Hybrid Differentiation Strategies for Region-Specific SOX10 Lineages

PROJECT SUMMARY/ABSTRACT During development, coordinated formation of key structures in the nervous and musculoskeletal systems are mediated through a specific "HOX code" that confers positional identity and contributes to cell subtype diversification and connectivity. To understand human nervous system diversity and subtype-specific neurodegenerative pathologies, it's essential to efficiently derive these region-specific cells from human pluripotent stem cells (hPSCs) in vitro. Yet, current small molecule-based approaches fall short of achieving desired efficiency while transcription factor (TF) overexpression methods fail to capture regional identity. Therefore, our goal is to develop a hybrid strategy to produce high-purity, region-specific cell populations. This project builds upon a recent innovation in our lab: a modular directed differentiation platform for generating spinal neurons from anywhere along the rostrocaudal or dorsoventral axes. To do this we go through intermediate neuromesodermal progenitors (NMPs), bipotent axial stem cells that are the first to acquire a distinct HOX code before diverging into mesodermal, neural tube, and neural crest progeny. Here we focus on expanding this platform to oligodendrocytes (OLs) and neural crest progenitors (NCPs), which share a common TF regulator, SOX10. We hypothesize that inducing SOX10 expression at key stages of differentiation will enable rapid generation of both lineages from the same region-specific NMP protocol. In Aim 1 we employ a TET-inducible SOX10 strategy to rapidly differentiate region-specific NCPs. In Aim 2, we seek to optimize the directed differentiation of region-specific OLs and determine whether the TET-inducible SOX10 strategy speeds differentiation and maturation to a myelinating phenotype. We will also determine whether the hybrid method enables generation of novel dorsal vs. ventral OL subtypes, which have not been generated from hPSCs previously. Thus, our project aims to define a next-generation system for producing region-specific cells that can be implemented using other TFs to create a translational region-specific toolbox. Future investigations may leverage this toolbox to advance our understanding of cell fate decision making, develop personalized cell therapies, or integrate into more accurate in vitro models of neurodegeneration, demyelination, and chronic pain. Ultimately we anticipate that this broad applicability will lay the groundwork for discovering novel therapeutic strategies for regenerative medicine.