Poster 126 - Photopatterning and Localization of Transdifferentiated Fibroblast Cells

Saturday, October 26, 2024

10:00 AM – 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Alexander V. Do

High School Student
Thomas Jefferson Highschool for Science and Technology, United States

Co-Author(s)

Stephanie Do

PhD Student
University of Southern California
Los Angeles, California, United States
LM

Leonardo Morsut

Assistant Professor, Stem Cell Biology and Regenerative Medicine, Biomedical Engineering
University of Southern California, United States

Primary Investigator(s)

Megan McCain

Associate Professor & Chonette Early Career Chair
Alfred E. Mann Department of Biomedical Engineering, United States

Introduction: One of the largest roadblocks in engineered muscle models is the spatial control of multiple cell types constituting the muscle. One solution is utilizing the Synthetic Notch receptor (SynNotch) technology, in which an engineered receptor recognizes a specified ligand to activate a user-defined gene. For example, a cell can be programmed to express MyoD (a myogenic regulator) after binding to Green Fluorescent Protein (GFP). Thus, we can transdifferentiate fibroblast cells into myoblasts and eventually myotubes at defined locations by spatially controlling the location of GFP. We propose a novel method to conjugate the target ligand, GFP, onto a hydrogel base using photocrosslinking to allow the spatial control of these locations.
In order to photo crosslink the target ligand with the hydrogel base, both materials need to be chemically modified with methacrylic anhydride, which replaces amine and hydroxyl groups to form methacrylate groups. These methacrylate groups covalently bond in the presence of UV light. By controlling the UV exposure to designated areas, we can then localize cellular activation via SynNotch to those areas.

Materials and

Methods: GFP is extracted via nickel column purification and is chemically modified with methacrylic anhydride to form the cross-linkable ligand. Gelatin is similarly methacrylated to form Gelatin-Methacrylate(Gelma), the crosslinkable hydrogel base. Coverslip bases are created from laser-cut petri dishes masked with tape surrounding a central hole for adding the hydrogel. After applying the hydrogel to the coverslip, either a smooth isotropic Polydimethylsiloxane(PDMS) stamp or a micromolded 10x10μm stamp is added to shape surface topography. The micromolded stamp was created by molding PDMS on a wafer template fabricated in a clean room via photolithography.
Next, photomasks were created in AutoCAD of horizontal slits 100x100μm and 250x250μm spacing apart. These photomasks block UV light in certain regions and allow photocrosslinking of the ligand in the specified exposed area. After the methacrylated GFP was added to the hydrogel, the photomask was added on top, and the coverslip was exposed to UV light. After the excess GFP was rinsed, anti-GFP MyoD SynNotch fibroblast cells were seeded onto the coverslip. Hydrogels were immunostained for dapi and alpha-actinin four days post-seeding.
The coverslips were then imaged with fluorescent microscopy and Orientation Order Parameter (OOP), a quantification of myotube alignment, was calculated through the ImageJ plugin. Binary masks were created through the Otsu thresholding algorithm with additional filtering through various Python packages. These were overlapped with the photopatterned GFP lanes to calculate the on-pattern myotube percentage.

Results, Conclusions, and Discussions: Micromolding leads to an increase in myotube alignment compared to the isotropic surface with both the 100μm and 250μm photopatterning spacing (Fig 1). 250μm photopatterning spacing increased alignment compared to the 100μm for the isotropic surface (Fig 2). On-Pattern myotube activation had no significant differences between the conditions, however, there was an observable increase in the on-pattern myotube percentage of the 250μm spacing compared to the 100μm in both surface types (Fig 3).
This study validates the combined use of hydrogel photopatterning and SynNotch technology as a method of localizing cellular activation to a distinct geometry. While photopatterning on isotropic surfaces had a significantly lower myotube alignment compared to micromolding, the 100μm photomask spacing was significantly more aligned than a surface without photopatterning. Micromolding changes surface topography over a large region and has low flexibility for growing multiple cell types with different topographical needs. Photopatterning on isotropic surfaces can stand alone for creating aligned myotubes and spatial control of future cell types. The next steps are to design a dual-ligand reporter cell allowing the activation of ETV2, an endothelial master regulator, and MyoD. Thus, we can get closer to physiologically representative vascularized tissue. Spatially controlling the growth of myotubes and their supporting cells is a huge step in the development of physiologically representative muscular tissue. More accurate human physiological samples are crucial to decreasing reliance on animal models and improving drug development.

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