br Introduction Neural tube defects NTDs are

Introduction Neural tube defects (NTDs) are still poorly understood, especially for human and non-human primates (NHPs) (Wallingford et al., 2013). In rhesus monkeys, at embryonic day 19–20 (E19–20), the neural tube (NT) contains multiple pseudostratified layers of neuroepithelial stem cells (NESCs) (Davignon et al., 1980). Proper cell division, establishment of polarity, and cell movement of NESCs are crucial for NT formation and NT closure (NTC) (Bush et al., 1990). However, abnormal growth of NESCs results in NTDs and, subsequently, defective oxytocin antagonist development (Fish et al., 2006). During development, formation of the NT is a time-dependent transient event, difficult to capture, which limits study of it. After NT formation, the NESC at E12 in mouse undergoes asymmetric divisions to generate one radial glial progenitor cell (RGPC), which exhibits residual neuroepithelial and astroglial properties (Kriegstein and Götz, 2003), and one migratory postmitotic daughter neuron (Kriegstein and Alvarez-Buylla, 2009). Thus, the transition of RGPCs is another fundamental event of brain development. Unfortunately, we still know very little about the RGPC transition process. Previous reports have demonstrated differentiation of human embryonic stem cells (ESCs) into primitive neural precursor cells (NPCs) or neural rosette cells, which are composed of a myriad of cells along with anterior to posterior cell types (Elkabetz et al., 2008; Koch et al., 2009a; Li et al., 2011). A myriad of cells renders it difficult to study NT development and RGPC transition, even though a few cells maintain clonal expansion. In addition, a human pluripotent stem cells (PSCs)-derived three-dimensional organoid culture system, termed cerebral organoids, was developed to generate various discrete brain regions and could be used to model microcephaly (Lancaster et al., 2013). With the demonstration that the cortex and brain development can be recapitulated in vitro using stem cells (Espuny-Camacho et al., 2013; Lancaster et al., 2013), it is now conceivable that NTC could also be modeled in this way. Although a 3D neural tube system was recently established using embryonic bodies from mouse ESCs (Meinhardt et al., 2014), it is unclear whether the system can be used to model NTC and study NTDs. Furthermore, the system is unable to definitely control NESC self-renewal and differentiation as well as RGPC transition. Therefore, developing a simple culture system, which supports single-ESC-derived NESCs to self-organize into NT-like structures and model the RGPC transition in a stable, controlled, and conserved manner, will be rather advantageous to unveil molecular mechanisms underlying primate NTC and NTDs as well as NESC self-renewal mechanisms.
Results
Discussion In summary, a simple culture system was successfully developed to generate expandable single NESCs derived from monkey ESCs in vitro. A large number of experiments were used to demonstrate that the system is suitable for studying NTC and related disorders. These cells were found to (1) be polarized neuroepithelial cells; (2) self-organize into miniature NT structures at a cellular level; (3) have robust expansion ability; (4) display IKNM; (5) produce functional neurons and integrate into monkey brains; (6) be dependent on FA for NT formation; (7) express many NT genes, such as LIN28A, ASNS, IL-6, LYAR, and ZNRF3, but not for RPGC markers, including GFAP, GLAST, and TBR2; and (8) turn into RGPCs once deprived of Wnt signaling. These characteristics show these cells behave similarly as in vivo NT NESCs. The ability to reproducibly generate NT structures and clonal expansion from single NESCs, as demonstrated in this study, offers several advantages for studying brain development and disease: (1) stability—our NESCs faithfully self-renew. Their cell-cycling parameters, differentiation potentials, gene expression profiles, and NT formation process remain stable for over 50 passes. (2) Single cell function—individual NESCs self-organize into NTs, complete RGPC transition, and give rise to functional neurons. These special properties render the power to reveal how highly complex neural developmental processes organize at a cellular level. (3) Clonal growth—the clonal expansion feature of single NESCs will allow for targeted gene editing facilitated by TALEN (Liu et al., 2014) or CRISPR-Cas9 technologies (Niu et al., 2014). Acquisition of mutant or genetically corrected clones of cells is ideal for disease modeling or mutation corrections using NESCs.