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Theink4a/Arflocus Is a Barrier to Direct Neuronal Transdifferentiation

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Theink4a/Arflocus Is a Barrier to Direct Neuronal Transdifferentiation 12560 • The Journal of Neuroscience, September 10, 2014 • 34(37):12560–12567 Brief Communications The Ink4a/Arf Locus Is a Barrier to Direct Neuronal Transdifferentiation James D. Price,1,2,3* Ki-Youb Park,1,3* Jiadong Chen,1,4 Ryan D. Salinas,1,3 Mathew J. Cho,1,3 Arnold R. Kriegstein,1,4 and Daniel A. Lim1,2,3,5 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, 2Developmental and Stem Cell Biology Graduate Program, 3Department of Neurological Surgery, 4Department of Neurology, and 5San Francisco Veterans Affairs Medical Center, University of California, San Francisco, San Francisco, California 94143 Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that the Ink4a/Arf (also known as Cdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passage in vitro, wild-type postnatal cortical astroglia become progressively resistant to Dlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence of Ink4a/Arf- deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced from Ink4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16 Ink4a and p19Arf indicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient for Ink4a/Arf also exhibit greatly enhanced transcription factor- induced neuronal induction. These data indicate that Ink4a/Arf is a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes. Key words: astroglia; induced neuron; Ink4a/Arf; transcription factor; transdifferentiation Introduction suppression (Kim and Sharpless, 2006), Ink4a/Arf gene products Neurons can be directly converted from non-neuronal cell types impede the generation of induced pluripotent stem cells (iPSCs) via the enforced expression of specific transcription factors (Vi- from fibroblasts: Ink4a/Arf is rapidly silenced during iPSC pro- erbuchen and Wernig, 2012). Other than tissue-specific stem cell duction, and Ink4a/Arf-deficient fibroblasts generate iPSCs more populations, cells in the adult mammal generally do not change efficiently (Banito et al., 2009; Li et al., 2009; Utikal et al., 2009). their cellular identities, and this inherent phenotypic stability Whether Ink4a/Arf also constitutes a barrier to direct neuronal represents a barrier to cell transdifferentiation (Zhou and reprogramming is not known. Melton, 2008). Thus, discovering the molecular-genetic mecha- During postnatal development, astrocytes in the mouse cere- nisms that facilitate direct neuronal transdifferentiation provides bral cortex divide symmetrically, greatly expanding their num- insight into how cell fates normally become restricted, and may bers (Ge et al., 2012); and when isolated from postnatal day 7 (P7) also inform strategies that enhance cell fate conversion for the mice, astroglial cells still exhibit developmental plasticity: these purpose of disease-modeling and cell-based therapeutics. young astroglia can give rise to multipotent neurospheres Ink4a The Ink4a/Arf tumor suppressor locus encodes the p16 (Palmer et al., 1999; Laywell et al., 2000) and can also be con- Arf and p19 cell cycle inhibitors, which are expressed at basal levels verted into mature neurons via enforced expression of neuro- in differentiated cell types. In addition to their function in tumor genic transcription factors, such as Dlx2 (Heins et al., 2002; Berninger et al., 2007; Heinrich et al., 2010). However, cortical astrocytes from P15 mice no longer give rise to neurospheres Received July 24, 2013; revised Aug. 1, 2014; accepted Aug. 6, 2014. Author contributions: J.D.P., K.-Y.P., and D.A.L. designed research; J.D.P., K.-Y.P., J.C., R.D.S., and M.J.C. per- (Laywell et al., 2000), and transcription factor-mediated neu- formed research; J.D.P., K.-Y.P., J.C., R.D.S., M.J.C., A.R.K., and D.A.L. analyzed data; J.D.P., K.-Y.P., and D.A.L. wrote ronal transdifferentiation is greatly hindered in cells isolated the paper. from later postnatal mice (Heinrich et al., 2011; Robel et al., ThisworkwassupportedbyNationalInstitutesofHealthDP2-OD006505-01,theSontagFoundation,aNorthern 2011). Thus, during this postnatal period of symmetric glial California Institute for Research and Education/Department of Defense subaward to D.A.L., the Shurl and Kay Curci Foundation, and the San Francisco Veterans Affairs Medical Center for resources. cell proliferation, cortical astrocytes appear to lose their neu- The authors declare no competing financial interests. rogenic “competence.” *J.D.P. and K.-Y.P. contributed equally to this work. Here, we used transcription factor-mediated neuronal trans- Correspondence should be addressed to Dr. Daniel A. Lim, University of California, San Francisco, 35 Medical differentiation to gain insight into the molecular-genetic mecha- Center Way, San Francisco, CA 94143. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.3159-13.2014 nisms that normally restrict neurogenic competence. We found Copyright © 2014 the authors 0270-6474/14/3412560-08$15.00/0 that Ink4a/Arf is a potent barrier to direct neuronal transdiffer- Price, Park et al. • Ink4a/Arf Inhibits Neuronal Transdifferentiation J. Neurosci., September 10, 2014 • 34(37):12560–12567 • 12561 entiation of mouse astroglia isolated from non-neurogenic brain Lentiviral production. To acutely knock-down Ink4a/Arf, one short regions as well as mouse fibroblasts. Our findings suggest a gen- hairpin RNA (shRNA) sequence against Ink4a/Arf (targeting sequence: eralizable approach to enhance direct cell conversion methodol- AATGGCTGGATTGTTTAAA) (Fasano et al., 2007) and one control ogies and further indicate a role for Ink4a/Arf in the postnatal sequence targeting luciferase described in targeting sequence: GAGCT- glial-fate restriction of parenchymal astrocytes. GTTTCTGAGGAGCC (Ventura et al., 2004) was cloned into the lenti- viral vector pSicoR-mCh (a gift from Dr. Miguel Ramalho-Santos). The efficiency of viral transduction ranged from 95 to 98%, and no significant Materials and Methods differences were observed between wild-type (WT) and null glia. Brn2, Animals. Ink4a/Arf-null mice were maintained and genotyped as in Ser- Ascl1, and Myt1l lentiviral vectors have been described previously (Vier- rano et al. (1996). All experiments were performed with mice of either sex buchen et al., 2010) and were obtained through Addgene. VSV-G and and in accordance to protocols approved by the Institutional Animal EnvA pseudotyped lentiviruses were produced in HEK 293T cells as in Care and Use Committee at University of California, San Francisco. Lewis et al. (2001). Cell culture and neuronal transdifferentiation. Postnatal cortical astro- glial cultures were established essentially as in Berninger et al. (2007). Results Briefly, cortical tissue above the corpus callosum was microdissected Loss of neurogenic competence of cortical astroglia through from P5–P7 mouse brain sections, dissociated by treatment with 0.25% serial passage in vitro trypsin 0.5 mM EDTA as in Lim et al. (2009), and plated into 6-well P5–P7 cortical astroglial cultures infected with lentivirus express- culture plates (Corning) coated with 0.1 mg/ml poly-D-lysine (Sigma- ing Dlx2 and the GFP marker (LV-Dlx2-GFP) but not GFP alone Aldrich) in astrocyte growth medium described by Heinrich et al. (2010). (LV-GFP) produced GFP-positive neuronal cells immunoposi- Glial cultures were replated 5–7 d later into 16-well poly-D-lysine-coated ␤ Nunc Lab-Tek chamber slides (Thermo Scientific) at a density of 50,000 tive for III Tubulin (Tuj1-positive) after7dofdifferentiation cells per well (passage 1) or passaged into culture plates for continued (Fig. 1A,B). We investigated whether this “neurogenic compe- culture in astrocyte growth medium. Cells in chamber slides were in- tence” is maintained through serial passage in vitro. Cortical as- fected with lentivirus and switched to astrocyte differentiation medium troglia were serially passaged 5 times over 15 d, infected with (DMEM/F12, B27, glutamine, and penicillin/streptomycin) (Heinrich et LV-Dlx2-GFP or LV-GFP at passages 1, 2, 3, and 5, and neuronal al., 2010) 24 h later. differentiation was quantified 8 d after infection. While the effi- Mouse embryonic fibroblasts (MEFs) were isolated and cultured in N3 ciency of LV-Dlx2-GFP induced neuronal transdifferentiation medium (DMEM/F12, apotransferrin, insulin, sodium selenite, proges- was ϳ7% at passage 1 (Fig. 1B, Pass 1), the percentage of Tuj1- terone, putrescine, andpenicillin/streptomycin) (Vierbuchen et al., positive LV-Dlx2-GFP transduced cells was reduced 2.7-fold by 2010). Passage 3 MEFs were transduced with lentivirus expressing reverse passage 2 (Fig. 1B, Pass 2), becoming 8.5-fold reduced (0.8% tetracycline transactivator (FUW-M2rtTA (Hockemeyer et al., 2008) and the transcription factors Myt1l and Brn2 (Vierbuchen et al., 2010), and efficiency) at passage 5 (Fig. 1B, Pass 5). Thus, with serial in
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