Periodic Reporting for period 2 - NSC-Reconstruct (Novel Strategies for Cell-based Neural Reconstruction)
Período documentado: 2021-07-01 hasta 2022-12-31
i. develop cellular products, reprogramming methods and research tools with broad potential for brain repair;
ii. develop novel strategies for cell-based repair to pave the way for the treatment of major neurodegenerative and traumatic diseases;
iii. achieve integration and reconstruction of neural circuit using transplants of diverse subtypes of stem cell-derived neurons;
iv. translate and commercialize new cell and gene products, research tools and therapies for clinical trials and market approval.
In WP2, we assess and maximize the therapeutic potential of the A9 subtype of dopaminergic (DA) neurons obtained through stem cells or direct conversion for PD cell therapy. We've conducted an analysis of behavioral recovery and graft performance in xenograft models of PD using state-of-the-art cells, setting a benchmark for comparing the cells developed in WP1. As the significance of non-DA cells for graft survival and function is increasingly recognized, we developed a methodology for studying graft composition in a high-throughput manner. Our ongoing work involves enhancing cell function and repair capacity.
WP3 aims to improve the maturation, innervation, and function of optimized human medium spiny neurons (hMSNs) derived from hPSCs and transplanted into rodent models of HD. We've achieved substantial progress in confirming the enhanced differentiation of MSNs using a new protocol from WP1 through in vivo grafts. We've established enriched housing conditions to support human graft survival, maturation, and integration and developed molecular tools for modulating the activity of grafted MSNs. Additionally, we've devised a new methodology for deriving astrocytes from hPSCs, tested the second-generation hMSN protocol, and identified instructive TFs for generating new neurons from striatal rodent astrocytes.
WP4 centers on understanding how transplanted hPSC-derived or directly converted neural cells integrate into the rodent central nervous system. We've made progress in developing protocols for imaging of immunolabeled structures in 2D and 3D, optimizing viral transduction for assessing neuronal functionality and synapse formation, and characterizing the composition of DA grafts. Our current computational pipeline enables comprehensive 2D and 3D immunofluorescence analysis. We've also worked on tools and models to study the capacity of transplanted human cortical neurons to restore motor and sensory function. In order to test various combinations of TFs to directly generate MSNs from other cell types, a highly pure culture of mouse striatal astrocytes was established and showed that they can be reprogrammed into neurons.
WP5 aims to determine and reduce the immunogenicity of hPSC-derived neurons. We've established protocols for in vitro assays to assess the immunogenicity of stem cell-derived neurons and generated cell lines that cannot be recognized by the host immune response. Humanized mouse models have been developed for PD. When examining the immunogenicity of hESC derived dopamine neural progenitors (NPCs), no immunogenicity was detected in any functional assay in vitro and a small T cell infiltration into graft sites in vivo. Ongoing experiments involve immunodeficient mice for longer-term studies without the risk of graft rejection. Overall, the NPCs have not displayed much evidence of immunogenicity either in vitro or in vivo.
In WP6, activities aiming at ensuring efficient translation of the cell-based repair strategies, to communicate the project goals, developments and results and to further promote networking and training within and outside the Consortium were intensified after overcoming the pandemic-related hurdles: whilst it was possible to organize the activities in person, NSCR increased their impact on the scientific community and the lay public by adopting tailored use of hybrid and online formats. In WP7 NSCR teams worked to maintain and continuously improve the established project structure and to create the most collaborative environment within the network as well as to manage ethical aspects which were addressed in WP8.