The advent of cell reprogramming and the prospect of analyzing the molecular pathogenesis of neurological disorders in iPSC-derived neurons and glia have created a demand for protocols that reproducibly and quantitatively yield defined neural populations. The identification and dissection of disease-relevant mechanisms will depend critically on the ability to compare consistently differentiated neural cell populations generated from genetically distinct pluripotent starting cells. Based on the findings in this study we suggest that lt-NES cells represent a valuable step in this direction.
The successful generation of lt-NES cells from iPSCs was independent of the parental cell type used for reprogramming, the factors used to induce pluripotency, culture conditions used for propagating the pluripotent cells and the variable propensity of the individual parental pluripotent stem cell lines to differentiate directly into neural cell types. Lt-NES cells show a highly characteristic morphology, differentiation potential and gene expression profile, which remain stable even after long-term propagation over more than 100 passages. Importantly, lt-NES cells exhibit a consistency that appears to override variations in initial differentiation efficiency, cell lines, and handling. Using a related approach to that applied here, Nemati et al. recently described the generation of an expandable neural precursor cell population from an iPS cell line 
. However, their cultures showed only partial suppression of neuronal and glial differentiation, possibly due to the use of a serum substitute and/or of very high concentrations of FGF (100 ng/ml) in the culture media. Importantly, in our hands lt-NES cells invariably exhibited negligible expression of markers associated with differentiation during expansion culture.
Lt-NES cells display a characteristic gene expression signature independent of their hESC or hiPSC origin. They appear to represent a developmentally early cell population as reflected by their rosette-like growth pattern and marker expression. Consistent with this, lt-NES cells have a distinct transcription profile from the more radial glia-like fetal NS cells. Multiple cell cycle genes are upregulated in lt-NES cells, which is in accordance with their faster proliferation rate. Fetal NS cells express markers of radial glial cells (e.g. GFAP, CD44, A2B5), whereas lt-NES cells preferentially exhibit neuroepithelial and neural rosette markers (e.g. PAX6, PROM1, SOX3, MMRN1, PLZF), indicating that they represent an earlier stage of CNS development. Furthermore, anterior regional identity markers are maintained in the cortically derived fetal NS cells, whilst lt-NES cells adopt a hindbrain regional specification.
It is interesting that these two molecularly and developmentally distinct neural cell types can be propagated long term in nearly identical culture conditions. This is particular striking considering that FGF2 is generally considered to exert a dominant respecifying effect on neural progenitors in vitro 
. Remarkably, the early developmental stage of lt-NES cells appears to make them more amenable to regional re-specification. Under standard culture conditions, lt-NES cells have a rather well-defined regional identity compatible with a hindbrain phenotype. Despite this overall posteriorization, data from this and previous studies 
suggest that these cells may be recruited into adjacent regional cell types, most provocatively into midbrain dopamine neurons. It will also be interesting to determine whether lt-NES cells may be differentiated in vitro into NS cells.
Considering their regional identity, lt-NES cells should constitute a particularly suitable tool for modeling a range of neurodegenerative diseases affecting neuronal and glial cells in midbrain, hindbrain and spinal cord. However, many pathophysiological pathways are based on common denominators such as oxidative stress response, axonal transport or synaptic plasticity, which are conserved among different regional subtypes. For that reason, we expect lt-NES cells to provide a useful tool for modeling diseases affecting many aspects of cellular neurobiology, and their advantages concerning highly reproducible derivation, easy handling, robust expandability and comparability might outweigh any limitations in regional plasticity. Notably, synaptic dysfunction is an early denominator of neurodegenerative and neurodevelopmental disease. The demonstration that lt-NES cells form spontaneously active neuronal networks provides prospects for employing them for functional studies of synaptic transmission in neurons with control and disease genotypes.
Once established, lt-NES cells are a robust population that can be subjected to repeated freeze-thawing, cryoconservation and automated spotting in multiwell formats. Based on their extensive self-renewal capacity, lt-NES cells are further amenable to various genetic modifications with high efficiency. This may be a stem cell reporter such as the Nestin-GFP employed here, or a neuron-specific selection marker as the one we previously used to generate virtually pure cultures of human neurons, which themselves can be subject to efficient cryopreservation 
. Lt-NES cells may thus provide a consistent source of human neurons for pharmaceutical screening against defined molecular targets or for neurotoxicity studies. These properties make lt-NES cells potentially suitable for high throughput applications involving genetic and compound library screening. Biomedical applications might range from the analysis of disease mechanisms to refined drug discovery using disease-relevant human neurons and glia.