252 | Journal of Molecular Cell Biology (2012), 4, 252–255 doi:10.1093/jmcb/mjs015 Published online April 15, 2012 Letter to the Editor Zic3 induces conversion of human fibroblasts to stable neural progenitor-like cells Dear Editor, with OSKZ after 8 days and the frequency RT-qPCR analysis revealed that the ecto- Recent advances in cellular reprogram- increased further by day 14 (Figure 1A). dermal TF PAX6 was expressed at much ming have opened the door to the On day 14, 50–60 ZiC colonies were higher levels in ZiCs and pZiCs than in Downloaded from https://academic.oup.com/jmcb/article/4/4/252/908007 by guest on 02 October 2021 generation of patient-specific cells for obtained from 1.5 × 105 BJ1 cells, with a hESCs/hiPSCs, whereas the endodermal regenerative medicine and disease model- reprogramming efficiency of 0.035%. In TFs, FOXA2 and SOX17, and the mesoder- ing (Takahashi and Yamanaka, 2006; Yu contrast, transduction of BJ1 cells with mal TFs, BRACHYURY and GSC, were not et al., 2007; Nakagawa et al., 2008). Apart OSK did not yield visible iPSC colonies by expressed (Supplementary Figures S2C from OCT4, NANOG,andSOX2, additional day 30, even though iPSC colonies and S3A). The combined expression of transcription factors (TFs) like zinc-finger appeared at later times. Omitting either SOX2, PAX6, and SSEA1 in strongly transcription factor in cerebellum-3 (Zic3) OCT4, SOX2,orKLF4 from the combination AP-positive cells suggested that the new have been shown to play a role in the main- completely abrogated the colony forma- cell lines were of the neuroectoderm fate. tenance of pluripotency (Lim et al., 2007; tion (Figure 1A). Similar results were We therefore compared the expression 2010). Zic3, a member of the GLI superfam- obtained when primary human fibroblasts levels of additional neuroectodermal TFs ily, has been implicated in the maintenance were transduced with OSKZ. Ten to in ZiCs and pZiCs with those in hiPSCs or of pluripotency in mouse and human embry- twenty colonies were generated from hESC-derived neural stem cells (NSCs), onic stem cells (ESCs) by directly controlling 1.5 × 105 human fibroblasts, 14 days fol- generated by dual homologs of drosophila the expression of Nanog (Lim et al., 2007; lowing transduction with a reprogramming mothers against decapentaplegic (MAD) 2010). Zic3 is also an immediate early efficiency of 0.009% (Supplementary and the Caenorhabditis elegans protein gene induced by fibroblast growth factor Figure S2A). ZiC lines generated from BJ1 SMA (SMAD) inhibition with noggin and (FGF) signaling during neural specification cells that were further characterized are SB431542 (Chambers et al., 2009). The (Marchal et al., 2009) and, by preventing clones ZiC1 and ZiC2, while ZiC lines gener- neuroectodermal genes, PAX6, OTX1, neuronal differentiation, plays a role in the ated from primary fibroblasts for further FOXG1, GLAST, p75, SOX1, and OLIG2, maintenance of the neural progenitor cell studies are clones pZiC1, pZiC2, and pZiC3. were expressed in hESCs and hiPSCs only fate (Inoue et al., 2007). These studies dem- In contrast to hiPSC or hESC lines, ZiCs upon differentiation towards NSCs, onstrate thusthat Zic3 plays important roles expressed very low levels of endogenous whereas in ZiCs and pZiCs, these genes in both the maintenance of ESC pluripo- OCT4 and NANOG mRNA. The expression of were highly expressed prior to any cytokine tency as well as commitment to and the endogenous SOX2 was lower compared treatment (Figure 1D and Supplementary maintenance of neuroprogenitor cells. We with hESCs or human induced pluripotent Figure S2D). Prior to transduction, both hypothesized that co-transduction of Zic3 stem cells (hiPSCs), whereas endogenous BJ1 and primary fibroblasts did not together with OCT4 (O), SOX2 (S), and KLF4 was expressed at levels similar to express significant levels of the above- KLF4 (K) might lead to improved generation those in hESCs or hiPSCs (Supplementary mentioned neuronal genes as well as pluri- of induced pluripotent stem cells (iPSCs) Figures S1BandS2B). Endogenous Zic3 potent genes. Treatment of ZiCs with the from human fibroblasts. Because forced ex- was expressed at lower levels in ZiCs two SMAD inhibitors did not further pression of SOX17 or SOX7 in human embry- compared with hESCs and hiPSCs induce the expression of onic stem cells (hESCs) creates stable (Supplementary Figure S1C). The OCT4, these neuroectoderm-specific genes endodermal and primitive endodermal cell SOX2,andKLF4 transgenes were silenced, (Figure 1D). Near homogenous expression fates (Seguin et al., 2008), the alternative whereas the Zic3 transgene remained of PAX6, OTX1, FOXG1, and OLIG2 in ZiCs outcome of these experiments could be expressed (Supplementary Figure S1C). The maintained on feeders was confirmed at the creation of stable neuroprogenitor lines. cell surface phenotype of ZiCs was similar the protein level by immunostaining To investigate the consequence of Zic3 to that of neuroprogenitor cells, i.e. alkaline (Supplementary Figure S4). during reprogramming, human BJ1 fibro- phosphatase (AP) (Figure 1B) and SSEA1 To investigate whether the continuous blasts were retrovirally transduced with positive (Figure 1C). However, ZiCs were expression of the Zic3 transgene is import- OSK with and without Zic3 (Z). Colonies negative for SSEA4 and TRA160,which ant for the maintenance of the ZiC fate, we of cells referred to as Zic3-induced cells expressed on hESCs (Figure 1C). Altogether, silenced the expression of Zic3 by two in- (ZiCs) (Supplementary Figure S1A) these results indicated that ZiCs were not dependent shRNAs cloned in a pTRIPZ appeared in the culture plates transduced reprogrammed to an ESC-like fate. doxycycline inducible lentiviral vector. # The Author (2012). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. Zic3 induces neural fate in human fibroblasts Journal of Molecular Cell Biology | 253 Downloaded from https://academic.oup.com/jmcb/article/4/4/252/908007 by guest on 02 October 2021 Figure 1 Transduction of Zic3 together with OCT4, SOX2, and KLF4 in human fibroblasts yields neural progenitor-like cells. (A) Quantification of the number of colonies 8 and 14 days after retroviral transduction of BJ1 fibroblasts with different combinations of retroviral vectors encoding for OCT4 (O), SOX2 (S), KLF4 (K), and Zic3 (Z) (n ¼ 3, P ¼ 0.02). No colonies could be observed after transduction with OSK, SKZ, OKZ, and OSZ until after 20 days following transduction. (B) AP staining of BJ1 fibroblasts transduced with OSK or OSKZ encoding retroviral vectors. (C) Immunostaining of the cell line ZiC1 for SSEA1, SSEA4, TRA160, and DAPI (×20, Nikon Eclipse Ti; n ¼ 3). Scale bar ¼ 50 mm. (D) Transcript levels of ectodermal genes in BJ1 fibroblasts, hESCs, hiPSCs, ZiCs, and neurospheres derived from those cells as determined by RT-qPCR (mean + SEM; n ¼ 3). (E) Transcript levels of pluripotency genes at different time points after retroviral transduction of BJ1 fibroblasts with OSKZ (mean + SEM; n ¼ 3). (F) Transcript levels of the epiblast marker FGF5 and the neuroprogenitor markers PAX6 and FOXG1 at different time points after retroviral transduction of BJ1 fibroblasts with OSKZ (mean + SEM; n ¼ 3). (G and H) The ZiC1 cell line was cultured under motor neuron differentiation conditions as described in Supplementary Materials and methods. (G) On day 14 of differentiation, RT-qPCR was used to evaluate the expression of motor neuron-specific marker genes (mean + SEM; n ¼ 3). Zic3Tg, Zic3 transgene. (H) Differentiated ZiC1 progeny were immunostained for the neuronal proteins NF200 kDa, MAP2 and the motor neuron proteins HB9 and ISL1 (n ¼ 3). Scale bar ¼ 50 mm. (I) ZiCs differentiate towards neuronal and astrocyte lineage cells upon injection in the striatum of adult mice. In total, 150000 green fluorescent protein (GFP)-transduced ZiC1 cells cultured as neurospheres were injected into the striatum of adult mice. The grafts were analyzed 4–5 weeks after transplantation. Colabeling of GFP with antibodies against the neuronal proteins NF200 kDa, NeuN, the neural progenitor protein OTX1/2, and the astrocyte protein GFAP (n ¼ 3). Scale bar ¼ 50 mm. ZiCs, passage 25, were transduced with (Supplementary Figure S5) without con- in hESCs-derived NSCs and 1876 probes shRNAs and selected by puromycin comitant increase in endodermal or meso- in ZiCs, whereas 1550 and 1977 probes (1 mg/ml) 1 day after transduction. dermal gene expression (data not shown). were expressed at lower levels in hESCs- Expression of Zic3-specific shRNA was To further ascertain the neuroprogenitor- derived NSCs and ZiCs, respectively. Of induced by adding doxycycline (1 mg/ml) like fate following OSKZ transduction of the significantly upregulated probes, 563 to the media. Induction of Zic3-shRNA fibroblasts, the global gene expression were common to hESCs-derived NSCs and expression in ZiCs induced an 82%–90% profile of ZiCs and hESCs-derived NSCs ZiCs (Supplementary Figure S3B and decrease in Zic3 expression. This was asso- was compared with microarray analysis. Table S1). Gene ontology analysis (http ciated with a 60%–80% decrease in ex- Compared with undifferentiated hESCs, ://david.abcc.ncifcrf.gov/) demonstrated pression levels for PAX6, OTX1, and OTX2 1617 probes were more highly expressed that many of these genes are involved in 254 | Journal of Molecular Cell Biology Kumar et al. neural differentiation and development progeny in vitro. ZiCs could be committed et al., 2010; Pang et al., 2011; Sekiya (Supplementary Figure S3C). to the astrocyte lineage, as shown by ex- and Suzuki, 2011). For instance, forced When cells were cultured as embryoid pression of the astrocyte-specific tran- combinatorial expression of the bodies, ensuing clusters resembled neuro- script, GFAP (Supplementary Figure S7A) neural-lineage-specific TF can rapidly and spheres. RT-qPCR analysis identified only and the astrocyte-specific proteins, efficiently convert mouse and human fibro- neuroectoderm-specific transcripts S100b and GFAP in 42% + 7% and blasts into functional neurons in vitro (Supplementary Figure S6A).