Neurons: Important Cells in the CNS

Neurons are very important cells in the CNS and are responsible for mood, language, memory, behavior and so on. Many incurable diseases like amyotrophic lateral sclerosis, hereditary spastic paraplegia and Parkinson's disease are caused by degenerative changes in certain neuron subtypes (Dauer and Przedborski, 2003; Dion et al., 2009). Besides genetic origins, exposure to environmental pollutants has been also proved to be a contributor to neuron disorders (Kanavouras et al., 2011; Sonnack et al., 2015; Weisskopf et al., 2009). To model the pathological processes and mechanisms in these neuron disorders, but also derive proper transplantable cells and identify either effective drugs or toxicants, scientists have relied more and more on hPSCs (Chang et al.

, 2018).

In fact, researchers have successfully generated in vitro, many neuron subtypes with specific biochemical and functional traits.The neurons in the CNS are all derived from the neural epithelium. The mimic of in vivo processes in combination with morphogens or their agonists/antagonists can define the identity of neural progenitors and thus neurons, in vitro (Tao and Zhang, 2016).

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FGF8, WNTs and RA (retinoic acid) are responsible for the rostral-caudal patterning (Tao and Zhang, 2016): FGF signaling is required for rostral cells, while RA plays opposite roles by attenuating FGF activity and specifying the caudal fate (Diez del Corral and Storey, 2004).

In addition, WNT signaling facilitates the switch from FGF to RA, leading to caudal cell differentiation (Olivera-Martinez and Storey, 2007). The dorsal-ventral patterning is defined by the cooperation of WNT, BMP, and SHH pathways (Campbell, 2003; Le Dreau and Marti, 2012). SHH plays a major role in patterning the ventral neural fate; the two synthetic small molecules purmorphamine and SAG are SHH agonists, frequently used as alternatives to SHH, to activate SHH downstream targets, such as GLI2, which then enters into the nucleus and participates in the regulating of genes including Gli1, Ptch1 and Hhip (Le Dreau and Marti, 2012; Sinha and Chen, 2006; Stanton and Peng, 2010).

Conversely, to specify the dorsal fate, GLI3, which is directly activated by the WNT signaling, suppresses SHH activity; the BMP pathway acts in more complex manner to favor the dorsal identity (Campbell, 2003; Le Dreau and Marti, 2012; Li et al., 2009).The neural epithelium derived from hPSCs expresses PAX6 and OTX2, shows an anterior identity and undergoes a forebrain fate in presence of FGF or WNT inhibitors, or in absence of morphogens (Tao and Zhang, 2016). By modulating SHH concentrations, neural epithelial cells further evolve into either lateral ganglionic eminence (LGE) expressing GSX2, CTIP2 and MEIS2, or medial ganglionic eminence (MGE) progenitors, positive for NKX2.1 and FOXG1 (Liu et al., 2013b; Ma et al., 2012; Tao and Zhang, 2016).

With certain neural trophic factors (e.g. cAMP, BDNF, GDNF and IGF1), LGE and MGE progenitors further differentiate into subtypes of GABA neurons (Liu et al., 2013a; Ma et al., 2012). To simplify the medium and reduce the length of GABA neuron induction protocols, researchers have also tried to forcibly express the transcription factors ASCL1, DLX2 and LHX6 in hPSCs (Sun et al., 2016; Yang et al., 2017).

For spinal cord motor neuron differentiation, numerous protocols based on co-culture with stromal feeders, EB-based induction and differentiation in monolayer conditions have been established (Faravelli et al., 2014). Once hPSCs are committed to neural epithelium, RA and SHH are needed for caudal and ventral patterning, respectively (Briscoe and Ericson, 2001; Durston et al., 1998; Maden, 2001). OLIG2-positive motor neuron progenitors are specified from spinal cord progenitors expressing HOXB4. Neural trophic factors (BDNF, GDNF, IGF1, cAMP, etc.) are also applied for the maturation and maintenance of motor neurons expressing HB9, ISL1, LHX3 and ChAT (Hu and Zhang, 2009; Qu et al., 2014). The WNT agonist CHIR99021 is employed to shorten the time of motor neuron progenitor generation and enhance motor neuron differentiation, and probably acts in cooperation with the WNT and SHH pathways (Du et al., 2015).

Different from other neurons in the CNS, midbrain dopaminergic neurons are derived from FOXA2/LMX1A-positive floor-plate precursors rather than PAX6-expressing neural epithelium (Kriks et al., 2011). As mentioned above, blocking both the BMP and TGFІ pathways is sufficient to generate PAX6-positive neural epithelium, while the addition of SHH (or its agonists), a GSK3І-inhibitor (CHIR99021) and FGF8, facilitates formation of floor-plate precursors (positive for FOXA2 and LMX1A) (Kriks et al., 2011; Studer, 2012; Xi et al., 2012). The terminal differentiation of midbrain dopaminergic neurons expressing TH, LMX1, FOXA2, EN1 and MAP2, occurs in the absence of any patterning factor but with neuron trophic factors, including BDNF, ascorbic acid, GDNF, db-cAMP, and DAPT (or TGFІ3) (Kriks et al., 2011; Niclis et al., 2017; Nolbrant et al., 2017).

Differentiation procedures for other neuron types, like glutamatergic neurons, from hPSCs have also been established (Riemens et al., 2018; Yuan et al., 2015). In conclusion, since all the above protocols have been developed based on in vivo studies, and generally lead to mature and functional neural cells, they not only serve as powerful tools for disease modeling but also offer promising alternatives to DNT assessments.