In vitro recapitulation of developmental transitions in human neural stem cells

Progress made in pluripotent stem cell (PSC) technology potentially enables the production of almost any cell type in unlimited quantities for human specific models. This is particularly interesting for the generation of neuronal cells as the human brain differs considerably from commonly used animal models and human brain tissue samples are scarce, difficult to obtain and to propagate <em>in vitro</em>.<br> In recent years it has become possible to isolate and expand neural stem cells (NSCs) from different sources using growth factor-based protocols. However, a still open question is to what extent these diverse stem cell systems reflect physiological stem cell states observed <em>in vivo</em> (Conti et al. 2010).<br> During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells (lt-NES cells) and the fetal human hindbrain (hbNES cells) (Tailor et al. 2013; Koch et al. 2009b).<br> In this study a sequential differentiation protocol was developed enabling the <em>in vitro</em> generation of RG-like neural stem cells from NES-like cells (lt-NES and hbNES cells). By employing differentiating conditions NES-like cells were coaxed into following the <em>in vivo</em> developmental transition of neural stem cells into a RG-like phenotype. Indeed, this newly established NSC population exhibits important features of multipotent neuro- and gliogenic RG cells. These RG-like NS cells could be expanded for at least 25 passages and express classical NSC markers such as Nestin and SOX2 as well as markers typically associated with RG cells including SOX9, CD44, AQP4 and HOP, while the NES cell markers PLZF, DACH1, ZO1 and MMRN1 were down-regulated.<br> Importantly, RG-like cells generated from PSC-derived lt-NES cells and from primary hbNES cells showed similar properties as primary RG cells obtained from fetal tissue, indicating that conversion of NES-like cells into RG-like cells recapitulates the developmental progression of early NES cells into radial glia cells occurring <em>in vivo</em>.<br> Key challenges associated with the biomedical application of PSC-derived neural stem cells are their controlled patterning towards distinct regional subtypes and the maintenance of an acquired regional phenotype across multiple passages of <em>in vitro</em> expansion.<br> Previous studies have shown that lt-NES cells, shortly after their derivation from PSC, undergo gradual posteriorization into an anterior hindbrain phenotype, which might be due to regionalizing effects of the growth factors employed for <em>in vitro</em> proliferation (Bithell et al. 2008; Koch et al. 2009b). This behavior is in line with their <em>in vivo</em> role in which they respond to morphogens in order to establish different regions of the brain. In a second step this regional identity is consolidated in RG cells which can no longer be influenced by regionalizing factors.<br> In order to harness this property of RG cells, RG-like NS cells were generated from dorsal forebrain-, ventral forebrain-, hindbrain- and spinal cord-patterned NES-like cells. The resulting regionalized RG-like NS cell populations express transcription factors appropriate for their positional identity across multiple passages of <em>in vitro</em> expansion while maintaining their differentiation potential into neurons and glia. Reminiscent of heterotopic transplantation studies, even under the influence of strong rationalizing factors <em>in vitro</em> the positional identity of dorsal forebrain-like NS cells remained stable (Onorati et al. 2011). Following differentiation RG-like cells generated region-specific neurons appropriate for their positional identity. These results indicate that conversion into RG-like cells may provide a route for conserving the regional identity of pre-patterned early NES cells and establishing regionally stable neural stem cell lines for biomedical application.