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    • br Introduction Human embryonic and induced pluripotent stem

    2018-10-20


    Introduction Human embryonic and induced pluripotent stem cells (h/iPSCs) are of considerable interest in developmental biology and regenerative medicine, representing an enormous opportunity for generating patient-specific cells for screening drugs and cell therapies for various diseases. Stem cell neuronal differentiation has been used as an in vitro model for a number of genetic conditions, such as spinal muscular atrophy (Ebert et al., 2009) and familial dysautonomia (Lee et al., 2009), as well as inherited and sporadic forms of various human neurodegenerative conditions, including motor neuron disease, Niemann-Pick disease (NPD), Huntington disease (HD), Parkinson\'s disease (PD) and Alzheimer\'s disease (AD) (Dimos et al., 2008; Park et al., 2008; Yagi et al., 2011; Shi et al., 2012a; Israel et al., 2012; Devine et al., 2011; Jeon et al., 2012). In all cases, h/iPSCs are being used to generate large populations of healthy neurons to explore the therapeutic potential of neurotransplantation. The two basic methods for generating neurons from h/iPSCs are adherent (neuroectoderm) (Chambers et al., 2009; Shi et al., 2012b) and non-adherent (embryoid body or neurosphere) (Matigian et al., 2010; Koehler et al., 2011; Bez et al., 2003) culture conditions. Adherent methods (neuroectoderm) using dual inhibition of SMAD signaling promote efficient neuronal differentiation (Chambers et al., 2009; Lindvall and Kokaia, 2010). Another method is to generate neurons from non-adherent neurospheres or embryoid bodies (Matigian et al., 2010; Koehler et al., 2011; Bez et al., 2003). In neural transplantation, neurospheres are the most commonly used neuroprogenitors that are injected into the brain, due to their easy delivery and ability to rapidly migrate to the neurogenic areas of the calculate molarity of a solution (Englund et al., 2002; Flax et al., 1998; Jensen and Parmar, 2006). Neurospheres, as dynamic three-dimensional physiological microincubators for human neural precursor cells (NPCs), have many advantages over the neuroectoderm (Reynolds and Weiss, 1992). In 1992, Reynold and Weiss showed that free-floating NPCs can divide and form multicellular spheres in vitro (Reynolds and Weiss, 1992). These neurospheres have self-renewal ability, can be cultured over 10 passages, and can be easily maintained and expanded without losing the expression of neural progenitor markers (Jensen and Parmar, 2006; Reynolds and Rietze, 2005). Neurospheres have the potential to generate sub-type or region-specific neurons (Liu et al., 2013). However, their tendency to clump in culture makes them very difficult to study and to identify the types of neurons that can be derived after neurosphere transplantation (Jensen and Parmar, 2006; Reynolds and Rietze, 2005). It is also difficult to precisely monitor the morphology of single neurons from neurosphere-derived neuronal aggregates. Moreover, generating sub-type-specific or region-specific functional neurons from h/iPSCs takes more than 6–8weeks with the traditional neuronal generation protocols (Shi et al., 2012b; Goulburn et al., 2012; Liu et al., 2013). Here, we present novel culture conditions and methods to rapidly and efficiently generate functional human sub-type or region-specific neurons from neurospheres. This method involves a combination of supplemented knockout serum replacement medium (SKSRM) with 10% CO2 and a mechanical procedure termed “AdSTEP,” which involves breaking the neurospheres into smaller fragments to increase the efficiency of neuronal production. Furthermore, we injected the fragmented neurospheres into the severe combined immunodeficiency (SCID) mouse brains to investigate the effect of AdSTEP on neurogenesis in vivo, which might have significant impacts on neuronal transplantation and regenerative medicine.
    Materials and methods
    Results
    Discussion The neurosphere-derived cultures for neuronal differentiation are a valuable model system for studying neurogenesis and understanding the molecular mechanisms associated with neurodegenerative diseases (Jensen and Parmar, 2006; Reynolds and Rietze, 2005). Recent studies on iPSC-derived neurospheres and 3D cultures showed a significant promise for the development of disease-specific cells with the desired genetic backgrounds, which would facilitate the study of many important diseases, such as Timothy syndrome, Fragile X syndrome or NPD (Kumari et al., 2015; Macauley et al., 2008; Pasca et al., 2011, 2015). Here, we present a new defined culture medium and conditions: SKSRM medium and 10% CO2. This new culture condition doubled the expression of the neuroprogenitor genes NESTIN, PAX6, and FOXG1 compared with the traditional 5% CO2 culture conditions. The molecular mechanism by which the higher CO2 levels facilitate neurogenesis is still not clear. It could be due to reduced oxygenation or hypoxia, as previously reported (Heinrich et al., 2011; Morrison et al., 2000; Putnam et al., 2004; Clarke and van der Kooy, 2009). There are several groups that have reported that hypoxia or reduced oxygenation enhances neural stem cell colony survival and increased NESTIN, SOX1, SOX2, and FOXG1 expression (Morrison et al., 2000; Clarke and van der Kooy, 2009; Xie et al., 2014), similar to our study (Fig. 4G).