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  • Pax is a well known neurogenic transcription factor


    Pax6 is a well-known neurogenic transcription factor that is essential for the development of multiple neural tissues (Gehring and Ikeo, 1999; Georgala et al., 2011; Zhang et al., 2010). Due to its important role in neuronal development, Pax6 was often included in the initial screening for essential factors required for iN reprogramming; however, it was always eliminated from the final recipes due to the lack of function, or sometimes inhibitory function in iN conversion (Ambasudhan et al., 2011; Kim et al., 2011; Pang et al., 2011; Son et al., 2011; Vierbuchen et al., 2010). In this study, Pax6-alone was not sufficient to induce neuronal traits from hRFLCs, which is consistent with previous reports in mouse and human dermal fibroblasts (Vierbuchen et al., 2010). However, when combining Pax6 with Ascl1, either simultaneously, or 1-day ahead of Ascl1, the iN reprogramming capacity of Ascl1 was significantly improved. This observation indicates that Ascl1 might function as a pioneer factor in initiating the hRFLC-to-iN reprogramming process as in MEF-to-iN reprogramming (Wapinski et al., 2013), while Pax6 promotes the reprogramming process of hRFLC-to-iN, either through cooperating with Ascl1 to enhance its ‘on target’ pioneer factor function, or through directly activating certain neurogenic loci that promote neuronal fate transition but are not accessible by Ascl1. Further molecular dissections are required to reveal the detailed mechanisms on how Pax6 is involved in the iN reprogramming process. Similarly to mouse-BAMN-iN reprogramming from human fetal and postnatal fibroblasts, mouse-AP-iN reprogramming of hRFLC was not efficient. With the attempt to assess whether human genes would give better results, we cloned human ASCL1 and PAX6 into lentivirus vectors to repeat the reprogramming experiment. Based on our results, human ASCL1 alone worked better than mouse Ascl1 alone, but adding human PAX6 caused significant cell death, resulting in a lower number of successfully reprogrammed proteasome inhibitor compared with the mouse genes (Fig. S6). Future studies are warranted to evaluate the reprogramming efficiency after combining Ascl1 and Pax6 with other reagents, such as microRNAs or small molecules. Furthermore, additional research is required to assess whether combining Ascl1, Pax6 with lineage specific transcription factors would direct hRFLC-iNs to specific neuronal types, such as retinal ganglion cells and photoreceptors.
    Funding information This work was supported by Guangzhou Science Technology and Innovation Commission (201504010030), Guangdong Provincial Department of Science and Technology (2015B020225003), Ministry of Science and Technology of China 973 program (2015CB964600).
    Conflict of interest
    Introduction Pluripotent stem cells hold great promise in the field of regenerative medicine because of their ability to grow indefinitely and give rise to all cells of the body (Takahashi and Yamanaka, 2006). Both embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively) have been invaluable tools in the investigation of in vitro disease modeling, drug testing, and in vivo cell replacement therapies, as human primary cells are nearly impossible to obtain and survivability in culture is low. Human iPSCs (hiPSCs) have now been generated from several human tissues using a variety of approaches (Takahashi and Yamanaka, 2006; Karakikes et al., 2014). Clinicians are exploring the use of stem cell therapy for many diseases, including neurodegenerative disease, diabetes, rheumatologies and hematological disease (Karussis et al., 2010; Matsumoto, 2010; Persons, 2010; Szodoray et al., 2010; Trounson et al., 2011), but since stem cells run the risk of forming teratomas in vivo, progenitor cells are being explored as a more realistic therapy option (Le and Chong, 2016). Recently, the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis has been proposed as a potential therapeutic target for ischemic heart disease. This led to the recent clinical trial (JVS-100) in which mobilizing heart failure patients\' own stem cells was considered using SDF-1 gene therapy as a homing signal (Penn et al., 2013). SDF-1/CXCR4 signaling is important to a variety of fundamental processes, including organogenesis, embryogenesis, cell mobilization, and trafficking (Busillo and Benovic, 2007). Recent work has demonstrated that SDF-1 also binds to the chemokine receptor CXCR7 (SDF-1 alternative receptor) (Balabanian et al., 2005); however, the roles of CXCR7 are poorly characterized, confounding the understanding of this signaling pathway.