Recent advances in induced pluripotent stem cell (iPSC) research have turned limitations of prior and current research into possibilities. growing BMS-536924 simple cells. In this review, we focus on the central nervous system (CNS) and describe the challenges faced in 2D and 3D differentiation research studies and the processes of overcoming them. We also discuss current studies and future perspectives on brain organoid researches. cultivation would be the best option. Adult stem cells recovered from various tissues have a limited self-renewal ability and differentiation potential (Hanna et al., 2007; Raya et al., 2009; Carvajal-Vergara BMS-536924 et al., 2010; Liu et al., 2010; Yang et al., 2010; Brennand et al., 2011; Itzhaki et al., 2011; Yazawa et al., 2011). In particular, by differentiating patient-derived iPSCs into a neural lineage, study and modeling on a neurological disease which would otherwise be arduous to perform can be easily conducted. iPSCs have been differentiated into neural stem cells (NSCs) in a 3 dimensional (3D) environment, including neurospheres, and 2 dimensional (2D) NSCs, including rosette-types (Elkabetz et al., 2008) and primitive NSCs (Shin et al., 2019). Continuing efforts on the differentiation technology to mimic brain tissue using pluripotent stem cells have led to technical advances, such as the formation of a mini brain-like structure or brain organoids (Lancaster et al., 2013). In this review, we discuss the technical advances on neural differentiation model systems using pluripotent stem cells toward mimicking the brain tissue and present the obstacles that need be overcome and the future directions in the field. 2D Neural Lineage Differentiation 2D Neural Lineage Differentiation From Pluripotent Stem Cells During gastrulation in mammals, the first neural structure that emerges is a form of neural tube consisting of a layer of neuroepithelial cells (Stiles and BMS-536924 Jernigan, 2010). Neuroepithelial cells are early neural stem cells that can further differentiate into radial glial cells (RGCs), which are bipolar-shaped neural progenitor cells (NPCs) that can in turn produce both neurons and glial cells, including astrocytes and oligodendrocytes (Malatesta et al., 2000; Noctor et al., 2001; Tamamaki et al., 2001; Merkle et al., 2004). NSCs are tripotent cells that can differentiate into 3 neural lineage cell subtypes: neurons, astrocytes, and oligodendrocytes (Glaser et al., 2007). In addition, NSCs are known to reside in the subventricular zone (SVZ) of the lateral ventricle and subgranular zone (SGZ) of the adult brain hippocampus (Alvarez-Buylla and Lim, 2004). Neural stem cells can be cultured by isolating cells from niches of brain tissues. The no new neuron hypothesis was first challenged in 1889 by reports claiming that NSCs capable of producing neuron and glia cells were isolated from an embryonic rat forebrain (Temple, 1989). Since then, isolation of NSCs from the adult central nervous system has been successfully performed in various species of mammals (Reynolds and Weiss, 1992). Both mouse and human NSCs can be isolated and maintained in the presence of extrinsic factors, such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) (Conti et al., 2005; Figure 1). Open in a separate window Figure 1 Morphological differences in diverse neural differentiation approaches. The differentiation of pluripotent stem cells into a neural lineage was developed in a stepwise manner: 2D, 3D, and brain organoid. A depiction of the BTD morphologies of growing neural stem cells and neural BMS-536924 rosettes in 2D monolayer cultures. A 3D neurosphere formed with the floating culture technique. Folded brain organoid structure formation after culture embedding in Matrigel and differentiation of pluripotent stem cells. Layer division of early neurons (Tuj-1 positive) and neural progenitors (Sox2 positive) identified by immunocytochemistry. This figure was modified with BMS-536924 permission from Stem Cell Biology, published by Life Science Publishing Co. Neural lineage differentiation from pluripotent stem cells can be generally achieved under serum-free conditions, which are important for maintaining neural cell cultures. In the past, numerous studies on the differentiation of pluripotent stem cells into nerve cells have been performed, and in this review we focus on representative cases of mouse and human studies (Table 1). The main types of nerve cells that comprise the central nervous system (CNS) are neurons, astrocytes, and oligodendrocytes. A study on the differentiation into each of these cell types and diverse protocols has been previously reported (Cazillis et al., 2006). Retinoic acid (RA) and sonic hedgehog (SHH) were the main factors initially used to drive differentiation of mouse embryonic stem cells (mESCs) into motor neurons (Wichterle et al., 2002). RA has long been known to be a pivotal factor in CNS development and has been.