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• W4246383450 abstract "Human induced pluripotent stem cells (hiPSCs) have been generated by reprogramming a number of different somatic cell types using a variety of approaches. In addition, direct reprogramming of mature cells from one lineage to another has emerged recently as an alternative strategy for generating cell types of interest. Here we show that a combination of a microRNA (miR-124) and two transcription factors (MYT1L and BRN2) is sufficient to directly reprogram postnatal and adult human primary dermal fibroblasts (mesoderm) to functional neurons (ectoderm) under precisely defined conditions. These human induced neurons (hiNs) exhibit typical neuronal morphology and marker gene expression, fire action potentials, and produce functional synapses between each other. Our findings have major implications for cell-replacement strategies in neurodegenerative diseases, disease modeling, and neural developmental studies. Human induced pluripotent stem cells (hiPSCs) have been generated by reprogramming a number of different somatic cell types using a variety of approaches. In addition, direct reprogramming of mature cells from one lineage to another has emerged recently as an alternative strategy for generating cell types of interest. Here we show that a combination of a microRNA (miR-124) and two transcription factors (MYT1L and BRN2) is sufficient to directly reprogram postnatal and adult human primary dermal fibroblasts (mesoderm) to functional neurons (ectoderm) under precisely defined conditions. These human induced neurons (hiNs) exhibit typical neuronal morphology and marker gene expression, fire action potentials, and produce functional synapses between each other. Our findings have major implications for cell-replacement strategies in neurodegenerative diseases, disease modeling, and neural developmental studies. miR-124 promotes direct neuronal conversion of human fibroblasts Reprogramming of both postnatal and fully adult human fibroblasts Robust conversion mediated by a cocktail of miR-124, BRN2, and MYT1L Formation of functional synapses between adult human fibroblast-derived neurons The differentiated cell state is often considered stable and resistant to changes in lineage identity. However, challenging this view, differentiated somatic cell types from humans and other organisms have been reprogrammed to the pluripotent state by forced expression of a set of transcription factors (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14962) Google Scholar), somatic cell nuclear transfer (Campbell et al., 1996Campbell K.H. McWhir J. Ritchie W.A. Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line.Nature. 1996; 380: 64-66Crossref PubMed Scopus (1468) Google Scholar, Gurdon et al., 1958Gurdon J.B. Elsdale T.R. Fischberg M. Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei.Nature. 1958; 182: 64-65Crossref PubMed Scopus (394) Google Scholar), or cell fusion (Cowan et al., 2005Cowan C.A. Atienza J. Melton D.A. Eggan K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells.Science. 2005; 309: 1369-1373Crossref PubMed Scopus (766) Google Scholar, Tada et al., 2001Tada M. Takahama Y. Abe K. Nakatsuji N. Tada T. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells.Curr. Biol. 2001; 11: 1553-1558Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). Additionally, by lineage reprogramming through ectopic expression of selected genes or cell fusion, a few studies have demonstrated that an adult cell type can be directly converted to another adult cell type (Cobaleda et al., 2007Cobaleda C. Jochum W. Busslinger M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors.Nature. 2007; 449: 473-477Crossref PubMed Scopus (380) Google Scholar, Davis et al., 1987Davis R.L. Weintraub H. Lassar A.B. Expression of a single transfected cDNA converts fibroblasts to myoblasts.Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2470) Google Scholar, Feng et al., 2008Feng R. Desbordes S.C. Xie H. Tillo E.S. Pixley F. Stanley E.R. Graf T. PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage-like cells.Proc. Natl. Acad. Sci. USA. 2008; 105: 6057-6062Crossref PubMed Scopus (274) Google Scholar, Ieda et al., 2010Ieda M. Fu J.D. Delgado-Olguin P. Vedantham V. Hayashi Y. Bruneau B.G. Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.Cell. 2010; 142: 375-386Abstract Full Text Full Text PDF PubMed Scopus (1884) Google Scholar, Zhou et al., 2008Zhou Q. Brown J. Kanarek A. Rajagopal J. Melton D.A. in vivo reprogramming of adult pancreatic exocrine cells to beta-cells.Nature. 2008; 455: 627-632Crossref PubMed Scopus (1634) Google Scholar, Zhou and Melton, 2008Zhou Q. Melton D.A. Extreme makeover: converting one cell into another.Cell Stem Cell. 2008; 3: 382-388Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). However, all these studies involve conversion of one cell type to another within the same lineage, a major limitation for many applications. For cell-replacement therapies, the idea of reprogramming across lineages is fascinating because of its potential to rapidly generate a variety of therapeutically important and immunologically matched cell types directly from one's own easily accessible tissues, such as skin or blood. In this context, Vierbuchen et al., 2010Vierbuchen T. Ostermeier A. Pang Z.P. Kokubu Y. Südhof T.C. Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors.Nature. 2010; 463: 1035-1041Crossref PubMed Scopus (2258) Google Scholar demonstrated that differentiated mouse cells have the capacity for changing lineage by showing direct conversion of dermal fibroblasts to functional “induced neurons” (iNs). Recently, the same group extended these findings to human fetal and postnatal fibroblasts to produce human iN (hiN) cells (Pang et al., 2011Pang Z.P. Yang N. Vierbuchen T. Ostermeier A. Fuentes D.R. Yang T.Q. Citri A. Sebastiano V. Marro S. Südhof T.C. Wernig M. Induction of human neuronal cells by defined transcription factors.Nature. 2011; Crossref PubMed Scopus (941) Google Scholar). Nonetheless, these hiNs formed functional synapses only when cocultured with mature mouse cortical neurons, which potentially provided additional differentiation factors. Here we show that a combination of a microRNA (miR-124) and two transcription factors (MYT1L and BRN2) is sufficient to directly reprogram postnatal and adult human primary dermal fibroblasts (mesoderm) to functional neurons (ectoderm) under precisely defined conditions without a requirement for “helper” cells from a different species. These hiNs exhibit typical neuronal morphology and marker gene expression, fire action potentials, and form functional synapses. Given the known critical roles of specific transcription factors, signaling molecules, and microRNAs in neuronal lineage determination during development or cell fate regulation, we selected 11 transcription factors (Table S1 available online) and a microRNA (miR-124) to test for their ability to convert primary postnatal human fibroblasts, BJ or CRL2097, to functional neurons. After confirming the absence of any contaminating neural or neuronal cells in BJ and CRL2097 cell cultures by immunostaining (Figure S1A available online) and RT-PCR (Figure S1B), we transduced them with lentiviruses carrying the 12 factors pooled together (12F pool, with equal representation of each). The subsequent culture conditions are depicted in the schematic diagram in Figure 1A and in the Supplemental Experimental Procedures section. Infected cells were monitored by the presence of red fluorescent protein (RFP) coexpressed with the miRNA vector (pLemiR). Immunocytochemistry for the early neuronal marker βIII-tubulin (Tuj1), performed 18 days after infection, revealed a few Tuj1/RFP double-positive cells that manifested typical neuronal morphology in the 12F-infected cultures (Figure 1B), while no such cells were observed in uninfected or GFP-control cultures (Figures S1A and S1C). These findings suggested that at least some of the factors in the 12F pool had the capacity to convert fibroblasts to human neuronal-like cells. To test whether any single factor among the 12F pool was sufficient to induce the neuronal phenotype, we transduced BJ cells with the individual factors. Although on day 18 none of these cultures displayed cells with distinctive neuronal morphology, in cultures transduced with miR-124 alone we observed a few Tuj1-positive cells with multiple, small processes that resembled neurites emanating from the soma (Figures S1D and S1E). However, even after continued culturing for over 1 month, these cells did not mature further to produce a characteristic neuronal phenotype (data not shown). Such changes resembled those observed previously when miR-124 was overexpressed in nonneuronal cells (Yu et al., 2008Yu J.Y. Chung K.H. Deo M. Thompson R.C. Turner D.L. MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation.Exp. Cell Res. 2008; 314: 2618-2633Crossref PubMed Scopus (267) Google Scholar). Next, we tested two-factor combinations and found that cultures transduced with miR-124 plus BRN2 (designated miB) or PAX 6 (miP) exhibited cells that were morphologically similar to those observed with miR-124 alone, but with >20-fold better efficacy (Figure S1F). Cells infected with miR-124 plus MYT1L (miM) showed a distinctive elongated morphology, unique among the combinations that we used (Figure S1G). Reasoning that some of these four factors might complement one other, we tested three-factor (3F) combinations. Remarkably, within 3 days of transduction with miBM, many RFP-positive BJ cells exhibited small, compact cell bodies with monopolar or bipolar projections and weak βIII-tubulin expression (Figure 1C). A characteristic neuronal morphology, consisting of multiple neuritic extensions and elaborate branching, became progressively obvious when these cells were allowed to mature for an additional 15 days (Figures 1D–1F). The majority of these cells displayed positive immunoreactivity for the mature neuronal markers MAP2 (55%, n = 100) (Figure 1G) and NeuN (46%, n = 100) (Figure 1H). In contrast, we did not observe any neuronal-like cells in cultures transduced with any other 3F combinations (data not shown) or in control cultures where miR-124 was replaced with scrambled, nonspecific small RNAs (Figure 1I). Also, the addition of more factors to miBM did not substantially improve the process. CRL2097 fibroblasts produced similar results to BJ cells (data not shown). An EdU incorporation assay suggested that fibroblasts destined to become hiN cells were most likely postmitotic within 24 hr of transgene induction, and thus it is likely that hiN conversion occurred in the absence of a mitotic progenitor cell stage (Figures S1H–S1P). By dividing the number of Tuj1-positive hiN cells on day 18 by the total number of cells in the starting fibroblast population, we estimated an efficiency of 4%–8% for hiN generation from BJ or CRL2097 human fibroblast cells (Figure S1Q). Furthermore, and importantly, using a doxycycline or cumate-inducible system, we found that expression of miBM for 7 days was sufficient to produce hiN cells at a frequency comparable to that of our earlier experiments (Figure 1A and Figures S2A–S2G). To functionally characterize hiN cells derived with miBM, we examined their electrophysiological properties on day 25 when the vast majority of cells displayed synapsin immunoreactivity (Figures 2A and 2B ), a marker associated with functional maturation of neuronal synapses. During whole-cell recording in voltage-clamp mode, the majority of hiN cells (60%, n = 10) exhibited rapidly inactivating inward current with a rise time of 2–3 ms, followed by outward currents, most likely corresponding to opening of voltage-dependent Na+ and K+ channels, respectively (Figure 2C). The inward current was inhibited by the sodium channel blocker tetrodotoxin (Figure 2D). In current-clamp mode, the resting membrane potential averaged about −45 mV, and with increasing time in culture the majority of cells (81%, n = 29) fired action potentials with amplitudes of ∼110 mV in response to injection of 10 to 20 pA currents (Figure 2E). Some hiN cells (∼15%) exhibited spontaneous action potentials (Figure 2F, left panel), and others (∼20%), repetitive trains of evoked action potentials (Figure 2F, right panel). We also monitored additional electrophysiological parameters, including membrane capacitance, access resistance, and total membrane resistance (Table S2A); these values are consistent with the notion that the hiN cells were maturing neurons. We next examined whether the hiN cells manifested functional neurotransmitter properties by testing for specific markers and corresponding ligand-gated currents. Immunostaining revealed that about 8% of hiN cells (n = 50) were positive for the inhibitory neurotransmitter GABA (Figures 2G and 2H), and 12% manifested punctuate staining for VGAT (data not shown), a protein involved in vesicular transport of GABA. Importantly, hiN cells responded to exogenous application of GABA (60%, n = 5), producing whole-cell currents (Figure 2I). Furthermore, we recorded slowly decaying NMDA-evoked current in 67% (n = 6) of hiN cells (Figure 2J). Moreover, a high percentage of hiN cells displayed presynaptic properties of excitatory glutamatergic neurons, as indicated by positive VGLUT1 staining (44%, n = 50) (Figures 2K and 2L), a marker for the vesicular glutamate transporter. While we observed rare hiN cells of dopaminergic-like phenotype, as evidenced by tyrosine hydroxylase staining (Figures 2M and 2N), none stained for peripherin, choline acetyltransferase, or serotonin (data not shown). By day 30 postinfection, patch-clamp recordings revealed miniature excitatory postsynaptic currents (mEPSCs), indicative of functional synapses, in 25% (n = 8) of hiN cells (Figure 2O, top panel). These currents were sensitive to NBQX, an AMPA-type glutamate receptor antagonist, but not bicuculline, a competitive inhibitor of GABAA receptors (Figure 2O, bottom panel), thus further confirming their excitatory nature. These results further substantiate the functional neurotransmitter properties of hiN cells derived from postnatal human fibroblasts, and collectively provide strong evidence that the cells had become functional neurons that generated synaptic connections. Finally, to examine whether adult human fibroblasts could generate hiN cells, we tested dermal fibroblasts derived from abdominal skin of a 55-year-old Caucasian female (aHDF-1) and breast skin of a 41-year-old Caucasian female (aHDF-2). After confirming that these cells were free of contaminating neural or spinal progenitor cells, neurons, or epidermal keratinocytes (Figures S1A and S1B), we transduced them with viruses carrying the miBM transgenes. Similar to our observations on postnatal fibroblasts, these adult human fibroblasts were converted to hiN cells with an efficiency of 1.5%–2.9% (aHDF-1) or 9.5%–11.2% (aHDF-2) (Figure S1Q), and displayed characteristic mature neuronal morphology and marker gene expression (Figures 2P–2R). When tested on day 25 postinfection, hiN cells derived from adult fibroblasts also exhibited rapidly inactivating Na+ currents (47%, n = 17) and fired action potentials (12%, n = 25) (Figures 2S and 2T). The mean action potential amplitude was 73.4 ± 18.3 mV (mean ± SEM). Values for resting membrane potential, membrane capacitance, access resistance, and total membrane resistance (Table S2B) were comparable to those of BJ or CRL2097-derived hiN cells, except that the greater membrane resistance probably reflected their more immature nature. Moreover, many of these cells exhibited VGLUT1 immunoreactivity (28%, n = 50) (Figure 2U) and NMDA-evoked responses (60%, n = 5) (Figure 2V), thus displaying excitatory neuronal properties. Additionally, when plated at high density, but not low density, aHDF-hiN cells displayed excitatory synaptic currents (43%, n = 7) (Figure 2W), reflecting functional contacts with neighboring hiNs. Therefore, we have demonstrated here that a combination of miR-124, BRN2, and MYT1L can induce rapid reprogramming of postnatal and adult human fibroblasts into functional neurons. While other combinations of transcription factors have recently been reported to produce neuronal-like cells from human fibroblasts (Pang et al., 2011Pang Z.P. Yang N. Vierbuchen T. Ostermeier A. Fuentes D.R. Yang T.Q. Citri A. Sebastiano V. Marro S. Südhof T.C. Wernig M. Induction of human neuronal cells by defined transcription factors.Nature. 2011; Crossref PubMed Scopus (941) Google Scholar) and dopaminergic neurons (Pfisterer et al., 2011Pfisterer U. Kirkeby A. Torper O. Wood J. Nelander J. Dufour A. Björklund A. Lindvall O. Jakobsson J. Parmar M. Direct conversion of human fibroblasts to dopaminergic neurons.Proc. Natl. Acad. Sci. USA. 2011; 108: 10343-10348Crossref PubMed Scopus (596) Google Scholar, Caiazzo et al., 2011Caiazzo M. Dell'anno M.T. Dvoretskova E. Lazarevic D. Taverna S. Leo D. Sotnikova T.D. Menegon A. Roncaglia P. Colciago G. et al.Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.Nature. 2011; Crossref PubMed Scopus (780) Google Scholar), an important development with our combined microRNA/transcription factor approach is that we have been able to produce neurons with mature functional synapses between adult fibroblast-derived hiNs in the absence of other cell types. Along these lines, in our study reprogramming was ineffective for combinations of factors lacking miR-124. miR-124, the most abundant microRNA in the mammalian CNS (Lagos-Quintana et al., 2002Lagos-Quintana M. Rauhut R. Yalcin A. Meyer J. Lendeckel W. Tuschl T. Identification of tissue-specific microRNAs from mouse.Curr. Biol. 2002; 12: 735-739Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar), is markedly upregulated in differentiating and mature neurons (Deo et al., 2006Deo M. Yu J.Y. Chung K.H. Tippens M. Turner D.L. Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides.Dev. Dyn. 2006; 235: 2538-2548Crossref PubMed Scopus (246) Google Scholar), where it modulates the activity of major antineuronal differentiation factors, including REST/SCP1 (Conaco et al., 2006Conaco C. Otto S. Han J.J. Mandel G. Reciprocal actions of REST and a microRNA promote neuronal identity.Proc. Natl. Acad. Sci. USA. 2006; 103: 2422-2427Crossref PubMed Scopus (605) Google Scholar, Visvanathan et al., 2007Visvanathan J. Lee S. Lee B. Lee J.W. Lee S.K. The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development.Genes Dev. 2007; 21: 744-749Crossref PubMed Scopus (565) Google Scholar), PTBP1 (Makeyev et al., 2007Makeyev E.V. Zhang J. Carrasco M.A. Maniatis T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.Mol. Cell. 2007; 27: 435-448Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar), SOX9 (Cheng et al., 2009Cheng L.C. Pastrana E. Tavazoie M. Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche.Nat. Neurosci. 2009; 12: 399-408Crossref PubMed Scopus (817) Google Scholar), and npBAF complex (Yoo et al., 2009Yoo A.S. Staahl B.T. Chen L. Crabtree G.R. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development.Nature. 2009; 460: 642-646Crossref PubMed Scopus (467) Google Scholar). Moreover, miR-124 targets more than 1000 genes, many of which are downregulated during neuronal differentiation (Lewis et al., 2005Lewis B.P. Burge C.B. Bartel D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9835) Google Scholar). Interestingly, introduction of miR-124 to HeLa cells could transform their gene expression pattern to resemble that of brain tissue (Lim et al., 2005Lim L.P. Lau N.C. Garrett-Engele P. Grimson A. Schelter J.M. Castle J. Bartel D.P. Linsley P.S. Johnson J.M. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.Nature. 2005; 433: 769-773Crossref PubMed Scopus (3995) Google Scholar). However, overexpression of miR-124 by itself seems to be insufficient to induce neurogenesis, but rather ensured that nonneuronal genes were posttranscriptionally inhibited in neurons (Cao et al., 2007Cao X. Pfaff S.L. Gage F.H. A functional study of miR-124 in the developing neural tube.Genes Dev. 2007; 21: 531-536Crossref PubMed Scopus (298) Google Scholar). In line with these observations, in our experiments miR-124 only effected neuronal reprogramming in the presence of other key factors. The reprogramming platform described here provides rapid conversion of adult cell types directly into neurons, as opposed to more time-consuming and labor-intensive iPSC or ESC-based methods. Our findings also provide a technology that can use patient-specific cells for the rapid production of models of various human neurodegenerative “diseases in a dish,” including diseases that are only manifest later in life. We would like to thank Marius Wernig (Stanford University) for the generous gift of pBrn2-TetO-FUW and pMyt1l-TetO-FUW plasmids. We thank all members of the Ding lab for helpful discussions, and Wenlin Li and Jem Efe for critical reading of the manuscript. M.T. and S.A.L. were supported in part by NIH grants P01 HD29587, P01 ES016738, P30 NS057096, and R01 EY05477, and by the California Institute for Regenerative Medicine. S.D. was supported by funding from NICHD, NHLBI, NEI, and NIMH/NIH, California Institute for Regenerative Medicine, Prostate Cancer Foundation, Fate Therapeutics, and the Gladstone Institutes. R.A. conceived the study. R.A., M.T., S.A.L., and S.D. designed experiments. R.A. performed viral infections, generated hiN cells, conducted cellular and molecular characterization, and interpreted the results. R.C. assisted in constructing viral vectors. X.Y. assisted in qPCR analysis and the cell culture. X.Y. and R.C. contributed equally to this work. S.Z. assisted in the cell culture. M.T. and S.A.L. designed, performed, and/or analyzed the electrophysiological assays. R.A. and M.T. produced the figures. R.A., S.A.L., and S.D. wrote and/or edited the manuscript. Download .pdf (.82 MB) Help with pdf files Document S1. Supplemental Figures, Tables, Experimental Procedures, and Primers" @default.
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• W4246383450 title "Direct Reprogramming of Adult Human Fibroblasts to Functional Neurons under Defined Conditions" @default.
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