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Generation of high-performance cellular model for Huntington’s disease

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Universitat de Barcelona. Facultat de Medicina i Ciències de la Salut

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[eng] Huntington’s disease (HD) is an autosomal dominant inherited neurological disease, which is caused by mutation in the HTT gene that encodes for the huntingtin protein. The disease leads to
involuntary movements, progressive deterioration of a person’s functional abilities, and eventually cognitive and movement disorders. Pathologically, HD is characterized by the loss of medium spiny neurons (MSNs) in the striatum. Although there are medical interventions available for improving the symptoms, there is no treatment to slow down the progression or cure the disease.
Induced pluripotent stem cells (iPSCs) are capable of self-renewal and can be differentiated into diverse cell types with specialized functions. iPSC-derived MSNs would provide a more relevant in vitro human cellular model to recapitulate and elucidate the pathogenesis of HD in human. iPSC- derived cells could also be used in cell-based therapy to treat HD. However, existing iPSC differentiation protocols often result in mixed cell population with a relatively low yield of MSNs, limiting their utility for comprehensive studies.
The objectives of the present thesis were: i) to develop a robust and specific differentiation protocol that can produce iPSC-derived D1 Dopamine Receptor (DR)- or D2DR-MSN subtypes with improved yield and purity and; ii) to assess the potential of iPSC-derived neurons for cell therapy in HD.
Using CombiCult® technology, we identified that modulation of the FGF, SHH and RA signalling pathways could give cells with higher level of lateral ganglionic eminence (LGE) marker gene expression compared to a previously published protocol (the control protocol). A combination of TGFβ inhibitor, BMP inhibitor, Wnt inhibitor, along with FGF8, SHH agonist, activin A and RXR agonist, could enhance the differentiation of iPSCs into LGE-like neural progenitor cells (NPCs). These LGE-like NPCs were able to mature into neurons with higher level of MSN marker gene expression compared to the control protocol. Unfortunately, we failed to identify any protocol that was able to differentiate iPSCs restrictively into either D1DR- or D2DR-subtype of MSNs. iPSC-derived neurons differentiated using the newly defined protocol survived, matured, and integrated in vivo in mouse brain. iPSC-derived neurons showed no xenograft overgrowth when transplanted intra-striatally.
In conclusion, we developed a new protocol for differentiating iPSCs into LGE-like NPCs, that can further mature into neurons with improved MSN gene expression profile. This protocol can be used to generate iPSC-derived MSNs for in vitro HD modelling and potentially used for cell therapy in HD.

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Tesis Doctorals - Facultat - Medicina i Ciències de la Salut

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Generation of high-performance cellular model for Huntington’s disease