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  • br Results br Discussion This study

    2018-10-20


    Results
    Discussion This study describes a simplified protocol for the efficient generation of hiNSCs via direct reprogramming. hiNSC lines grow as colonies on MEF feeder layers. Like hESCs, hiNSCs stain positive for pluripotent transcription factors OCT4, SOX2, and NANOG, but do not express cell-surface markers SSEA4 and TRA-1-81, suggesting that these hiNSC clonal lines are not truly pluripotent. In a previous study using fluorescence-activated cell sorting to derive NSCs from H9 hESCs, only TRA-1-60−/SSEA4− ICG-001 could form neurospheres, suggesting that hESCs lacking these markers specifically adopt a neuroectodermal cell fate (Chaddah et al., 2012). All clonal lines tested showed an increase in endogenous SOX2 expression, but not endogenous OCT4 expression. Previous studies of various types of NSCs have demonstrated that the maintained expression of SOX2 and the absence of OCT4 is one of the hallmarks of NSCs (Graham et al., 2003; Mistri et al., 2015). Clonal lines generated by our method result in a highly pure population of hiNSCs lacking these pluripotent markers, providing further evidence of their NSC identity. Studies have shown that the introduction of pluripotent factors under certain conditions yields iPSCs and not iNSCs. This protocol of directly reprogramming somatic cells into hiNSCs differs from the protocol used to generate iPSCs in several ways. First is the use of xeno-free serum replacement (SR). Rajala et al. (2007) tested nine different types of xeno-free culture media and found that none were able to maintain undifferentiated growth of hESCs, demonstrating a lack of pluripotency. While the xeno-free knockout (KO)-SR may contribute to the adoption of a stable non-pluripotent cell fate, another likely reason is the relatively high levels of bFGF. Neural induction from iPSCs has been shown to require very high levels of FGF (up to 100 ng/mL) (Nemati et al., 2011). Our method of reprogramming hiNSCs results in the formation of colonies that resemble the neurosphere stage, which relies mostly on high levels of FGF. The third factor is the use of MEFs in conjunction with xeno-free KO-SR and high FGF. MEF feeder layers have been shown to be crucial in maintaining the proliferation of undifferentiated stem cells (Thomson et al., 1998). It is plausible that the MEF feeder layers are helping to promote continuous self-renewal of hiNSCs while the xeno-free SR, which lacks the ability to maintain pluripotency of cells, and the high levels of FGF work in combination to promote the neural stem cell fate. We are not the first investigators to describe a protocol for generating hiNSCs. Most protocols to generate hiNSCs that utilize similar reprogramming genes use them as separate factors (Wang et al., 2013a, 2013b). As such, there is an increased chance of variability with respect to controlling the relative expression of exogenously introduced genes that can accompany the use of multiple expression vectors. By using a singular polycistronic lentivirus, the relative expression and stoichiometry of the introduced factors is held constant in every round of reprogramming. Lastly, because our method could be used to successfully reprogram somatic cells from two different tissues, it is possible that it may also be utilized for other starting cell types. Previous studies have described the generation of hiNSCs; however, few have demonstrated the applicability of their ICG-001 respective protocols in reprogramming multiple cell types (Lee et al., 2015; Wang et al., 2013a; Zhu et al., 2015). For example, a method was described to reprogram urine-derived epithelial cells, but the same protocol could not be used to reprogram fibroblasts (Wang et al., 2013a). We fully acknowledge that herein we have only described the generation of hiNSC lines from two different starting cell types. It remains to be seen whether this method can effectively reprogram other starting cell types or whether it is restricted to human fibroblasts and adipose-derived stem cells.