Zic3 induces conversion of human fibroblasts to stable neural progenitor-like cells.


Dear Editor, Recent advances in cellular reprogramming have opened the door to the generation of patient-specific cells for regenerative medicine and disease modeling (Takahashi and Yamanaka, 2006; Yu et al., 2007; Nakagawa et al., 2008). Apart from OCT4, NANOG, and SOX2, additional transcription factors (TFs) like zinc-finger transcription factor in cerebellum-3 (Zic3) have been shown to play a role in the maintenance of pluripotency (Lim et al., 2007; 2010). Zic3, a member of the GLI superfamily, has been implicated in the maintenance of pluripotency in mouse and human embryonic stem cells (ESCs) by directly controlling the expression of Nanog (Lim et al., 2007; 2010). Zic3 is also an immediate early gene induced by fibroblast growth factor (FGF) signaling during neural specification (Marchal et al., 2009) and, by preventing neuronal differentiation, plays a role in the maintenance of the neural progenitor cell fate (Inoue et al., 2007). These studies demonstrate thus that Zic3 plays important roles in both the maintenance of ESC pluripotency as well as commitment to and the maintenance of neuroprogenitor cells. We hypothesized that co-transduction of Zic3 together with OCT4 (O), SOX2 (S), and KLF4 (K) might lead to improved generation of induced pluripotent stem cells (iPSCs) from human fibroblasts. Because forced expression of SOX17 or SOX7 in human embryonic stem cells (hESCs) creates stable endodermal and primitive endodermal cell fates (Seguin et al., 2008), the alternative outcome of these experiments could be the creation of stable neuroprogenitor lines. To investigate the consequence of Zic3 during reprogramming, human BJ1 fibroblasts were retrovirally transduced with OSK with and without Zic3 (Z). Colonies of cells referred to as Zic3-induced cells (ZiCs) (Supplementary Figure S1A) appeared in the culture plates transduced with OSKZ after 8 days and the frequency increased further by day 14 (Figure 1A). On day 14, 50–60 ZiC colonies were obtained from 1.5 × 10 BJ1 cells, with a reprogramming efficiency of 0.035%. In contrast, transduction of BJ1 cells with OSK did not yield visible iPSC colonies by day 30, even though iPSC colonies appeared at later times. Omitting either OCT4, SOX2, or KLF4 from the combination completely abrogated the colony formation (Figure 1A). Similar results were obtained when primary human fibroblasts were transduced with OSKZ. Ten to twenty colonies were generated from 1.5 × 10 human fibroblasts, 14 days following transduction with a reprogramming efficiency of 0.009% (Supplementary Figure S2A). ZiC lines generated from BJ1 cells that were further characterized are clones ZiC1 and ZiC2, while ZiC lines generated from primary fibroblasts for further studies are clones pZiC1, pZiC2, and pZiC3. In contrast to hiPSC or hESC lines, ZiCs expressed very low levels of endogenous OCT4 and NANOG mRNA. The expression of endogenous SOX2 was lower compared with hESCs or human induced pluripotent stem cells (hiPSCs), whereas endogenous KLF4 was expressed at levels similar to those in hESCs or hiPSCs (Supplementary Figures S1B and S2B). Endogenous Zic3 was expressed at lower levels in ZiCs compared with hESCs and hiPSCs (Supplementary Figure S1C). The OCT4, SOX2, and KLF4 transgenes were silenced, whereas the Zic3 transgene remained expressed (Supplementary Figure S1C). The cell surface phenotype of ZiCs was similar to that of neuroprogenitor cells, i.e. alkaline phosphatase (AP) (Figure 1B) and SSEA1 positive (Figure 1C). However, ZiCs were negative for SSEA4 and TRA160, which expressed on hESCs (Figure 1C). Altogether, these results indicated that ZiCs were not reprogrammed to an ESC-like fate. RT-qPCR analysis revealed that the ectodermal TF PAX6 was expressed at much higher levels in ZiCs and pZiCs than in hESCs/hiPSCs, whereas the endodermal TFs, FOXA2 and SOX17, and the mesodermal TFs, BRACHYURY and GSC, were not expressed (Supplementary Figures S2C and S3A). The combined expression of SOX2, PAX6, and SSEA1 in strongly AP-positive cells suggested that the new cell lines were of the neuroectoderm fate. We therefore compared the expression levels of additional neuroectodermal TFs in ZiCs and pZiCs with those in hiPSCs or hESC-derived neural stem cells (NSCs), generated by dual homologs of drosophila mothers against decapentaplegic (MAD) and the Caenorhabditis elegans protein SMA (SMAD) inhibition with noggin and SB431542 (Chambers et al., 2009). The neuroectodermal genes, PAX6, OTX1, FOXG1, GLAST, p75, SOX1, and OLIG2, were expressed in hESCs and hiPSCs only upon differentiation towards NSCs, whereas in ZiCs and pZiCs, these genes were highly expressed prior to any cytokine treatment (Figure 1D and Supplementary Figure S2D). Prior to transduction, both BJ1 and primary fibroblasts did not express significant levels of the abovementioned neuronal genes as well as pluripotent genes. Treatment of ZiCs with the two SMAD inhibitors did not further induce the expression of these neuroectoderm-specific genes (Figure 1D). Near homogenous expression of PAX6, OTX1, FOXG1, and OLIG2 in ZiCs maintained on feeders was confirmed at the protein level by immunostaining (Supplementary Figure S4). To investigate whether the continuous expression of the Zic3 transgene is important for the maintenance of the ZiC fate, we silenced the expression of Zic3 by two independent shRNAs cloned in a pTRIPZ doxycycline inducible lentiviral vector. 252 | Journal of Molecular Cell Biology (2012), 4, 252–255 doi:10.1093/jmcb/mjs015 Published online April 15, 2012

DOI: 10.1093/jmcb/mjs015

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@article{Kumar2012Zic3IC, title={Zic3 induces conversion of human fibroblasts to stable neural progenitor-like cells.}, author={Anujith Kumar and Jeroen Declercq and Kristel Eggermont and Xabier Agirre and Felipe Pr{\'o}sper and Catherine M. Verfaillie}, journal={Journal of molecular cell biology}, year={2012}, volume={4 4}, pages={252-5} }