Engineering human cortical brain organoid’s connections to restore brain functions

Project Information

COnNect

Grant agreement ID: 101077714

DOI

10.3030/101077714

EC signature date 29 June 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

UNIVERSITA DEGLI STUDI DI PADOVA
Italy

Periodic Reporting for period 1 - COnNect (Engineering human cortical brain organoid’s connections to restore brain functions)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

The human brain relies on highly organized neuronal networks to generate perception, memory, and behavior. When these networks are disrupted by stroke, trauma, or neurodegenerative disease, neurons are irreversibly lost and the adult central nervous system shows very limited regenerative capacity. As a result, millions of patients live with permanent neurological deficits for which no curative therapies exist.
Stem cell–based approaches, particularly human cortical brain organoids derived from pluripotent stem cells, offer a promising strategy for brain repair. These three-dimensional tissues generate diverse neuronal populations and partially recapitulate cortical architecture. After transplantation into rodent brains, organoids can survive, mature, and form connections with host tissue. However, current strategies rely largely on spontaneous graft–host integration, lacking spatial and temporal control. This can lead to inefficient integration, aberrant connectivity, and uncontrolled growth. Moreover, the injured brain presents an inhibitory environment that limits axonal regeneration, making structural guidance essential for effective circuit reconstruction.
COnNect addresses this limitation by introducing a bioengineering strategy to actively guide neuronal integration in vivo. The project combines human cortical organoids with light-responsive biomaterials that mimic key extracellular matrix properties while allowing precise spatial and temporal control through light-based fabrication. The central hypothesis is that transplanted organoid-derived neurons can reconstruct specific pathways when supported by a programmable, dynamic scaffold.
To test this, COnNect pursues four objectives: (1) develop human-specific, light-responsive hydrogels compatible with brain mechanics and supportive of survival and axonal growth; (2) establish advanced 3D bioprinting strategies enabling fabrication and selective removal of hydrogel structures in vitro and in vivo to create axon-guiding microchannels; (3) translate these tools to a cortical lesion model with spatially controlled integration; and (4) assess whether guided structural integration promotes functional network restoration.
By integrating stem cell biology, biomaterials engineering, and intravital light-based fabrication, COnNect moves beyond passive transplantation toward programmable neural integration. This approach establishes a conceptual and technological framework for controlled brain repair and may contribute to the long-term development of targeted regenerative strategies for focal brain lesions.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the reporting period, the project focused on establishing and validating the technological and biological platform required to guide the integration of human cortical brain organoids (CBOs) within the injured mouse cortex.
Significant progress was achieved in Objective 1 through the development and characterization of coumarin-functionalized hydrogels based on PEG, hyaluronic acid (HA), and decellularized extracellular matrix (dECM). These formulations demonstrated tunable mechanical properties, efficient light-induced crosslinking and multiphoton-mediated decrosslinking with micrometric resolution. HA-based hydrogels were optimized for CNS applications and validated for biocompatibility with neural stem cells and CBOs in vitro. In parallel, functionalized dECM hydrogels were generated and characterized, providing a biomimetic and potentially tissue-specific alternative to conventional matrices.
Under Objective 2, in vitro guidance of neuronal projections was demonstrated. By combining HA-based hydrogels with selective multiphoton decrosslinking, defined microchannels (1–4 µm) were generated. Narrow channels promoted directional neurite elongation, and 1 µm channels enabled structural bridging between two distinct CBOs, establishing proof-of-principle for guided neuronal connectivity. A complementary PDMS-based microfluidic system was developed to study collective axonal growth and engineered assembloid formation.
For Objective 3, a reproducible cortical resection model in the mouse visual cortex was established and combined with a PDMS-based cranial window to allow intravital access. HA-based hydrogels were implanted and their persistence, mechanical properties, and biocompatibility were evaluated at 14 and 30 days post-implantation. Mechanical tuning strategies (increased HA concentration and HA/PEG blends) were implemented to improve in vivo stability. Preliminary data indicate absence of significant microglial activation or glial scar formation under selected conditions. In parallel, anatomical mapping of projections between V1 and higher visual areas enabled depth-controlled intravital microchannel fabrication using reversible intravital 3D bioprinting (Ri3DB). Initial organoid implantation experiments revealed the need to prioritize vascular integration to sustain graft survival.
Under Objective 4, structural prerequisites for functional restoration were established, including confinement of transplanted CBOs within defined matrices and optimization of multiphoton live imaging protocols for monitoring matrix remodeling and graft–host interactions.
Overall, the project has successfully established the material platform, demonstrated in vitro guided connectivity, stabilized the in vivo lesion model, and laid the foundation for subsequent functional validation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

COnNect advances regenerative neuroscience by introducing a programmable and reversible strategy to guide neuronal integration in vivo. Unlike current brain organoid transplantation approaches, which rely on spontaneous graft–host integration without spatial or temporal control, COnNect establishes a dynamic biomaterial platform enabling active modulation of the extracellular environment within the living brain.
A major achievement is the development of biomimetic, coumarin-functionalized hydrogels based on PEG, HA, and decellularized ECM, combining biological relevance with reversible light responsiveness. Orthogonal crosslinking and multiphoton-mediated decrosslinking with micrometric precision allow spatially controlled matrix remodeling inside implanted scaffolds—an approach not achievable with conventional static biomaterials.
The project further demonstrates that defined microchannel geometries (1–4 µm) can guide axonal projections between human cortical organoids in vitro, establishing a strategy for engineered neuronal connectivity rather than passive integration. In parallel, intravital reverse 3D bioprinting within an injured cortical environment proved feasible, enabling anatomically informed guidance strategies and representing a conceptual step toward targeted neural circuit reconstruction.
Finally, the identification of early vascular integration as a critical determinant of organoid survival refines the translational framework, emphasizing guided vascularization as a prerequisite for stable neuronal reconnection.
Together, these results provide a technological and conceptual foundation for programmable neural repair, with potential long-term applications in minimally invasive, spatially controlled treatment of focal brain lesions and broader relevance for dynamic tissue engineering strategies.