A Giorgetti

E Lujan

GH Liu

K Liu

M Li

M Thier

MA Rasmussen

N Fan

NM Lind

T Ezashi

T Vierbuchen

Z Wu

Xiu-Ling Xu

Ji-Ping Yang

Li-Na Fu

Ruo-Tong Ren

Fei Yi

Keiichiro Suzuki

Kai Liu

Zhi-Chao Ding

Jing Qu

Wei-Qi Zhang

Ying Li

Ting-Ting Yuan

Guo-Hong Yuan

Li-Na Sui

Di Guan

Shun-Lei Duan

Hui-Ze Pan

Ping Wang

Xi-Ping Zhu

Nuria Montserrat

Ming Li

Rui-Jun Bai

Lin Liu

Juan Carlos Izpisua Belmonte

Guang-Hui Liu

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LETTERDirect reprogramming of porcine ﬁbroblaststo neural progenitor cellsDear Editor,Neural progenitor cells (NPCs) have proven potential tofacilitate mechanistic studies of neurological disordersin vitro, as well as the discovery of new medicines. In addi-tion, NPCs have been proposed as promising cell sourcesfor cell replacement therapy of neurological diseases (Liuet al., 2012b). For these areas of study, experimental ani-mals are indispensable models. Among the possible animalspecies, pigs are advantageous compared to rodentsbecause of their physiological and anatomical similarities tohumans (Lind et al., 2007). Despite the shown advantages ofporcine models in different ﬁelds, their applications are sig-niﬁcantly restricted due to the limited access of porcine cells,including NPCs. To date, encouraging breakthroughs havebeen made in obtaining NPCs from a series of species bydifferent methods, including primary cell isolation from tis-sues, differentiation from pluripotent stem cells, and directreprogramming from other somatic cells (Vierbuchen et al.,2010; Giorgetti et al., 2012; Lujan et al., 2012; Thier et al.,2012; Zhang et al., 2013).Here, we report the successful generation of inducedporcine NPCs (ipNPCs) from porcine fetal ﬁbroblasts (PFFs)(Fig. S1A, upper panel). Using our method, functional ip-NPCs can be readily obtained via direct cell reprogrammingwithout going through a pluripotent state. We show that ip-NPCs retain the ability for long-term culture and efﬁcientneural differentiation in vitro. Moreover, ipNPCs couldeffectively integrate into the local neural network after celltransplantation in vivo.In order to initiate the direct cell reprogramming, wesought to prime PFFs using non-integrative episomal vectorsexpressing reprogramming factors (Oct4, Sox2, Klf4, Lin28,and L-Myc) (Li et al., 2011) and then subjected the cells tohuman embryonic stem cell-amenable culture conditions (Liuet al., 2011). Three weeks later, ∼ 10 colonies emerged from5 × 105 transduced PFFs plated on mouse embryonicﬁbroblasts (MEFs). These colonies did not exhibit charac-teristic morphology of porcine induced pluripotent stem cells(iPSCs) including high nucleus to cytoplasm ratio and clearcolony boundaries, as reported in porcine iPSCs generatedby viral vector-mediated methods (Ezashi et al., 2009; Wuet al., 2009). Instead, they exhibited irregular and unclearboundaries with ﬁlament-like cells spreading out (Fig. S1A,bottom panel). When a treatment of 50 μmol/L sodiumbutyrate (an inhibitor of histone deacetylases) was appliedduring the reprogramming, a slightly higher number of col-onies with similar morphology emerged (data not shown).Immunoﬂuorescence staining showed that these coloniesexpressed Sox2 at low levels and were negative for pluri-potency markers Oct4 and Nanog (Fig. S1B). To initiate theneural commitment, these colonies were mechanicallypicked and seeded to MEFs supplied with neural stem cellculture medium (NSM) (Liu et al., 2012a). After a 10-dayinduction in NSM, the formation of neural rosettes wasobserved (Fig. 1A), which resembles an early stage ofneurodevelopment. Immunoﬂuorescence staining of neuralrosettes conﬁrmed the presence of an ipNPC population byexpression of both Sox2 and Nestin (Fig. 1A). These neuralrosette ipNPCs were then individualized, and subcultured inNSM on Matrigel. Under this condition, ipNPCs expanded inmonolayer and over 95% of them were positive for Pax6(Fig. 1B). Quantitative real-time PCR (qPCR) analysis furtherdemonstrated the induction of NPC markers (NCAM, Nestin,and Pax6) in ipNPCs compared to their parental PFFs(Fig. 1C). We further determined the possible presence ofresidual or integrated episomal vectors in ipNPCs by geno-mic qPCR analysis of EBNA-1, a viral element of episomalvectors originating from Epstein-Barr virus (Li et al., 2011).The results showed that ipNPCs and PFFs contained almostundetectable levels of EBNA-1 (∼ 0.0001–0.001 copies percell), while episomal vector-transfected PFFs showed nearly100 copies per cell, implying the absence of EBNA-1 in theipNPC genome (Fig. S1C). The negative readout from thisanalysis relieved safety concerns to use ipNPCs for celltransplantation in the future. Also, ipNPCs had been robustlymaintained for over 12 passages without signiﬁcant signs oflosing potency, suggesting an ability of long term self-renewal of ipNPCs in vitro. Meanwhile, when cultured onlow-attachment plates, ipNPCs formed neurospheres spon-taneously (Fig. S1D), further demonstrating neural stem cellidentity and robust viability.Next, in order to assess the neural differentiation potencyof ipNPCs, in vitro spontaneous neural differentiation wasperformed. After three weeks of culturing in spontaneousneural differentiation medium (NDM), the cell bodies of mostcells were clustered and long neurites protruded.© The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cnProtein Cell 2014, 5(1):4–7DOI 10.1007/s13238-013-0015-y Protein&CellProtein&CellDNAPax6A B CDEFGCurrent (pA)01500100050050010001500150  100  0 50  200  250  Time (ms) Time (ms) Time (s) Voltage (mV)0 50  100  150  200  20010050050150100Voltage (mV)60204020040600 20  40  80  120  60  100  140  160  Sox2NestinDNADNA/Sox2/NestinDNA GFP DNA/GFP DNA/GFPTuj1         GFAP          MBPPFF PFF PFFipNeuron ipNeuron ipNeuron806040200Relative mRNA  expression(Fold induction)********NCAM Nestin Pax6PFF PFF PFFipNPC ipNPC ipNPC3020100Relative mRNA  expression(Fold induction)*********DNA Tuj1 DNA/Tuj1DNA GFAP DNA/GFAPGeneration of induced neural progenitor cells from porcine ﬁbroblasts LETTER© The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn 5Protein&CellImmunoﬂuorescence staining showed that the majority ofdifferentiated cells were Tuj1 positive neurons while GFAPpositive glial cells were also present (Fig. 1D). Correspond-ingly, the mRNA levels of Tuj1 and GFAP signiﬁcantlyincreased after ipNPCs spontaneous differentiation(Fig. 1E). In addition, up-regulation of myelin basic protein(MBP), which is enriched in oligodendrocytes, was alsoobserved by qPCR (Fig. 1E), suggesting a possible exis-tence of oligodendrocytes in the differentiated derivatives.Taken together, our data indicated multipotent neural differ-entiation potential of ipNPCs.Subsequently, in order to assess the functional membraneproperties of ipNPC-derived neurons (ipNeurons), electrophys-iology recordings were performed after thirty days of spontane-ous differentiation. Whole-cell patch-clamp recordings onipNeurons revealed voltage-dependent currents including rap-idly inactivating inward currents and persistent outward currentsin response to depolarization voltage steps, which reﬂectedopen and closed states of sodium channels and potassiumchannels respectively (Fig. 1F, left). Current clamp recordingsdemonstrated that the ipNeurons could generate action poten-tials, an evident membrane characteristic of excitable cells(Fig. 1F, middle). Meanwhile, spontaneous action potentialswere also observed in differentiated ipNeurons (Fig. 1F, right).Collectively, these data demonstrated that ipNPCs are capableof differentiating into excitable neurons in vitro.Finally, we explored the neural differentiation potency ofipNPCs in vivo. The ipNPCs were labeled with GFP bylentiviral vectors, and then transplanted into the dentategyrus (DG) of NOD/SCID mice. Four weeks after trans-plantation, brains of recipient mice were sectioned andanalyzed. We found that most GFP-labeled cells werelocalized in the DG region, indicating a robust survival ofipNPCs in vivo (Fig. 1G). We further observed GFP positiveneurons with complex branching morphology that werepresent at neighboring zones of the DG region (Fig. 1G),which suggested that ipNPCs were able to effectively inte-grate into the local neural network after transplantation. Noteratoma formation was observed in any mouse brainsexamined, which further supports the safety of ipNPCsin vivo. (Data not shown)In summary, we report here a new strategy to obtainintegration-free functional porcine neural progenitor cells bydirect reprogramming of porcine fetal ﬁbroblasts in vitro. Forthe ﬁrst time, porcine neural progenitor cells were directlygenerated from somatic cells, and functionally characterizedboth in vitro and in vivo. Considering the importance of pigsas a model species, a sufﬁcient supply of functional porcineneural progenitor cells are of great interest in translationalmedicine studies of neuroscience. However, the difﬁcultiesto establish porcine pluripotent stem cells including embry-onic stem cells and integration-free iPSCs limit the produc-tion of porcine NPCs through traditional cell differentiationapproaches (Wu et al., 2009; Rasmussen et al., 2011; Liuet al., 2012c; Fan et al., 2013). Therefore, how to obtainporcine NPCs directly from the somatic cells is attracting alot of attention in the ﬁeld. Similar to many other directreprogramming methods, our strategy bypassed obstacles inestablishing porcine pluripotent stem cells. Moreover, ourmethod provided a robust and efﬁcient way of generatingporcine NPCs with low risk of tumor formation. To ourknowledge, this is the ﬁrst attempt to direct reprogramsomatic cells into neural progenitor cells using the porcinespecies. As a promising species of model animals, the ip-NPCs generated in our study may provide an exciting tool tobridge the present gaps in neuroscience studies betweenrodents and humans.FOOTNOTESWe are grateful to April Goebl for manuscript editing. GHL is sup-ported by the Strategic Priority Research Program of the ChineseAcademy of Sciences (XDA01020312), National Basic ResearchProgram (2014CB964600), the National Natural Science Foundationof China (Grant Nos. 81271266, 31222039, and 81330008), Thou-sand Young Talents program of China, National Laboratory of Bio-macromolecules (2012kf02, 2013kf05, 2013kf11), and State KeyLaboratory of Drug Research (SIMM1302KF-17). JQ is supported byNational Basic Research Program (2014CB910500) and theNational Natural Science Foundation of China (81371342). XLX issupported by the National Natural Science Foundation of China(31201111). WQZ is supported by the National Natural ScienceFoundation of China (81300261). LNS is supported by the NationalNatural Science Foundation of China (81300677). DG is supportedFigure 1. Direct reprogramming of porcine fetal ﬁbroblasts(PFFs) to induced porcine neural progenitor cells (ipNPCs).(A) Immunoﬂuorescence staining of ipNPCs derived on MEFsshowing rosette morphology and expressions of neural pro-genitor cell markers Nestin (in green) and Sox2 (in red). Scalebars, 75 μm. (B) Immunoﬂuorescence staining of Pax6 positiveipNPCs cultured on matrigel. Scale bars, 25 μm. (C) Geneexpression analysis showed that the neural progenitor cellmarkers (NCAM, Nestin, Pax6) were induced in the directconversion of PFFs to ipNPCs. All values are relative to PFFsand shown as mean ± s.e.m. (n = 3). ***P < 0.001. (D) Differ-entiation of ipNPCs into neurons (Tuj1, Green) and glial cells(GFAP, Yellow) in vitro. Scale bars, 100 μm. (E) QuantitativePCR analysis showed the induction of neuronal speciﬁc marker(Tuj1), glial speciﬁc marker (GFAP), and oligodendrocytemarker (MBP) after spontaneous differentiation of ipNPCs. Allvalues are relative to PFFs and shown as mean ± s.e.m. (n = 3).**P < 0.01, ***P < 0.001. (F) Representative traces ofelectrophysiology recording of differentiated neurons at day30. Inward fast inactivating sodium currents and outwardcurrents were observed in differentiated neurons by whole cellcurrent recording (left). Action potentials (APs) were elicited bystep-current injections (middle). Spontaneous APs ﬁring fromipNeuron were recorded (right). (G) In vivo transplantation ofipNPCs. Overview of transplanted ipNPCs (GFP + , Green)cells in the dentate gyrus (DG) of brain from NOD/SCID mice at4 weeks after ipNPCs transplantation. Blue, DNA; Green, GFP.sLETTER Xiu-Ling Xu et al.6 © The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cnProtein&Cellby China Postdoctoral Science Foundation (2013M530751). K.S issupported by a California Institute for Regenerative Medicine fel-lowship. LL is supported by the National Basic Research Program(973 Program) (Nos. 2009CB941000 and 2011CBA01002) and theNational Natural Science Foundation of China (31271587). JCIBwas supported by G. Harold and Leila Y. Mathers Charitable Foun-dation, The Leona M. and Harry B. Helmsley Charitable Trust, TheGlenn Foundation and The Ellison Medical Foundation.All the authors (Xiu-Ling Xu, Ji-Ping Yang, Li-Na Fu, Ruo-TongRen, Fei Yi, Keiichiro Suzuki, Kai Liu, Zhi-Chao Ding, Jing Qu,Wei-QiZhang, Ying Li, Ting-Ting Yuan, Guo-Hong Yuan, Li-Na Sui, Di Guan,Shun-Lei Duan, Hui-Ze Pan, Ping Wang, Xi-Ping Zhu, Nuria Mont-serrat, Ming Li, Rui-Jun Bai, Lin Liu, Juan Carlos Izpisua Belmonte,and Guang-Hui Liu) declare that they have no conﬂict of interest.All institutional and national guidelines for the care and use oflaboratory animals were followed.Xiu-Ling Xu1&, Ji-Ping Yang1, Li-Na Fu1, Ruo-Tong Ren1, Fei Yi2,Keiichiro Suzuki3, Kai Liu4, Zhi-Chao Ding1, Jing Qu1, Wei-QiZhang1, Ying Li1, Ting-Ting Yuan1, Guo-Hong Yuan1, Li-Na Sui1,Di Guan1, Shun-Lei Duan1, Hui-Ze Pan1, PingWang1, Xi-Ping Zhu1,Nuria Montserrat5, Ming Li1, Rui-Jun Bai1, Lin Liu4, Juan CarlosIzpisua Belmonte3, Guang-Hui Liu1,6&1 National Laboratory of Biomacromolecules, Institute of Biophysics,Chinese Academy of Sciences, Beijing 100101, China2 Department of Molecular and Cellular Physiology, StanfordUniversity School of Medicine, Stanford, CA 94305, USA3 Gene Expression Laboratory, Salk Institute for Biological Studies,10010 North Torrey Pines Road, La Jolla, CA 92037, USA4 State Key Laboratory of Medicinal Chemical Biology, College ofLife Sciences, Nankai University, Tianjin 300071, China5 Center for Regenerative Medicine in Barcelona, Dr. Aiguader 88,08003 Barcelona, Spain6 Beijing Institute for Brain Disorders, Beijing 100069, China& Correspondence: xiulingxu@hotmail.com (X.-L. Xu),ghliu@ibp.ac.cn (G.-H. Liu)OPEN ACCESSThis article is distributed under the terms of the Creative CommonsAttribution License which permits any use, distribution, andreproduction in any medium, provided the original author(s) andthe source are credited.REFERENCESEzashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S, Roberts RM(2009) Derivation of induced pluripotent stem cells from pigsomatic cells. Proc Natl Acad Sci USA 106:10993–10998Fan N, Chen J, Shang Z, Dou H, Ji G, Zou Q, Wu L, He L, Wang F,Liu K et al (2013) Piglets cloned from induced pluripotent stemcells. Cell Res 23:162–166Giorgetti A, Marchetto MC, Li M, Yu D, Fazzina R, Mu Y, Adamo A,Paramonov I, Cardoso JC, Monasterio MB et al (2012) Cordblood-derived neuronal cells by ectopic expression of Sox2 andc-Myc. Proc Natl Acad Sci USA 109:12556–12561Li M, Suzuki K, Qu J, Saini P, Dubova I, Yi F, Lee J, Sancho-MartinezI, Liu GH, Izpisua Belmonte JC (2011) Efﬁcient correction ofhemoglobinopathy-causing mutations by homologous recombi-nation in integration-free patient iPSCs. Cell Res 21:1740–1744Lind NM, Moustgaard A, Jelsing J, Vajta G, Cumming P, Hansen AK(2007) The use of pigs in neuroscience: modeling braindisorders. Neurosci Biobehav Rev 31:728–751Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD,Suzuki K, Kurian L, Walsh C et al (2011) Recapitulation ofpremature ageing with iPSCs from Hutchinson-Gilford progeriasyndrome. Nature 472:221–225Liu GH, Qu J, Suzuki K, Nivet E, Li M, Montserrat N, Yi F, Xu X, RuizS, Zhang W et al (2012a) Progressive degeneration of humanneural stem cells caused by pathogenic LRRK2. Nature 491:603–607Liu GH, Yi F, Suzuki K, Qu J, Izpisua Belmonte JC (2012b) Inducedneural stem cells: a new tool for studying neural development andneurological disorders. Cell Res 22:1087–1091Liu K, Ji G, Mao J, Liu M, Wang L, Chen C, Liu L (2012c) Generationof porcine-induced pluripotent stem cells by using OCT4 andKLF4 porcine factors. Cell Reprogram 14:505–513Lujan E, Chanda S, Ahlenius H, Sudhof TC, Wernig M (2012) Directconversion of mouse ﬁbroblasts to self-renewing, tripotent neuralprecursor cells. Proc Natl Acad Sci USA 109:2527–2532Rasmussen MA, Hall VJ, Carter TF, Hyttel P (2011) Directeddifferentiation of porcine epiblast-derived neural progenitor cellsinto neurons and glia. Stem Cell Res 7:124–136Thier M, Worsdorfer P, Lakes YB, Gorris R, Herms S, Opitz T,Seiferling D, Quandel T, Hoffmann P, Nothen MM et al (2012)Direct conversion of ﬁbroblasts into stably expandable neuralstem cells. Cell Stem Cell 10:473–479Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, WernigM (2010) Direct conversion of ﬁbroblasts to functional neurons bydeﬁned factors. Nature 463:1035–1041Wu Z, Chen J, Ren J, Bao L, Liao J, Cui C, Rao L, Li H, Gu Y, Dai Het al (2009) Generation of pig induced pluripotent stem cells witha drug-inducible system. J Mol Cell Biol 1:46–54Zhang K, Liu GH, Yi F, Montserrat N, Hishida T, Rodriguez EstebanC, Izpisua Belmonte JC (2013) Direct conversion of humanﬁbroblasts into retinal pigment epithelium-like cells by deﬁnedfactors. Protein Cell. doi:10.1007/S13238-013-0011-2Electronic supplementary material The online version of thisarticle (doi:10.1007/s13238-013-0015-y) contains supplementarymaterial, which is available to authorized users.Generation of induced neural progenitor cells from porcine ﬁbroblasts LETTER© The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn 7Protein&Cell

Protein & Cell

Direct reprogramming of porcine fibroblasts to neural progenitor cells

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Direct reprogramming of porcine fibroblasts to neural progenitor cells

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