Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors
© Zhou et al.; licensee BioMed Central Ltd. 2014
Received: 12 December 2013
Accepted: 28 March 2014
Published: 3 April 2014
The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets.
Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state.
Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.
KeywordsCommon marmoset Direct reprogramming Induced neuronal cells Transcription factor Regenerative medicine Disease modeling Cell-fate plasticity Transdifferentiation
The common marmoset (Callithrix jacchus) is a New World primate that has recently attracted considerable attention as a non-human primate model for biomedical research . Specific features of the common marmoset are its small size, ease of handling, high fertility, early sexual maturity, its similarity of physiological properties with humans, drug metabolism, and neurophysiological functions . Thus far, transgenic mice modeling human neurodegenerative diseases have contributed to disease research and drug development. However, none of them have succeeded in faithfully recapitulating the full spectrum of disease pathologies observed in humans [2, 3]. In a recent report by our group, transgenic marmosets with germline transmission were successfully generated for the first time by lentiviral vector-mediated gene transfer . For these reasons, our novel transgenic non-human primate models may be suitable for studying human diseases, particularly those that are neurodegenerative, such as Alzheimer’s and Parkinson’s disease. These in vivo models are expected to faithfully recapitulate pathophysiology of human diseases, and thus provide for the missing link between mouse and human disease research with subsequent drug development. However, results of studies from these models may require an extended period because of the longer lifespan of common marmosets compared with mice . Moreover, detailed in vitro analyses using primary neuronal cultures of the affected area of the common marmoset transgenic models are not realistic.
Recent studies using human neuronal cells derived from either pluripotent stem cells or somatic cells have succeeded in modeling human neurological disorders in vitro[6, 7]. These results prompted us to develop a convenient and rapid method for obtaining common marmoset neuronal cells from accessible somatic cells. Therefore, we focused on somatic cell reprogramming technology, including induced pluripotent stem (iPS) cell and direct conversion technology [8, 9]. However, few studies have succeeded in generating common marmoset iPS cells from neonatal skin fibroblasts, fetal liver cells, and adult bone marrow-derived cells [10–12]. Moreover, only a few protocols exist for obtaining functional common marmoset neuronal cells from pluripotent stem cells . Furthermore, little attention has been given to the direct conversion technology of common marmoset dermal fibroblasts into neuronal cells thus far. Therefore, in the present study, we aimed to generate common marmoset neuronal cells directly from dermal fibroblasts. Our results provide the first line of evidence for the generation of electrophysiologically functional neuronal cells from common marmoset somatic cells by defined neuronal transcription factors.
Results and discussion
Validation of the lentivirus-mediated overexpression of neuronal transcription factors
Recently, generation of induced neuronal (iN) cells was reported using mouse and human dermal cells [9, 14]. In the present study, we used a set of neuronal transcription factors for human iN cells on common marmoset embryonic skin fibroblasts (cjF) isolated from embryonic day 91 (E91) embryos to determine whether common marmoset somatic cells could be converted into neuronal cells. We first confirmed transgene expression in the mouse fibroblast cell line, NIH3T3, by infecting these cells with lentiviral vectors coding the neuronal transcription factors: ASCL1, BRN2, MYT1L, and NEUROD1, under the control of tetracycline response element (TRE) together with reverse tetracycline transactivator (rtTA)-expressing vector. Upregulation of transgenes in NIH3T3 cells after doxycycline (dox) treatment was confirmed by immunocytochemistry (Additional file 1).
Generation of neuron-like cells from common marmoset somatic cells
The duration of dox treatment was critical for neuronal conversion
Next, we investigated the required duration of exogenous neuronal transcription factor expression to drive neuronal transdifferentiation. Therefore, we examined the exposure time of dox and the cjiN induction efficiency (Figure 2D). Treatment with dox from 1–3 div promoted the conversion of cjFs into synapsin reporter-positive cells with neuronal morphology (Figure 2E). Similarly, a previous study has shown that the endogenous neuronal transcriptional factor network is activated 48 h after dox treatment . Our findings also revealed that the induction efficiency at 23 div depended on the exposure time of dox (Figure 2F). The treatment of cjFs with dox from 1–11 div followed by a culture without dox treatment from 11–23 div generated 112.1 ± 28.1 cjiN cells/cm2 (Figure 2F). However, cells cultured with dox from 1–3 div followed by a culture without dox treatment from 3–23 div generated only 10.6 ± 5.4 cjiN cells/cm2 (Figure 2F). The efficiency of the former group was significantly (P*** < 0.001, n = 4) higher (more than 10-fold) compared with the latter group (Figure 2F). These results indicate that a longer expression of exogenous neuronal transcriptional factors is required for efficient lineage conversion of somatic cells.
cjiN cells expressed a subset of neuronal genes and proteins
Primer sets used in RT-PCR analysis
Product size (bp)
The majority of cjiN cells were glutamatergic
To characterize the neurotransmitter phenotype of iN cells, we examined the expression of neurotransmitters in MAP2-positive cjiN cells. Immunostaining at 38 div for vesicular glutamate transporter 1 (vGlut1) and gamma aminobutyric acid (GABA) revealed the presence of both excitatory glutamatergic and inhibitory GABAergic neuronal cells, respectively (Figure 4B). Counts of vGlut1 and GABA-positive cjiN cells showed a significantly (P*** < 0.001, n = 4) greater number of vGlut1-positive cells (39.2 ± 1.9/cm2) than GABA-positive cells (16.4 ± 2.8 cells/cm2) (Figure 4C). These results are in accordance with previous studies showing that the majority of iN cells are excitatory cells [7, 14, 22]. Our findings thus indicate that iN induction may be feasible in the future for the in vitro analysis of the transgenic common marmoset model of Alzheimer’s disease, in which forebrain excitatory neurons are expected to be affected. Moreover, our cjiN cell induction protocol is likely advantageous over the previously reported neuronal differentiation protocol which used common marmoset embryonic stem (ES) cells and iPS cells, because the ES/iPS cell-derived neural precursor cells showed caudal identity . Thus far, several groups have succeeded in generating reprogrammed neuronal cells with specific neuronal subtypes, such as dopaminergic neurons and motor neurons [23–26], which implicate the cell fate plasticity of terminally differentiated somatic cells. Our success in reprogramming common marmoset somatic cells into excitatory and inhibitory neuronal cells using defined iN factors may therefore provide great promise in the future for generating specific subtypes of neuronal cells with specific sets of neuronal transcription factors.
cjiN cells were functional as matured neurons
cjiN cells were directly converted from cjFs without passing through a proliferative state
In the present study, we established an in vitro method to convert common marmoset somatic cells into functional neuronal (i.e. cjiN) cells. The majority of the cjiN cells were vGlut1-positive excitatory neuronal cells and expressed the neuronal marker genes: TUBB3, DCX, MAP2, SYN1, VGLUT1, SCN1A, GRIN1 and GRIA1. Importantly, cjF-derived cjiN cells exhibited functional neuronal properties and responded to exogenous stimulation. Overall, these findings suggest that direct conversion technology may be beneficial in rapid and robust screening of neuronal phenotypes of transgenic common marmoset models of human diseases and analyzing underlying molecular mechanisms of diseases.
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Central Institute for Experimental Animals (CIEA), and was performed in accordance with CIEA and Keio University guidelines.
Common marmoset embryonic fibroblasts, NIH3T3 cells and 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin (10% FP medium) at 37°C with 5% CO2 incubation.
Molecular cloning and lentivirus production
cDNA entry clone of human ASCL1 [GenBank: NM_004316] was purchased from DNAFORM (clone ID: 100006383, Japan). cDNAs of human BRN2 [GenBank: NM_005604.3], MYT1L [GenBank: NM_015025.2] and NEUROD1 [GenBank: NM_002500.4] were cloned into pENTR-D-TOPO vector (Invitrogen, USA). Then cDNAs were inserted into a self-inactivation human immunodeficiency virus-1-based lentivirus construct, CSIV-TRE-RfA (CSIV-TRE-RfA-CMV-KT was kindly provided by Dr. Hiroyuki Miyoshi (RIKEN BRC, Japan), and then modified by Dr. Takuji Maeda (Nagoya University, Japan)), by LR reaction (Invitrogen, USA). Similarly, reverse tetracycline transactivator (rtTA) gene was inserted into CSII-EF1α-RfA-TK-HygR construct . The human synapsin I reporter constructs, pCSC-hSynI-GFP  and pHIV7-hSynI-DsRed , were kindly provided by Dr. Fred H. Gage, Salk Institute, USA, and Dr. Alysson R. Muotri, University of California, USA, respectively. Besides, CSIV-hSynI-GFP-IRES2-NeoR and CSIV-hSynI-DsRed-IRES2-NeoR were constructed in-house. These reporters were constructed using CSIV-TRE-RfA-CMV-KT, pCSC-hSynI-GFP, pHIV7-hSynI-DsRed and pIRESneo3 (Clontech, USA) with PCR- and restriction enzyme-based method. For lentivirus production, 293T cells were transfected with lentivirus plasmid, pCAG-HIVgp and pCMV-VSV-G-RSV-Rev  (kindly provided by Dr. Hiroyuki Miyoshi, RIKEN BRC, Japan). After 16-20 h, supernatant was replaced by fresh media followed by 48-72 h incubation. The virus containing media were then collected and 0.45 μm-filterated followed by ultracentrifugation. The concentrated virus was suspended in PBS and used in subsequent experiments.
Induction of common marmoset iN cells
Common marmoset embryonic fibroblasts were seeded directly on culture ware at 1 × 104 cells/cm2. Twenty-four hours later, the cells were infected with lentivirus in 10% FP media containing polybrene (8 μg/mL) (Sigma-Aldrich, USA). After 16–20 h in media containing lentivirus, the cells were switched into fresh 10% FP medium containing doxycycline (dox) (2 μg/mL) to drive transgene expression. Procedures for experiments determining the sufficient concentration and duration of dox are shown in the main text. After 48 h in 10% FP media with dox, the media was replaced with dox-containing neural media composed of N2B27 media , brain-derived neurotrophic factor (BDNF) (10 ng/mL, R&D systems, USA) and neurotrophin-3 (NT-3) (10 ng/mL, R&D systems, USA). For calcium imaging, 8-(4-Chlorophenylthio) adenosine 3′, 5′-cyclic monophosphate (8-CPT, 100 μM, Sigma-Aldrich, USA), one of the cAMP analogs , was also supplemented to promote neuronal maturation. The media was changed every 2–3 days during culture period. BrdU incorporation assay was performed as previously described  incubating cells with BrdU (10 μΜ, BD, USA) until cells were fixed.
Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then processed for immunocytochemistry . Samples were rinsed with PBS three times. Then, samples were incubated at 4°C overnight with the primary antibodies diluted in PBS containing 5% of fetal bovine serum and 0.3% Triton X-100. The primary antibodies used were as follows; Synaptophysin (1:50000, Millipore, USA), MAP2 (1:1000, Sigma-Aldrich, USA), MAP2 (1:500, Millipore, USA), vGlut1 (1:2000, Synaptic systems, Germany), GABA (1:1000, Sigma-Aldrich, USA), PSD95 (1:500, Millipore, USA), α-SMA (1:500, Sigma-Aldrich, USA), Sox2 (1:500, R&D, USA), Ascl1 (1:200, BD, USA), Brn2 (1:200, Santa Cruz, USA), Myt1L (1:500, Abcam, England) and NeuroD1 (1:500, Santa Cruz, USA). After three washes with PBS, samples were incubated with secondary antibodies conjugated with Alexa-488, Alexa-555 and Alexa-647 (Invitrogen, USA). Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI; 1:1000, Dojindo, Japan). After washing with PBS, samples were mounted on slides with FluorSave reagent (Calbiochem, Germany) and examined under a universal fluorescence microscope (Axioplan 2; Carl Zeiss, Germany). For anti-BrdU staining (1:500, Abcam, England), cells were treated with 1 N HCl in PBS for 30 minutes at 37°C and then rinsed with PBS three times before primary antibody incubation.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated with RNeasy Micro Kit with DNase I treatment (QIAGEN, Germany) and was used to synthesize cDNA with ReverTraAce qPCR RT Kit (TOYOBO, Japan) according to the manufacturer’s instruction. RT-PCR was conducted using Ex Taq HS (TAKARA, Japan) according to the manufacturer’s instruction. Common marmoset ES (cjES) cells and cjES-derived neurons were used as control . The primer sets used are listed in Table 1.
Calcium imaging and electrical stimulation
Calcium imaging analyses were performed as described previously . To load the calcium imaging dye, cells were incubated with 1 μM Fluo-4 AM (Invitrogen, USA) in imaging solution consisting of 117 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 25 mM HEPES and 30 mM D-(+)-glucose, (pH 7.4), at 37°C for 20 minutes, followed by washing for 30 minutes in imaging solution. Coverslips were placed on a custom-made field stimulation chamber and mounted on the stage of a Nikon Eclipse microscope with a 20× (NA 0.45) objective. Cells were perfused at 2 ml/minute with the imaging solution at room temperature with or without 0.2 μM tetrodotoxin (TTX; Alomone Labs Ltd., Israel). Images were acquired at 2 Hz (500 millisecond exposure time) with a cooled CCD camera (Andor iXon, DU897). Extracellular field stimulation was performed with two parallel platinum wires at 25 V/cm. Each stimulation was a train of 500 microsecond pulses at 40 Hz for 5 seconds. Images were analyzed with ImageJ software (NIH, Bethesda, MD).
Electrophysiological recordings were performed as described previously . Synapsin reporter-positive cjiN cells were identified under an inverted microscope (Diaphot-TMD 200; Nikon, Japan) and whole cell patch clamp recordings were done using Axopatch 200B (Axon Instruments, USA) at room temperature. The extracellular solution composed of 117 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 15 mM D-Glucose and 20 mM HEPES (pH 7.4 adjusted with NaOH, 304 mOsm) was continuously perfused during recordings. Patch pipettes had a resistance of 5-6 MΩ filled with the intracellular solution containing 130 mM K-gluconate, 1 mM CaCl2, 1 mM MgCl2, 10 mM EGTA, 10 mM Sucrose and 20 mM HEPES (pH adjusted with KOH, 305 mOsm). In voltage-clamp recordings, iN cells were held at -70 mV and voltage steps (10 mV, 300 msec) were applied to elicit voltage-activated currents. Action potentials were evoked by injecting step currents (20-40 pA, 500 msec) in the current-clamp mode. Data were digitized at 10 kHz with a 2 kHz low-pass filter. Liquid junction potential was corrected.
All data were expressed as means ± SEM. The statistical significance of differences was analyzed by Student’s t-test, Mann-Whitney U-test or one-way ANOVA followed by Tukey’s test using Graph Pad Prism5 software. Differences of P < 0.05 were considered statistically significant.
- cjiN cells:
Common marmoset induced neuronal cells
Common marmoset embryonic fibroblast
Reverse transcription-polymerase chain reaction
Reverse tetracycline transactivator.
We are grateful to memebers of Okano laboratory, Keio Unversity school of medicine for helpful advice and discussions. Dr. Hiroyuki Miyoshi, RIKEN BRC, Japan, kindly provided us with lentiviral vectors. Dr. Takuji Maeda, Nagoya University, Japan, kindly provided us with modified lentiviral vector. Dr. Fred H. Gage, Salk Institute, USA, and Dr. Alysson R. Muotri, University of California, USA, kindly provided us with synapsin reporter lentiviral vectors. This work is supported in part by a Grants-in Aid to Keio University from the Global COE Program of the Ministry of education, Culture, Sports, Science and Technology, Japan, Keio University Grant-in-Aid for Encouragement of Young Medical Scientists, and Funding Program for World-leading Innovative R&D on Science and Technology (FIRST) program of the Cabinet Office, Government of Japan and the Japan Society for the Promotion of Science (JSPS).
- Mansfield K: Marmoset models commonly used in biomedical research. Comp Med. 2003, 53: 383-392.PubMedGoogle Scholar
- Gotz J, Ittner LM: Animal models of Alzheimer’s disease and frontotemporal dementia. Nat Rev Neurosci. 2008, 9: 532-544. 10.1038/nrn2420.PubMedView ArticleGoogle Scholar
- Lim K-L, Ng C-H: Genetic models of Parkinson disease. Biochim Biophys Acta. 2009, 1792: 604-615. 10.1016/j.bbadis.2008.10.005.PubMedView ArticleGoogle Scholar
- Sasaki E, Suemizu H, Shimada A, Hanazawa K, Oiwa R, Kamioka M, Tomioka I, Sotomaru Y, Hirakawa R, Eto T, Shiozawa S, Maeda T, Ito M, Ito R, Kito C, Yagihashi C, Kawai K, Miyoshi H, Tanioka Y, Tamaoki N, Habu S, Okano H, Nomura T: Generation of transgenic non-human primates with germline transmission. Nature. 2009, 459: 523-527. 10.1038/nature08090.PubMedView ArticleGoogle Scholar
- Nishijima K, Saitoh R, Tanaka S, Ohsato-Suzuki M, Ohno T, Kitajima S: Life span of common marmoset (Callithrix jacchus) at CLEA Japan breeding colony. Biogerontology. 2012, 13: 439-443. 10.1007/s10522-012-9388-1.PubMedView ArticleGoogle Scholar
- Imaizumi Y, Okada Y, Akamatsu W, Koike M, Kuzumaki N, Hayakawa H, Nihira T, Kobayashi T, Ohyama M, Sato S, Takanashi M, Funayama M, Hirayama A, Soga T, Hishiki T, Suematsu M, Yagi T, Ito D, Kosakai A, Hayashi K, Shouji M, Nakanishi A, Suzuki N, Mizuno Y, Mizushima N, Amagai M, Uchiyama Y, Mochizuki H, Hattori N, Okano H: Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue. Mol Brain. 2012, 5: 35-10.1186/1756-6606-5-35.PubMedPubMed CentralView ArticleGoogle Scholar
- Qiang L, Fujita R, Yamashita T, Angulo S, Rhinn H, Rhee D, Doege C, Chau L, Aubry L, Vanti WB, Moreno H, Abeliovich A: Directed Conversion of Alzheimer’s Disease Patient Skin Fibroblasts into Functional Neurons. Cell. 2011, 146: 359-371. 10.1016/j.cell.2011.07.007.PubMedPubMed CentralView ArticleGoogle Scholar
- Takahashi 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: 861-872. 10.1016/j.cell.2007.11.019.PubMedView ArticleGoogle Scholar
- Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M: Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010, 463: 1035-1041. 10.1038/nature08797.PubMedPubMed CentralView ArticleGoogle Scholar
- Wu Y, Zhang Y, Mishra A, Tardif SD, Hornsby PJ: Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts. Stem Cell Res. 2010, 4: 180-188. 10.1016/j.scr.2010.02.003.PubMedPubMed CentralView ArticleGoogle Scholar
- Tomioka I, Maeda T, Shimada H, Kawai K, Okada Y, Igarashi H, Oiwa R, Iwasaki T, Aoki M, Kimura T, Shiozawa S, Shinohara H, Suemizu H, Sasaki E, Okano H: Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28. Genes Cells. 2010, 15: 959-969. 10.1111/j.1365-2443.2010.01437.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Wiedemann A, Hemmer K, Bernemann I, Göhring G, Pogozhykh O, Figueiredo C, Glage S, Schambach A, Schwamborn JC, Blasczyk R, Müller T: Induced pluripotent stem cells generated from adult bone marrow-derived cells of the nonhuman primate (Callithrix jacchus) using a novel quad-cistronic and excisable lentiviral vector. Cell Reprogram. 2012, 14: 485-496.PubMedGoogle Scholar
- Shimada H, Okada Y, Ibata K, Ebise H, Ota S-I, Tomioka I, Nomura T, Maeda T, Kohda K, Yuzaki M, Sasaki E, Nakamura M, Okano H: Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells. PLoS One. 2012, 7: e49469-10.1371/journal.pone.0049469.PubMedPubMed CentralView ArticleGoogle Scholar
- Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang TQ, Citri A, Sebastiano V, Marro S, Südhof TC, Wernig M: Induction of human neuronal cells by defined transcription factors. Nature. 2011, 476: 220-223.PubMedPubMed CentralGoogle Scholar
- Adler AF, Grigsby CL, Kulangara K, Wang H, Yasuda R, Leong KW: Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells. Mol Ther Nucleic Acids. 2012, 1: e32-10.1038/mtna.2012.25.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim J-E, O'Sullivan ML, Sanchez CA, Hwang M, Israel MA, Brennand K, Deerinck TJ, Goldstein LSB, Gage FH, Ellisman MH, Ghosh A: Investigating synapse formation and function using human pluripotent stem cell-derived neurons. Proc Natl Acad Sci U S A. 2011, 108: 3005-3010. 10.1073/pnas.1007753108.PubMedPubMed CentralView ArticleGoogle Scholar
- Kucharova K, Hefferan MP, Patel P, Marsala S, Nejime T, Miyanohara A, Marsala M, Drummond JC: Transplantation of rat synapsin-EGFP-labeled embryonic neurons into the intact and ischemic CA1 hippocampal region: distribution, phenotype, and axodendritic sprouting. Cell Transplant. 2011, 20: 1163-1178. 10.3727/096368910X564544.PubMedView ArticleGoogle Scholar
- Marchetto MCN, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR: A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell. 2010, 143: 527-539. 10.1016/j.cell.2010.10.016.PubMedPubMed CentralView ArticleGoogle Scholar
- Yoo AS, Sun AX, Li L, Shcheglovitov A, Portmann T, Li Y, Lee-Messer C, Dolmetsch RE, Tsien RW, Crabtree GR: MicroRNA-mediated conversion of human fibroblasts to neurons. Nature. 2011, 476 (7359): 228-231. 10.1038/nature10323.PubMedPubMed CentralView ArticleGoogle Scholar
- Ladewig J, Mertens J, Kesavan J, Doerr J, Poppe D, Glaue F, Herms S, Wernet P, Kögler G, Müller F-J, Koch P, Brüstle O: Small molecules enable highly efficient neuronal conversion of human fibroblasts. Nat Methods. 2012, 9: 575-578. 10.1038/nmeth.1972.PubMedView ArticleGoogle Scholar
- Wapinski OL, Vierbuchen T, Qu K, Lee QY, Chanda S, Fuentes DR, Giresi PG, Ng YH, Marro S, Neff NF, Drechsel D, Martynoga B, Castro DS, Webb AE, Südhof TC, Brunet A, Guillemot F, Chang HY, Wernig M: Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell. 2013, 155: 621-635. 10.1016/j.cell.2013.09.028.PubMedView ArticleGoogle Scholar
- Yang N, Ng YH, Pang ZP, Südhof TC, Wernig M: Induced neuronal cells: how to make and define a neuron. Cell Stem Cell. 2011, 9: 517-525. 10.1016/j.stem.2011.11.015.PubMedPubMed CentralView ArticleGoogle Scholar
- Pfisterer 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 U S A. 2011, 108: 10343-10348. 10.1073/pnas.1105135108.PubMedPubMed CentralView ArticleGoogle Scholar
- 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: Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature. 2011, 476 (7359): 224-227. 10.1038/nature10284.PubMedView ArticleGoogle Scholar
- Kim J, Su SC, Wang H, Cheng AW, Cassady JP, Lodato MA, Lengner CJ, Chung C-Y, Dawlaty MM, Tsai L-H, Jaenisch R: Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell. 2011, 9 (5): 413-419. 10.1016/j.stem.2011.09.011.PubMedPubMed CentralView ArticleGoogle Scholar
- Son EY, Ichida JK, Wainger BJ, Toma JS, Rafuse VF, Woolf CJ, Eggan K: Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell. 2011, 9: 205-218. 10.1016/j.stem.2011.07.014.PubMedPubMed CentralView ArticleGoogle Scholar
- Naka H, Nakamura S, Shimazaki T, Okano H: Requirement for COUP-TFI and II in the temporal specification of neural stem cells in CNS development. Nat Neurosci. 2008, 11: 1014-1023. 10.1038/nn.2168.PubMedView ArticleGoogle Scholar
- Miyoshi H, Blömer U, Takahashi M, Gage FH, Verma IM: Development of a self-inactivating lentivirus vector. J Virol. 1998, 72: 8150-8157.PubMedPubMed CentralGoogle Scholar
- Matsui T, Takano M, Yoshida K, Ono S, Fujisaki C, Matsuzaki Y, Toyama Y, Nakamura M, Okano H, Akamatsu W: Neural stem cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency. Stem Cells. 2012, 30: 1109-1119. 10.1002/stem.1091.PubMedView ArticleGoogle Scholar
- Tsuji O, Miura K, Okada Y, Fujiyoshi K, Mukaino M, Nagoshi N, Kitamura K, Kumagai G, Nishino M, Tomisato S, Higashi H, Nagai T, Katoh H, Kohda K, Matsuzaki Y, Yuzaki M, Ikeda E, Toyama Y, Nakamura M, Yamanaka S, Okano H: Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury. Proc Natl Acad Sci U S A. 2010, 107: 12704-12709. 10.1073/pnas.0910106107.PubMedPubMed CentralView ArticleGoogle Scholar
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