Signal requirement for cortical potential of transplantable human neuroepithelial stem cells

The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Initial expansion of the progenitor cell pool is followed by the generation of neurons of all the cortical layers and later, astrocytes and oligodendrocytes. However, the regulatory pathways that control the expansion and maintenance of the neuroepithelial progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-b1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate to lower and upper layer cortical neurons both in vitro and in vivo. Identifying the mechanisms that drive the self-renewal and fate of cNESCs decision of neuroepithelial stem cells is key to developing new stem cell-based therapeutic approaches to treat neurological conditions. In this study, we performed Oxford Nanopore technologies long-read RNA-sequencing (lrRNA-seq) of human cNESCs and of the human embryonic stem cells used to generate them. Two hESC cell lines were differentiated to cNESCs, which were then maintained in either six factors (6F : BMPRi, TGFBRi, GSK3i, FGF, TANKYRASEi, AKTi) or 4 factors (6F-2F : BMPRi, TGFBRi, GSK3i, FGF). All samples (hESCs, cNESCs in 6 factors, cNESCs in 4 factors) are submitted to lrRNA-seq as duplicates.