Enhancing human brain organoid complexity through biofabrication of a perfusable vasculature

Despite the developmental similarities between humans and model organisms, organs like the brain exemplify the existing species-specific differences, highlighting the need to develop novel tools. Human pluripotent stem cells (hPSCs) and brain organoids represent breakthrough technologies that allow the recapitulation of specific aspects of the human brain. However, one of the most striking shortcomings of hPSC-derived brain organoids is the lack of functional vascularization, which remains a major challenge in the development of increasingly complex architectures. Apart from providing nutrients to the organoid core (which becomes necrotic over time using current protocols), the specialized vasculature of the brain is also involved in developmental processes. Unfortunately, the effective integration of a functional brain vasculature with human brain organoids represents a milestone that has not yet been achieved.

By integrating our expertise in stem cells, developmental biology and bioengineering, we propose to design a robust methodology for the vascularization of hPSC-derived brain organoids that will be used to shed light on basic biological mechanisms driving the differentiation of neural progenitor cells and the development of more complex brain architectures.

To reach this goal, we propose to develop a mesoscale chip and its higher-throughput version hosting up to 24 millimeter-scale human brain organoids by integrating computer aided design, computational simulations and microfabrication approaches. This platform will be compatible with high-content imaging, automated seeding and liquid handling to reproducibly culture brain organoids and their vascular microenvironment (Work Package (WP)1 and WP2). Technical validation will guarantee reproducible seeding and compatibility with standard analytical instruments.

In parallel, we will derive endothelial cells, pericytes and astrocytes from hPSCs and compare them with human primary brain cells before and after integration with the organoids by RNAseq, flow cytometry and immunofluorescence, and benchmark them with public datasets (WP2).

A key task of the project is the design of a novel biofabrication strategy based on the fusion of single, pre-vascularized hPSC-derived brain organoids around a central channel that is meant to provide immediate perfusion to the brain tissue upon integration with the designed culture system. These brain organoids will be integrated with a biofabricated microcirculation surrounding them and sprouting towards their core, hence generating a functional brain vasculature nourishing each region of the tissue (WP3). The vessel functionality will be quantified through the measurement of barrier permeability and the activity of the efflux pump P-gp and GLUT1.

We will then combine spatial transcriptomics and single cell RNAseq to clarify the cellular crosstalk between the human brain vasculature and the brain microenvironment, and how this interaction finally regulates the heterogeneity of neural progenitor populations (e.g. different radial glia subtypes) and neurogenesis (WP4). After comparison with public datasets, the identified targets will be validated at the protein level using histological slides from fetal and adult tissues. The maturation of the organoids will be analyzed by calcium imaging, multielectrode arrays and manual patch clamping. Finally, the emerging molecular drivers will be validated in vitro using doxycycline-inducible knockouts and in vivo using mouse embryos.

Overall, our proposal will result in a novel methodology to develop long-lasting, vascularized human brain organoids derived from hPSCs and will provide a vasculature-focused, detailed overview of the molecular drivers of neurogenesis. The vascularized hPSC-derived brain organoids will lay the foundation for new research directions aimed at addressing biological questions beyond developmental biology, such as brain-related disorders ranging from neurodegeneration to tumors.