Characterization of optimized hESC-derived MSNs grafted in the intact striatum

Huntington’s disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss preferentially affecting striatal projection neurons. Affected patients display functional disability in the long term as a consequence of the impairment of cognitive and motor faculties resulting from HD-driven neurodegeneration. Cell replacement therapy using human embryonic stem cells (hESCs) is promising for achieving functional restoration of the affected tissue by mitigating the loss of striatal medium spiny neurons (MSNs). Importantly, the in vivo properties of transplanted cells may be affected by their time of in vitro differentiation at the moment of grafting. Here, we characterized cell grafts deriving from transplantation of hESC-derived MSNs in the striatum of athymic FOX-N1 adult rats to determine the optimal days in vitro (DIVs) to achieve successful grafting. Cells were obtained in vitro using a novel protocol developed by our collaborators (Conforti et al., 2022) for the generation of optimized striatal progenitors which underwent transplantation either at DIV13, DIV17, or DIV20. Upon animal sacrifice, we analyzed the grafts and concluded that the most suitable DIVs for grafting were DIV17 and DIV20. Since the functional outcome of the graft depends on the site of graft placement and integration in the rat striatum, we investigated these graft features by a three-dimensional brain reconstruction pipeline developed by us using TrackEM2 and Blender software. This pipeline constitutes an applicable method for further characterizing striatal grafts and aiding in the standardization of cell transplants.