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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster I2 - Synthetic morphogenesis to develop dorsal-ventral patterning in brain organoids

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Cheryl I. Quartey

Undergraduate
Vanderbilt University
Murfreesboro, Tennessee, United States

Co-Author(s)

CH

Catherine A. Hamann, BS

Graduate Student
Vanderbilt University, United States
EL

Ethan S. Lippmann, PhD

Associate Professor, Department of Chemical and Biomolecular Engineering
Vanderbilt University
Nashville, Tennessee, United States

Primary Investigator(s)

JB

Jonathan Brunger, PhD

Assistant Professor, Department of Biomedical Engineering
Vanderbilt University
Franklin, Tennessee, United States

Introduction: Brain organoid models derived from human embryonic stem cells (hESCs) aim to model early development of the human brain, but many current models lack native brain organization. Current differentiation strategies rely on the addition of small molecules and proteins that do not replicate morphogenesis programs, such as the patterning of the dorsal-ventral axis. In vivo, dorsal and ventral fates are determined by a concentration gradient of the morphogen Sonic Hedgehog (SHH) created by a polarized region of the neural tube known as the floor plate. To improve upon current brain organoid models, our objective is to mimic the SHH patterning of the dorsal-ventral axis in organoids by combining organoid spatial patterning methods and synthetic receptor technology.

Materials and

Methods: To mimic dorsal-ventral signaling programs in organoids, we use SynNotch, an artificial signaling platform modeled after native Notch signaling that can enable spatial control of programmed outputs. SynNotch receiver cells can be induced to express a transgene in response to juxtracrine contact with a ligand on the surface of a sender cell. To express the morphogen SHH, we engineered two hESC lines: (1) A receiver cell line with GFP-responsive SynNotch receptors which enable expression of SHH and mCherry and (2) a sender cell with constitutive expression of transmembrane GFP as an input for receiver cells. We used these SHH inducible receiver SynNotch cells to create signaling centers within brain organoids by polarizing receiver cells on one end of the organoid and surrounding them with sender cells. We also tested varying the number of receiver cells within this artificial signaling center to better understand how we can affect any resulting morphogen gradients. To polarize these signaling centers, we followed a modified organoid protocol where we seeded 500-4,000 SHH inducible SynNotch receiver cells and allowed them to aggregate for 24 hours before seeding 10,000 sender hESCs expressing extracellular GFP on top to induce differentiation. Organoids were embedded in gelatin after approximately seven days in culture before being placed on a shaker to allow for enhanced diffusion. Immunofluorescence was analyzed at days 20 and 30.

Results, Conclusions, and Discussions: Our results show GFP ligand-dependent expression of the fluorescent reporter mCherry and the morphogen SHH in SynNotch cells. These SynNotch cells can also induce gradients of dorsal, ventral, and floor plate marker expression in neighboring cells. SynNotch organoids show polarized expression of FOXA2, a marker of floor plate, along with expression of NKX2.1, a ventral marker, and PAX6, a dorsal marker with signaling centers consisting of 1,000 receiver cells (Fig 1A and B). We also demonstrate that the activation of signaling centers is dependent on signaling center size and surface-area to volume ratios (Fig 1C).

We plan on continuing to look at additional markers of dorsal-ventral patterning in organoids with varied signaling center sizes.

We have successfully developed SynNotch brain organoids and have seen some markers of dorsal-ventral patterning. By using a cell-based platform like SynNotch as a tool to coordinate brain morphogenesis in organoids, we hope to move towards more biomimetic ways of studying brain development in vitro.

Acknowledgements (Optional): The Brunger Lab
The Vanderbilt NIH MARC Program (NIH 5T34GM136451)
Vanderbilt University Department of Biomedical Engineering
Vanderbilt University Department of Biological Sciences
And a special thanks to my graduate student mentor, Catherine!