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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/215584
Development of a Safer and More Standardized Cell Therapy for Huntington’s Disease using Isolated Stem Cell-Derived Striatal Neuroblast Subpopulations
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[eng] Neurodegenerative diseases (NDs) are characterized by a selective and irreversible loss of neurons in a specific brain area. NDs are incurable and debilitating, becoming the greatest unmet clinical need of our time. Huntington’s Disease (HD) constitutes an example of a devastating ND, characterized by degeneration of striatal projection neurons (SPNs) leading to motor, cognitive and behavioral symptoms. Cell replacement therapies (CRTs) are distinguished by their potential to replace cells lost during the degenerative process and have shown a great potential to replace degenerated neurons in animal models and in clinical trials in Parkinson’s Disease (PD) and HD patients. In fact, CRT is the only approach currently focused on the structural and functional restoration of atrophied striatal tissue in HD, by replenishing the degenerating SPN population. In recent years, human pluripotent stem cells (hPSCs) have become a key cell source for CRTs given the scarcity and heterogeneity of previously used fetal-derived neural progenitors. However, hPSCs are prone to acquire genomic alterations in vitro, mainly due to suboptimal culture conditions and inappropriate routines to monitor genome integrity, which poses a challenge to both the safety of clinical applications and the reliability of basic and translational hPSC research. Furthermore, directing hPSCs into specific neuronal types in vitro is complex and the clinical translation of hPSC-derived neural progenitors is limited by the heterogeneity of cell products obtained using existing differentiation protocols. Therefore, clinical translation of CRTs for NDs requires standardization of production procedures for reproducible generation of defined functional cell products, by ensuring the presence of correctly specified target SPN progenitors and the absence of unwanted overly proliferative cell types
In this thesis, we have investigated if standardization of cell culture conditions and genomic screening strategies could decrease the prevalence of genomic alterations affecting hPSCs used as starting material in CRTs. We demonstrated that application of a Quality Management System (QMS) such as ISO9001:2015 to standardize cell culture conditions and genomic monitoring routines leads to a striking improvement of genomic stability in hPSCs cultured in vitro, as evidenced by a reduced probability of potentially pathogenic chromosomal aberrations and subchromosomal genomic alterations. We also described that CD200 is a cell surface marker suitable for the enrichment of hPSC-derived striatal neuroblasts (NBs). We set up and optimized an immunomagnetic sorting pipeline which allows for high-yield enrichment of striatal NBs in heterogenous cell populations
resulting from in vitro differentiation. Our data demonstrate that implementation of this approach leads to a reduction of the heterogeneity and batch-to-batch variation of the final cell population. Furthermore, we showed that sorted cell populations are composed of correctly specified, SPN-committed progenitors with the potential to generate different SPN subtypes, which result in a higher yield of neurons with an SPN phenotype upon terminal in vitro differentiation. We also provided evidence thar the use of CD200- based selection at different timepoints of in vitro differentiation results in obtention of cell compositions enriched in different subtypes of postmitotic striatal NBs. Finally, we demonstrate that transplanted selected NBs survive upon intra-striatal transplantation in adult mice with no evidence of graft overgrowth in vivo.
In conclusion, our findings underscore the critical need for implementing QMS in academic laboratories engaged in hPSC research to enhance the consistency and safety of applications of hPSC-derived cell products. Additionally, our results highlight the potential of the dual approach of combining standardization of hPSC culture conditions and CD200-based cell sorting of postmitotic striatal NBs before transplantation as a promising strategy to optimize the outcomes of hPSC-derived CRT products after transplantation. These strategies collectively advance the development and translation of safer, effective, and reproducible cell products to be used for clinical CRT applications in HD and potentially other NDs.
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MOLINA RUIZ, Francisco jose. Development of a Safer and More Standardized Cell Therapy for Huntington’s Disease using Isolated Stem Cell-Derived Striatal Neuroblast Subpopulations. [consulta: 13 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/215584]