Plasmid-based generation of neural cells from human fibroblasts using non-integrating episomal vectors
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摘要
Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, although some studies have been successful in directly inducing neurons through sustained expression of small molecule compounds, they have only been shown to be effective on mouse-derived cells. Thus, herein we delivered vectors containing Epstein-Barr virus-derived oriP/Epstein-Barr nuclear antigen 1 encoding the neuronal transcription factor, Ascl1, the neuron-specific microRNA, miR124, and a small hairpin directed against p53, into human fibroblasts. Cells were incubated in a neuron-inducing culture medium. Immunofluorescence staining was used to detect Tuj-1, microtubule-associated protein 2, neuron-specific nucleoprotein NeuN and nerve cell adhesion molecules in the induced cells. The proportion of Tuj1-positive cells was up to 36.7% after induction for 11 days. From day 21, these induced neurons showed neuron-specific expression patterns of microtubule-associated protein 2, NeuN and neural cell adhesion molecule. Our approach is a simple, plasmid-based process that enables direct reprogramming of human fibroblasts into neurons, and provides alternative avenues for disease modeling and neurodegenerative medicine.
Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, although some studies have been successful in directly inducing neurons through sustained expression of small molecule compounds, they have only been shown to be effective on mouse-derived cells. Thus, herein we delivered vectors containing Epstein-Barr virus-derived oriP/Epstein-Barr nuclear antigen 1 encoding the neuronal transcription factor, Ascl1, the neuron-specific microRNA, miR124, and a small hairpin directed against p53, into human fibroblasts. Cells were incubated in a neuron-inducing culture medium. Immunofluorescence staining was used to detect Tuj-1, microtubule-associated protein 2, neuron-specific nucleoprotein NeuN and nerve cell adhesion molecules in the induced cells. The proportion of Tuj1-positive cells was up to 36.7% after induction for 11 days. From day 21, these induced neurons showed neuron-specific expression patterns of microtubule-associated protein 2, NeuN and neural cell adhesion molecule. Our approach is a simple, plasmid-based process that enables direct reprogramming of human fibroblasts into neurons, and provides alternative avenues for disease modeling and neurodegenerative medicine.
Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, although some studies have been successful in directly inducing neurons through sustained expression of small molecule compounds, they have only been shown to be effective on mouse-derived cells. Thus, herein we delivered vectors containing Epstein-Barr virus-derived oriP/Epstein-Barr nuclear antigen 1 encoding the neuronal transcription factor, Ascl1, the neuron-specific microRNA, miR124, and a small hairpin directed against p53, into human fibroblasts. Cells were incubated in a neuron-inducing culture medium. Immunofluorescence staining was used to detect Tuj-1, microtubule-associated protein 2, neuron-specific nucleoprotein NeuN and nerve cell adhesion molecules in the induced cells. The proportion of Tuj1-positive cells was up to 36.7% after induction for 11 days. From day 21, these induced neurons showed neuron-specific expression patterns of microtubule-associated protein 2, NeuN and neural cell adhesion molecule. Our approach is a simple, plasmid-based process that enables direct reprogramming of human fibroblasts into neurons, and provides alternative avenues for disease modeling and neurodegenerative medicine.
引文
?kerblom M,Sachdeva R,Barde I,Verp S,Gentner B,Trono D,Jakobsson J(2012)MicroRNA-124 is a subventricular zone neuronal fate determinant.J Neurosci 32:8879-8889.
Caiazzo M,Dell’Anno MT,Dvoretskova E,Lazarevic D,Taverna S,Leo D,Sotnikova TD,Menegon A,Roncaglia P,Colciago G,Russo G,Carninci P,Pezzoli G,Gainetdinov RR,Gustincich S,Dityatev A,Broccoli V(2011)Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.Nature 476:224-227.
Capetian P,Azmitia L,Pauly MG,Krajka V,Stengel F,Bernhardi EM,Klett M,Meier B,Seibler P,Stanslowsky N,Moser A,Knopp A,Gillessen-Kaesbach G,Nikkhah G,Wegner F,Dobrossy M,Klein C(2016)Plasmid-based generation of induced neural stem cells from adult human fibroblasts.Front Cell Neurosci 10:245.
Chanda S,Ang CE,Davila J,Pak C,Mall M,Lee QY,Ahlenius H,Jung SW,Sudhof TC,Wernig M(2014)Generation of induced neuronal cells by the single reprogramming factor ASCL1.Stem Cell Reports 3:282-296.
Cheng LC,Pastrana E,Tavazoie M,Doetsch F(2009)miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche.Nat Neurosci 12:399-408.
Dell’Anno MT,Caiazzo M,Leo D,Dvoretskova E,Medrihan L,Colasante G,Giannelli S,Theka I,Russo G,Mus L,Pezzoli G,Gainetdinov RR,Benfenati F,Taverna S,Dityatev A,Broccoli V(2014)Remote control of induced dopaminergic neurons in parkinsonian rats.J Clin Invest 124:3215-3229.
Fiszbein A,Schor IE,Kornblihtt AR(2015)Chapter 11-Fundamentals of NCAM expression,function,and regulation of alternative splicing in neuronal differentiation.Neural Surf Antigens 112:131-140.
Hong H,Takahashi K,Ichisaka T,Aoi T,Kanagawa O,Nakagawa M,Okita K,Yamanaka S(2009)Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.Nature 460:1132-1135.
Jiang H,Xu Z,Zhong P,Ren Y,Liang G,Schilling HA,Hu Z,Zhang Y,Wang X,Chen S,Yan Z,Feng J(2015)Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons.Nat Commun 6:10100.
Karow M,Sánchez R,Schichor C,Masserdotti G,Ortega F,Heinrich C,Gascón S,Khan MA,Lie DC,Dellavalle A,Cossu G,Goldbrunner R,G?tz M,Berninger B(2012)Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells.Cell Stem Cell 11:471-476.
Kawamura T,Suzuki J,Wang YV,Menendez S,Morera LB,Raya A,Wahl GM,Izpisúa Belmonte JC(2009)Linking the p53 tumour suppressor pathway to somatic cell reprogramming.Nature460:1140-1144.
Kim SM,Kim JW,Kwak TH,Park SW,Kim KP,Park H,Lim KT,Kang K,Kim J,Yang JH,Han H,Lee I,Hyun JK,Bae YM,Scholer HR,Lee HT,Han DW(2016)Generation of integration-free induced neural stem cells from mouse fibroblasts.J Biol Chem291:14199-14212.
Krichevsky AM,Sonntag KC,Isacson O,Kosik KS(2006)Specific microRNAs modulate embryonic stem cell-derived neurogenesis.Stem Cells 24:857-864.
Liu ML,Zang T,Zou Y,Chang JC,Gibson JR,Huber KM,Zhang CL(2013)Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons.Nat Commun 4:2183.
Liu Y,Miao Q,Yuan J,Han S,Zhang P,Li S,Rao Z,Zhao W,Ye Q,Geng J,Zhang X,Cheng L(2015)Ascl1 converts dorsal midbrain astrocytes into functional neurons in vivo.J Neurosci 35:9336-9355.
Lombardo A,Cesana D,Genovese P,Di Stefano B,Provasi E,Colombo DF,Neri M,Magnani Z,Cantore A,Lo Riso P,Damo M,Pello OM,Holmes MC,Gregory PD,Gritti A,Broccoli V,Bonini C,Naldini L(2011)Site-specific integration and tailoring of cassette design for sustainable gene transfer.Nat Methods 8:861-869.
Nanbo A,Sugden A,Sugden B(2007)The coupling of synthesis and partitioning of EBV’s plasmid replicon is revealed in live cells.EMBO J 26:4252-4262.
Nienhuis AW,Dunbar CE,Sorrentino BP(2006)Genotoxicity of retroviral integration in hematopoietic cells.Mol Ther 13:1031-1049.
Niwa H,Yamamura K,Miyazaki J(1991)Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene108:193-199.
Okita K,Nakagawa M,Hyenjong H,Ichisaka T,Yamanaka S(2008)Generation of mouse induced pluripotent stem cells without viral vectors.Science 322:949-953.
Okita K,Yamakawa T,Matsumura Y,Sato Y,Amano N,Watanabe A,Goshima N,Yamanaka S(2013)An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.Stem Cells 31:458-466.
Pang ZP,Yang N,Vierbuchen T,Ostermeier A,Fuentes DR,Yang TQ,Citri A,Sebastiano V,Marro S,Südhof TC,Wernig M(2011)Induction of human neuronal cells by defined transcription factors.Nature 476:220-223.
Pfisterer U,Kirkeby A,Torper O,Wood J,Nelander J,Dufour A,Bj?rklund A,Lindvall O,Jakobsson J,Parmar M(2011)Direct conversion of human fibroblasts to dopaminergic neurons.Proc Natl Acad Sci U S A 108:10343-10348.
Son EY,Ichida JK,Wainger BJ,Toma JS,Rafuse VF,Woolf CJ,Eggan K(2011)Conversion of mouse and human fibroblasts into functional spinal motor neurons.Cell Stem Cell 9:205-218.
Vierbuchen T,Ostermeier A,Pang ZP,Kokubu Y,Sudhof TC,Wernig M(2010)Direct conversion of fibroblasts to functional neurons by defined factors.Nature 463:1035-1041.
Xu J,Du Y,Deng H(2015)Direct lineage reprogramming:strategies,mechanisms,and applications.Cell Stem Cell 16:119-134.
Zhang Y,Pak C,Han Y,Ahlenius H,Zhang Z,Chanda S,Marro S,Patzke C,Acuna C,Covy J,Xu W,Yang N,Danko T,Chen L,Wernig M,Sudhof TC(2013)Rapid single-step induction of functional neurons from human pluripotent stem cells.Neuron78:785-798.
Caiazzo M,Dell’Anno MT,Dvoretskova E,Lazarevic D,Taverna S,Leo D,Sotnikova TD,Menegon A,Roncaglia P,Colciago G,Russo G,Carninci P,Pezzoli G,Gainetdinov RR,Gustincich S,Dityatev A,Broccoli V(2011)Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.Nature 476:224-227.
Capetian P,Azmitia L,Pauly MG,Krajka V,Stengel F,Bernhardi EM,Klett M,Meier B,Seibler P,Stanslowsky N,Moser A,Knopp A,Gillessen-Kaesbach G,Nikkhah G,Wegner F,Dobrossy M,Klein C(2016)Plasmid-based generation of induced neural stem cells from adult human fibroblasts.Front Cell Neurosci 10:245.
Chanda S,Ang CE,Davila J,Pak C,Mall M,Lee QY,Ahlenius H,Jung SW,Sudhof TC,Wernig M(2014)Generation of induced neuronal cells by the single reprogramming factor ASCL1.Stem Cell Reports 3:282-296.
Cheng LC,Pastrana E,Tavazoie M,Doetsch F(2009)miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche.Nat Neurosci 12:399-408.
Dell’Anno MT,Caiazzo M,Leo D,Dvoretskova E,Medrihan L,Colasante G,Giannelli S,Theka I,Russo G,Mus L,Pezzoli G,Gainetdinov RR,Benfenati F,Taverna S,Dityatev A,Broccoli V(2014)Remote control of induced dopaminergic neurons in parkinsonian rats.J Clin Invest 124:3215-3229.
Fiszbein A,Schor IE,Kornblihtt AR(2015)Chapter 11-Fundamentals of NCAM expression,function,and regulation of alternative splicing in neuronal differentiation.Neural Surf Antigens 112:131-140.
Hong H,Takahashi K,Ichisaka T,Aoi T,Kanagawa O,Nakagawa M,Okita K,Yamanaka S(2009)Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.Nature 460:1132-1135.
Jiang H,Xu Z,Zhong P,Ren Y,Liang G,Schilling HA,Hu Z,Zhang Y,Wang X,Chen S,Yan Z,Feng J(2015)Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons.Nat Commun 6:10100.
Karow M,Sánchez R,Schichor C,Masserdotti G,Ortega F,Heinrich C,Gascón S,Khan MA,Lie DC,Dellavalle A,Cossu G,Goldbrunner R,G?tz M,Berninger B(2012)Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells.Cell Stem Cell 11:471-476.
Kawamura T,Suzuki J,Wang YV,Menendez S,Morera LB,Raya A,Wahl GM,Izpisúa Belmonte JC(2009)Linking the p53 tumour suppressor pathway to somatic cell reprogramming.Nature460:1140-1144.
Kim SM,Kim JW,Kwak TH,Park SW,Kim KP,Park H,Lim KT,Kang K,Kim J,Yang JH,Han H,Lee I,Hyun JK,Bae YM,Scholer HR,Lee HT,Han DW(2016)Generation of integration-free induced neural stem cells from mouse fibroblasts.J Biol Chem291:14199-14212.
Krichevsky AM,Sonntag KC,Isacson O,Kosik KS(2006)Specific microRNAs modulate embryonic stem cell-derived neurogenesis.Stem Cells 24:857-864.
Liu ML,Zang T,Zou Y,Chang JC,Gibson JR,Huber KM,Zhang CL(2013)Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons.Nat Commun 4:2183.
Liu Y,Miao Q,Yuan J,Han S,Zhang P,Li S,Rao Z,Zhao W,Ye Q,Geng J,Zhang X,Cheng L(2015)Ascl1 converts dorsal midbrain astrocytes into functional neurons in vivo.J Neurosci 35:9336-9355.
Lombardo A,Cesana D,Genovese P,Di Stefano B,Provasi E,Colombo DF,Neri M,Magnani Z,Cantore A,Lo Riso P,Damo M,Pello OM,Holmes MC,Gregory PD,Gritti A,Broccoli V,Bonini C,Naldini L(2011)Site-specific integration and tailoring of cassette design for sustainable gene transfer.Nat Methods 8:861-869.
Nanbo A,Sugden A,Sugden B(2007)The coupling of synthesis and partitioning of EBV’s plasmid replicon is revealed in live cells.EMBO J 26:4252-4262.
Nienhuis AW,Dunbar CE,Sorrentino BP(2006)Genotoxicity of retroviral integration in hematopoietic cells.Mol Ther 13:1031-1049.
Niwa H,Yamamura K,Miyazaki J(1991)Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene108:193-199.
Okita K,Nakagawa M,Hyenjong H,Ichisaka T,Yamanaka S(2008)Generation of mouse induced pluripotent stem cells without viral vectors.Science 322:949-953.
Okita K,Yamakawa T,Matsumura Y,Sato Y,Amano N,Watanabe A,Goshima N,Yamanaka S(2013)An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.Stem Cells 31:458-466.
Pang ZP,Yang N,Vierbuchen T,Ostermeier A,Fuentes DR,Yang TQ,Citri A,Sebastiano V,Marro S,Südhof TC,Wernig M(2011)Induction of human neuronal cells by defined transcription factors.Nature 476:220-223.
Pfisterer U,Kirkeby A,Torper O,Wood J,Nelander J,Dufour A,Bj?rklund A,Lindvall O,Jakobsson J,Parmar M(2011)Direct conversion of human fibroblasts to dopaminergic neurons.Proc Natl Acad Sci U S A 108:10343-10348.
Son EY,Ichida JK,Wainger BJ,Toma JS,Rafuse VF,Woolf CJ,Eggan K(2011)Conversion of mouse and human fibroblasts into functional spinal motor neurons.Cell Stem Cell 9:205-218.
Vierbuchen T,Ostermeier A,Pang ZP,Kokubu Y,Sudhof TC,Wernig M(2010)Direct conversion of fibroblasts to functional neurons by defined factors.Nature 463:1035-1041.
Xu J,Du Y,Deng H(2015)Direct lineage reprogramming:strategies,mechanisms,and applications.Cell Stem Cell 16:119-134.
Zhang Y,Pak C,Han Y,Ahlenius H,Zhang Z,Chanda S,Marro S,Patzke C,Acuna C,Covy J,Xu W,Yang N,Danko T,Chen L,Wernig M,Sudhof TC(2013)Rapid single-step induction of functional neurons from human pluripotent stem cells.Neuron78:785-798.