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  • The following are the supplementary related

    2018-11-12

    The following are the supplementary related to this article.
    Acknowledgment We would like to thank for the financial support by the ALF Research Grant, Stockholm County Council; Chinese Scholarship Council, PR China; Craig Hospital Foundation, CO, USA; Magnus Bergvall Foundation, Svenska Läkarsällskapet; the research funds of Karolinska Institutet and the Stockholms Sjukhem Foundation, Stockholm, Sweden; and the Swedish Research Council. The presented data covering hfNPCs has been supported by funding from the European Union Seventh Framework Program (FP//2007–2013), grant agreement no. 276130.
    Introduction Recent studies have reported that iPSCs are capable of differentiating into various functional cell types, including neurons, cardiomyocytes and hematopoietic adiponectin receptor (Karumbayaram et al., 2009; Raya et al., 2009; Zwi et al., 2009). Transplantation of neurospheres derived from murine iPS cell clones into the injured spinal cord was found to be able to promote functional recovery without tumor formation (Tsuji et al., 2010). Grafted dopaminergic neurons derived from iPSCs could alleviate the disease phenotype in a rat model of Parkinson\'s disease (Wernig et al., 2008). These studies suggest that iPSCs could be used for regenerative and therapeutic purposes. One of major concerns about the therapeutic use of iPSCs is the low efficiency and high variability in their neural differentiation. In the present study, we proposed a novel approach which drives mouse iPSCs to efficiently differentiate into neural cells and searched for critical molecules which are responsible for the neural differentiation and patterning of iPSCs.
    Methods
    Results
    Discussion Recent studies have reported that human iPSCs generate neuroepithelia and functional neuronal subtypes with significantly reduced efficiency and increased variability compared to ESCs (Hu et al., 2010). Here, we reported that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation (Wichterle et al., 2002; Ying et al., 2003), although mouse iPSCs are considered to be truly pluripotent because they can produce viable mice through tetraploid complementation (Boland et al., 2009; Zhao et al., 2009). Previous studies reported that mouse iPSCs could generate various types of neural cells (Castiglioni et al., 2012; Tsuji et al., 2010; Wernig et al., 2008). However, all these studies did not specify the efficiency of neural differentiation of mouse iPSCs when they were induced to differentiate. A number of studies suggest that there is existence of an intrinsic difference in gene expression between iPSCs and ESCs (Gore et al., 2011; Hussein et al., 2011; Lister et al., 2011; Mayshar et al., 2010; Stadtfeld et al., 2010), which may account for the difference of their differentiation propensity. A very interesting finding in the present study is that the differentiation efficiency of mouse iPSCs can be improved by modulating the EB formation. All iPS cell lines tested in the study showed a remarkable enhancement in neural differentiation using the whole cell clone-derived EB method in that some iPS cell lines improved the differentiation efficiency to a level comparable to ESCs. The difference in neural differentiation potential among the three EB methods enables us to dig out the critical regulators which control mouse iPSCs to differentiate. We found that EBs initiated from the whole cell clones expressed much higher levels of Cdh2 than those from aggregating by dissociated single cells. The whole cell clone-derived EB method preserves the microenvironment of iPSCs, especially the cell–cell interaction between one and another, which possibly facilitates to trigger the activation of Cdh2. Cdh2 plays an important role in regulating nervous system development by providing key molecular cues in many biological processes across species (Lele et al., 2002; Miyatani et al., 1989; Radice et al., 1997) and is essential for maintaining the normal architecture of neuroepithelial cells during brain development (Kadowaki et al., 2007). It has been reported that precursor cell interactions can activate Cdh2 expression and generate a self-supportive niche to regulate their own number and cell fate (Zhang et al., 2010). Gene screening analyses, forced expression and knockdown studies suggest that the early activation of Cdh2 underlies the potent neural differentiation-promoting effect of the whole cell clone EB method and is essential for mouse iPSCs to undergo efficient neural differentiation.