muscle, a parameter for differentiation (Fig E). However, even on the micromolded platform, we see a decrease in myotube count over time, indicating a need to further optimize the platform to improve long-term myotube adhesion and maturation (Figure F-G).

Gelatin hydrogels hold future potential for the spatial patterning of GFP, allowing for cellular scale control over synNotch activation. The synNotch platform has also been expanded to engineer dual receptor cells, which activate either MyoD or EVT2 in the presence of GFP or mCherry, respectively. By spatially patterning both ligands, muscle and endothelial transdifferentiation can be accomplished towards engineering a multicellular skeletal muscle model. Thus, this technology can generate more sophisticated tissue models that incorporate multiple cell types with precise spatial control to better recapitulate human physiological conditions, allowing for improved insight into tissue development and disease and new approaches for regenerative medicine.




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