Periodic Reporting for period 3 - NSC-Reconstruct (Novel Strategies for Cell-based Neural Reconstruction)
Reporting period: 2023-01-01 to 2023-12-31
Debilitating and incurable age-associated neurodegenerative diseases are on the rise worldwide as a result of our continuously increasing life expectancy. The focus of our project is on cell-based repair and neural circuit reconstruction for the treatment of neurodegenerative diseases such as Parkinson’s disease (PD) and Huntington’s Disease (HD). We envision strategies to repair complex cellular networks and therefore we expect this work will have implications for an even wider number of brain conditions, such as stroke and trauma. In summary, NSC-Reconstruct (NSCR) aims at four overarching goals:
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.
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.
WP1 focuses on developing advanced cells and vectors for cell replacement therapy in PD, HD, and cortical damage. For these diseases, we've identified the various neuronal cell types which need to be replaced specifically in three brain regions - the ventral midbrain, striatum, and cerebral cortex. In addition, we've explored the diversity of glial cells in the adult brain. Our approach involves engineering human pluripotent stem cells (hPSCs), creating reporter lines, and utilizing CRISPR and transcription factors (TF) to guide hPSCs differentiation into specific cell types. We've also designed new viral vectors and employed computational tools to define transcriptional networks in these cells. Our work has led to novel protocols for generating midbrain dopaminergic, striatal, cortical, and forebrain cholinergic neurons from human embryonic stem cells (hESCs). Additionally, we've made progress in converting somatic cells into induced neurons (iN).
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 as well as in vivo assessment of the functionality of second-generation DA neurons developed in WP1 using new methodologies developed within WP2.
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, started assessing the second-generation hMSNs therapeutical efficacy, and performed first in vivo reprogramming experiments to generate MSNs from 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 implemented a combined optogenetic/behavioral system for assessing functional integration of transplanted human neurons in the mouse visual cortex. Combinations of TFs to generate cortico-striatal projection neurons are being tested.
WP5 aims to determine and reduce the immunogenicity of hPSC-derived neurons. We've closed the in vitro analysis of the immunogenicity of stem cell-derived neural progenitor cells (NPCs) and concluded that they are not inherently immunogenic, and in fact that they have immunosuppressive qualities. Humanized mouse models have been developed for PD. We have now focused on co-cultures with human microglia and in vivo experiments using the humanized mouse model. Preliminary in vivo experiments have been performed, and the experimental design has now been optimized. We have started collecting blood and CSF from the STEM-PD clinical trial participants and have begun single cell sequencing to measure immune response in vivo. We have also generated less immunogenic lines, which we are now testing 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, organizing activities in person and adopting tailored use of hybrid and online formats to increase the impact on the scientific community and the lay public by. 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.
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 as well as in vivo assessment of the functionality of second-generation DA neurons developed in WP1 using new methodologies developed within WP2.
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, started assessing the second-generation hMSNs therapeutical efficacy, and performed first in vivo reprogramming experiments to generate MSNs from 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 implemented a combined optogenetic/behavioral system for assessing functional integration of transplanted human neurons in the mouse visual cortex. Combinations of TFs to generate cortico-striatal projection neurons are being tested.
WP5 aims to determine and reduce the immunogenicity of hPSC-derived neurons. We've closed the in vitro analysis of the immunogenicity of stem cell-derived neural progenitor cells (NPCs) and concluded that they are not inherently immunogenic, and in fact that they have immunosuppressive qualities. Humanized mouse models have been developed for PD. We have now focused on co-cultures with human microglia and in vivo experiments using the humanized mouse model. Preliminary in vivo experiments have been performed, and the experimental design has now been optimized. We have started collecting blood and CSF from the STEM-PD clinical trial participants and have begun single cell sequencing to measure immune response in vivo. We have also generated less immunogenic lines, which we are now testing 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, organizing activities in person and adopting tailored use of hybrid and online formats to increase the impact on the scientific community and the lay public by. 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.
After many years of preclinical work, stem cell-based cell replacement therapies for neurological disorders have progressed beyond the lab bench and are now entering clinical trials in several countries. These first-generation stem cell therapies consist of single-cell-type products aimed to either replace or provide trophic support to a dying population of endogenous neurons. Our ambition is to move beyond the current state-of-the-art and work towards goal-oriented, improved second-generation cell therapy products with more advanced properties, clinical relevance and commercial potential. If successful, NSCR will have a direct impact on health care systems, strengthen the European (bio)medical industry and boost European competitiveness in the field by offering innovative, efficient and cost-effective regenerative stem cell-based therapies for chronic and debilitating diseases where, today, there is very little to offer in terms of effective treatments. In fact, our restorative strategies have the potential to result in an entirely new approach to treatment of a broader range of chronic and debilitating brain injury caused by neurodegeneration or trauma that promises to increase the quality of life for a large number of affected individuals, with obvious economic benefits to our societies. Cell therapies could lead ultimately to allow the patients to live a fuller life and potentially make all current anti-PD therapies redundant.