(W1168) GENERATION OF LGE-COMMITTED NEURAL PROGENITOR CELLS WITH AFFINITY TOWARD DRD2+ MEDIUM SPINY NEURON POPULATION FROM HUMAN STEM CELLS USING A NOVEL SMALL MOLECULE-BASED RECIPE
Scientific Director Trailhead Biosystems, United States
Abstract: Medium spiny neurons originate from the LGE region of developing forebrain and later migrate to the striatum where they mature into DRD1 and DRD2 neuronal subtypes. Loss of DRD2+ cells due to HTT gene mutation is the cause of Huntington’s disease (HD). HD is a devastating neurodegenerative disease with life altering symptoms including motor, cognitive and psychological disorders. To this day there is no cure for HD disease. Effective differentiation of human stem cells to DRD2+ subtype in vitro can be the key to finding a cure for Huntington’s disease by providing access to correct cell type with minimal contamination from other fates that can faithfully reflect the mechanisms involved in the onset and advancement of the disease, hence, helping the researchers to gain better understanding of the disease and facilitate drug discovery efforts. Moreover, stem cell derived medium spiny neural progenitors can be transplanted into patients to replace the lost cell population. Here, we implemented HD-DoE technology to develop a small molecule-based differentiation protocol that can target optimization of specific genes at each stage of differentiation, closely following embryonic development. We successfully developed a 10-day protocol comprised of 3 chemically defined small molecule-only recipes that guide the hiPSCs to LGE-committed neural progenitor cells. These cells express forebrain markers FOXG1, SIX3 and OTX2, ganglionic eminence markers DLX5 and ASCL1 and LGE specific markers FOXP1 and FOXP2. The purity of culture was evaluated by immunocytochemistry, and it’s estimated that more than 80% of cells are committed to LGE region. When NPCs were differentiated for an additional three days, we were able to confirm expression of DRD2 protein along with early neuronal markers such as TUBB3 and DCX in the culture, while majority of cells express SIX3 and CTIP2 proteins. Importantly, expression of NKX2-1 protein, which is a MGE marker, is successfully suppressed. Bulk RNA sequencing was performed during differentiation, and it was confirmed that generated cells express DRD2 gene as early as day 10 while DRD1 gene is absent in culture. The significant advantage of this novel protocol is that it can be translated to large scale production of cells in a cost-effective way that makes the cells easily accessible.
