University of Wisconsin–Madison

Randolph Ashton, PhD

Randolph Ashton Slide of the Week 2017

Title: Engineering Human Central Nervous System (CNS) Morphogenesis: Controlled Induction of Singular Neural Rosette Emergence

Legend: A) Images of human pluripotent stem cell (hPSC)-derived forebrain neuroepithelium forming neural rosette structures within micropatterned circular tissues of varying size. Tissues were formed over a 5-day culture period in the presence and absence of ROCK inhibitor, which attenuates cytoskeletal contractility. B) Quantification of tissues that contain 0, 1, and 1+ rosettes on microarrays from which the former images were taken. Number of tissues analyzed on each array is denoted (n). C) In situ modification of array substrates enables biomimetic radial tissue expansion of forebrain tissues. Images taken after 4 days of outgrowth, show a polarized neuroepithelial tissue (N-cadherin+) with basement membrane protein deposition (Laminin+). D) Neuronal differentiation (Tuj1+) occurs at the periphery of the neuroepithelial (Pax6+) tissues by day 7 of outgrowth. Scale bars are 200mm.

Citation: Knight GT et al., In preparation. 2017.

Abstract:  Human pluripotent stem cell-derived neural organoids provide unprecedented potential to recapitulate human brain and spinal cord tissues in vitro. However, organoid morphogenesis relies upon cell-intrinsic self-assembly of biomimetic tissue structures in the absence of normal developmental constraints and morphogen signaling centers. This inherently limits the reproducibility and biomimicry of organoid cytoarchitecture and potentially impedes maturation and interconnectivity of developing tissue structures. As an initial step in overcoming these limitations, ­we have begun investigating how spatial and temporal control of tissue morphology can be used to instruct controlled neural organoid morphogenesis in 2.5-D tissues. Using micropatterned culture of hPSC-derived neural stem cells (NSCs), we have identified biophysical parameters necessary to induce the emergence of tissues with a singular neural rosette cytoarchitecture (>80% efficiency), which resembles a 2D cross-section of the developing neural tube. Additionally, we have engineered culture substrates that enable spatiotemporal control of­­ the tissues’ radial expansion while maintaining an apical, polarized neuroepithelial zone. Ultimately, we aim to meld engineered culture platforms and organoid derivation protocols to enable reproducible derivation of brain and spinal cord tissues with organotypic cell phenotype diversity and biomimetic cytoarchitecture.

About the Lab: Our goal is to understand, model, and recapitulate in vitro the instructive signals utilized by human embryos to pattern tissue-specific differentiation of pluripotent stem cells, and apply this knowledge toward the rational design of tissue-engineered scaffolds and other regenerative therapeutic strategies. Currently, we primarily focus on generating tissues and therapies for the CNS.

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