Identification of Protogenin as a novel surface marker for early cortical neural stem cells

Abstract

During mammalian corticogenesis, a wide diversity of neural stem cells (NSCs) orchestrate the development and organization of the cortex. The pool of NSCs initially expands through proliferative symmetric divisions, and sequentially starts dividing asymmetrically to give rise to the diverse cell types residing within the cortical layers. Throughout this process, cortical NSCs undergo extensive modifications in their transcriptomic profile and chromatin landscape contributing to the formation of heterogeneous progenitor populations. Although much progress has been made towards understanding cell-fate specification during human corticogenesis the mechanisms responsible for the temporal lineage specification of NSCs remain largely unknown. Understanding the variability of these distinct NSC populations is key for developing an in vitro system that allows for the homogeneous and unlimited culture of the desired NSC type which is crucial for cell replacement-based therapies. Hence, one of the main aims in our lab is to identify and discern these distinct NSC types which sequentially appear during cortical development with the objective to better understand these cell stages and, eventually, being able to manipulate them in vitro. In order to address this question, my project is focused on developing a strategy to isolate the early cortical NSC population for its characterization and potential manipulation. The main approach is to identify a cell surface marker to enable the isolation of these cells from our in vitro culture by fluorescence-activated cell sorting. By profiling our hiPSC-derived cortical progenitors at different stages by means of single-cell RNA sequencing, we selected potential candidate markers that were validated using immunofluorescence and sequencing methods. In this study, we identify Protogenin (PRTG) as a novel surface marker for early human cortical NSCs that can be used to isolate this population in vitro. We provide evidence that early expression of the novel marker correlates with cortical lineage specification. Furthermore, by sorting for such marker at early stages of neural induction we can prospectively isolate three distinct cortical subpopulations, resulting in highly pure subtype-specific NSC cultures. These findings illustrate the utility of PRTG cell-surface sorting for enriching early cortical NSCs in culture and, thus, aiding to develop a more robust and homogenous differentiation protocol. Ultimately, such knowledge should facilitate the generation of highly pure stage- and region-specific NSC populations from patient-derived samples which would provide a reliable source for cell replacement and regenerative therapies.