Background

Human iPSCs were first established in 2007 through the reprogramming of dermal fibroblasts using four transcription factors (Oct4, Sox2, Klf4, and c-Myc) and exhibited similar characteristics of embryonic stem cells (ESCs), including their pluripotency and self-renewal (Takahashi et al., 2007; Yu et al., 2007). Since iPSCs overcome the limitation of the ethical problems and the allogenic immune rejection, which ESCs also have, they are highly expected to be applied to regenerative medicine. Importantly, researchers can establish disease-specific iPSCs from patients’ somatic cells, including skin fibroblasts, keratinocytes, cord blood-derived endothelial cells, peripheral blood mononuclear cells, and surprisingly tumor cells (Raab et al., 2014; Marin Navarro et al., 2018; Saha et al., 2018).

Driving disease iPSCs into tissue-specific cells enables us to obtain target cells, study the diseases' etiology and pathogenesis, and evaluate the efficacy or toxicity of drugs using the patients’ cells (Inoue et al., 2014). Before establishing iPSC technology, it was almost impossible to analyze the affected neural cells in the central nervous system from patients with neurodegenerative diseases as the biopsy is not always available for the human brain. Therefore, the iPSC-based disease models are expected to be a powerful approach in elucidating the pathogenesis of neurodegenerative diseases and in drug discovery because they mimic pathological conditions that have not been easily reproduced in animal models and cultured cell lines (Dimos et al., 2008; Soga et al., 2015; Matsushita et al., 2019; Kajihara et al., 2020; Kido et al., 2020). So far, neuronal cells derived from disease-specific iPSCs in vitro have been used to study the pathology of neurodegenerative diseases including Alzheimer’s disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), and they accurately recapitulated the pathological changes that occur in the patients’ brain (Dimos et al., 2008; Inoue et al., 2014).

The generation of disease-derived iPSCs was first established in 2008 by Dimos et al. using fibroblasts obtained from patients with familial ALS (Dimos et al., 2008). They induced ALS-specific iPSCs into motor neurons via EB formation. For many years, the major methods for neural induction from human ESCs and iPSCs have been either co-culture with a stromal cell line (Kawasaki et al., 2000; Lee et al., 2007) or the formation of EBs (Nemati et al., 2011; Falk et al., 2012; Shi et al., 2012), which involve the time-consuming steps, resulting in low-efficiency derivation of NSCs/NPCs. In the EB-based protocol, iPSCs are plated on a low adherence plate to form EBs, where EBs gradually aggregate over 5-7 days. These EBs are then transferred to a plate that supports cell adhesion, allowing the EBs to adhere to the bottom of the plate and spread in a neural rosette shape. From these rosettes, NSCs can be generated and passaged to form relatively stable NPCs. NPCs can then be plated in a neuron induction medium to be differentiated into mature neurons. Each of these methods (i.e., co-culture with a stromal cell line, or the formation of EBs) has contributed significantly to the field's advancement. However, some of these methods are not easily reproducible due to the variability of the iPSC lines or are not scalable to the extent needed for disease modeling, high-throughput screening, or cell therapy. Therefore, a simplified NSCs/NPCs generation protocol needs to be developed to the demands of research in brain development and neurological disorders.

Recent studies have shown that human iPSCs can be induced even into a monolayer neural culture in which iPSCs are plated on a defined matrix and exposed to growth factors (Chambers et al., 2009; Li et al., 2011; Yan et al., 2013). The monolayer-based protocols differ primarily in that iPSC colonies are maintained as a monolayer during neural induction and are differentiated directly into NSCs/NPCs without aggregation. Moreover, studies also demonstrated that dual SMAD inhibition enhances the efficiency of neural induction from iPSCs, in which small-compound inhibitors, SB431542 and dorsomorphin, block TGFβ/activin/nodal and BMP signaling, respectively (Chambers et al., 2009; Morizane et al., 2011). It is well-known that TGFβ/activin/nodal signaling activates SMAD2/3 and promotes mesoderm induction (Dunn et al., 2004; Nishikawa et al., 2007; Era et al., 2008), while BMP signaling activates SMAD1/5/8 and induces mesendoderm differentiation (Tada et al., 2005; Wang and Chen, 2016). Thus, inhibiting these two SMAD signals results in promoting the ectoderm differentiation, including neural cells.

Herein, we describe a methodology for rapid and reproducible induction to the NSCs/NPCs from iPSCs within a week in a monolayer culture system without the process of EB formation. Our method yields a PAX6-positive homogeneous cell population, a cortical NSCs/NPCs (Matsushita et al., 2019; Kajihara et al., 2020). Moreover, differentiated mature neurons via the NSCs/NPCs by our protocol are capable of neurotransmission revealed by FM1-43 imaging assay.