Research Article Efficient Reprogramming of Mouse ...downloads.hindawi.com/journals/tswj/2014/957548.pdf · Direct Conversion of Fibroblasts to Pan-Neuronal Cells. Since we conrmed

Research ArticleEfficient Reprogramming of Mouse Fibroblasts to NeuronalCells including Dopaminergic Neurons

Seung-ick Oh123 Hang-soo Park12 Insik Hwang123 Han-kyul Park12 Kyung-Ah Choi1

Hyesun Jeong123 Suhng Wook Kim2 and Sunghoi Hong123

1 Laboratory of Stem Cell Biology School of Biosystem and Biomedical Science College of Health Science Korea UniversityJeongneung-dong Seongbuk-gu Seoul 136-703 Republic of Korea

2Department of Biomedical Science College of Health Science Korea University Jeongneung-dong Seongbuk-guSeoul 136-703 Republic of Korea

3 Department of Health Science College of Health Science Korea University Jeongneung-dong Seongbuk-guSeoul 136-703 Republic of Korea

Correspondence should be addressed to Sunghoi Hong shong21koreaackr

Received 14 January 2014 Accepted 8 May 2014 Published 1 June 2014

Academic Editor Enoch Y Park

Copyright copy 2014 Seung-ick Oh et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Somatic cells were directly converted to functional neurons through the use of a combination of transcription factors includingAscl1 Brn2 and Myt1l However a major limitation is the lack of a reliable source of cell-replacement therapy for neurologicaldiseases Here we show that a combination of the transcription factors Ascl1 and Nurr1 (AN) and neurotrophic factors includingSHH and FGF8b directly reprogrammed embryonic mouse fibroblasts to induced neuronal (iN) cells pan-neuronal cells anddopaminergic (DA) neurons under our systematic cell culture conditions Reprogrammed cells showed the morphologicalproperties of neuronal cells Additionally cells were analyzed using variousmarkers including Tuj1 andMap2 for neuronal cells andLmx1a Th Aadc and Vmat2 for DA neurons in our immunostaining and reverse transcription (RT)-PCR experiments We foundthat a combination of transcription factors and neurotrophic factors could directly reprogramfibroblasts to neuronal cells includingDA neurons Various types of reprogrammed cells are promising cell sources for cell-based therapy of neurological disorders likeParkinsonrsquos disease and spinal cord injury

1 Introduction

Cellular reprogramming by which somatic cells can beconverted to induced pluripotent stem cells (iPSCs) andsubsequently differentiated into mature cells is a break-through for disease modeling and cell-based therapy [1ndash4] However major limitations such as low reprogrammingefficiency and lengthy procedures restrict the use of iPSCs[2 5ndash7] Moreover clinical applications require subsequentredifferentiation into a specific cell type and undifferentiatediPSCsmay become tumorigenic by incomplete differentiationof iPSCs Recently it was shown that combined expressionof defined factors could convert somatic cells into othersomatic cell types such as brown fat [8] cardiomyocytes [9]hepatocyte-like cells [10 11] hematopoietic progenitors [12]neural progenitors or neural precursor cells [13] neural stem

cells [14 15] glutamatergic neurons or GABAergic neurons[16] motor neurons [17] and neurons or dopaminergic(DA) neurons [18 19] Reprogrammed cells that do not passthrough the pluripotent state may not be tumorigenic andmay serve as a potential alternative to iPSCs for generatingpatient- andor disease-specific neurons However publishedreprogramming protocols involve different combinations ofvarious transcription factors to convert iPSCs into othermature cell types making it difficult to generate a desired celltype

Here we showed that mouse embryonic fibroblasts couldbe directly reprogrammed into pan-neurons andDAneuronsusing a combination of the Ascl1 and Nurr1 transcriptionfactors and various neurotrophic factors under our systematiccell culture conditions However our approach should befurther optimized for use as a cell source for cell-based

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 957548 8 pageshttpdxdoiorg1011552014957548

2 The Scientific World Journal

therapy to treat neurological disorders such as Parkinsonrsquosdisease

2 Materials and Methods

21 Cell Culture MEFs were isolated and cultured asdescribed previously [18] from embryonic day (E) 145wild-type BALBc mice embryos Mouse experiments wereapproved by the Institutional Animal Care and Use Com-mittee of Korea University (KUIACUC-2012-111) and wereperformed in accordance with government and institutionalguideline and regulations Briefly MEFs were expanded upto passage 2 in an MEF medium consisting of DMEM con-taining 10 FBS 1 NEAA and 1 penicillinstreptomycin(all from Gibco Grand Island NY USA) at 37∘C 5 CO

2in

95 humidity At passage number 2 theMEF phenotype wasconfirmed by immunocytochemical analysis with a positivemarker (vimentin) and negative markers (Sox1 Nestin orTuj1)

22 Retroviral Vectors Construction Production and Titra-tion Human Nurr1 cDNAs were amplified with primers foreach gene using high-fidelity cloned Pfu DNA polymerase(Stratagene La Jolla CA USA) and subcloned into theEcoRV site of the vector pUC19 Retroviral vectors expressingNurr1 were constructed by inserting the respective cDNAderived from pUC19 into the monocistronic retroviral vectorpCL Retroviral vectors expressing Ascl1 were gifts fromCH Park (Department of Microbiology Hanyang UniversitySeoul Korea) Retroviral vectors were introduced into theretrovirus packaging cell line 293 GPG by transient trans-fection with Lipofectamine 2000 (Invitrogen Carlsbad CAUSA) Supernatants were respectively collected each dayfor two weeks and stored in a deep freezer at minus80∘C [20]Mouse embryonic fibroblasts (MEFs) (5 times 104 cellswell) weretransduced with viruses at multiplicity of infection (MOI) of10 in the presence of polybrene (2 120583gmL) for 18 h at passage3 At 2 days after transduction the transduced cells wereseeded onto Cellstart-coated (Invitrogen) 24-well (5 times 104cellswell) or 6-well (25 times 105 cellswell) plates and werecultured for 1 day in Dulbeccorsquos modified Eagle medium(DMEM) containing 10 fetal bovine serum (FBS) and1 penicillinstreptomycin The next day the medium wasreplaced with DMEMF12-based neuronal medium (NM)supplemented with 1 ITS 1 N2 supplement 1 B27 1ascorbic acid and 1 penicillinstreptomycin The mediumwas replaced every other day

23 Neuronal Precursor Cells To induce neuronal precursor(NP) cells the MEF medium was replaced with NM con-taining 20 ngmL bFGF and 20 ngmL EGF (RampD SystemsMinneapolis MN USA) at 3 days after transduction MEFcells were cultured for an additional 17 days and NP-like cellswere analyzed by immunofluorescence and semiquantitativereverse transcription (RT)-RCR

24 Neurons To induce neurons the medium was replacedand cultured with NM containing 20 ngmL bFGF and

20 ngmL EGF (RampD Systems) for 12 days at 3 days aftertransduction The NM was changed to bFGF-EGF-free NMfor a further 16 days Neuron-like cells were analyzed byimmunofluorescence and semiquantitative RT-PCR

25 DA Neurons To induce DA neurons the medium wasreplaced with NM containing 20 ngmL bFGF and 20 ngmLEGF at 3 days after transduction for 3 days Next 200 ngmLShh and 100 ngmL FGF8b (RampD Systems) were added to themedium and the cells were cultured for an additional 9 daysThe medium was changed with NM supplemented with onlyboth Shh (200 ngmL) and FGF8b (100 ngmL) and then cellswere cultured for an additional 10 days DA neuron-like cellswere induced bywithdrawing both Shh and FGF8b fromNMOver the next 25 days reprogrammed cells began to changeto neuron-like cell morphology DA neuron-like cells wereanalyzed by immunofluorescence and semiquantitative RT-PCR

26 Immunofluorescence Assay Cells were fixed in 4paraformaldehyde and were stained with the following pri-mary antibodies rabbit 120572-rat Nurr1 (sc-991 1 200 SantaCruz Biotechnology Santa Cruz CA USA) goat 120572-humanAsc1 (sc-13219 1 200 Santa Cruz Biotechnology) mouse 120572-human vimentin (V5255 1 200 Sigma St Louis MO USA)rabbit 120572-mouse Sox1 (ab22572 1 200 Abcam CambridgeUK) mouse 120572-human Nestin (MAB5326 1 200 MilliporeBillerica MA USA) rabbit 120572-rat Tuj1 (MRB-435p 1 1000Covance Princeton NJ USA) mouse 120572-human MAP2(M2320 1 200 Sigma St Louis MO USA) rabbit 120572-ratTH (AB152 1 200 Millipore) Appropriate Alexa Fluor 488or 594 F (ab1015840)2 fragment of goat 120572-rabbit or 120572-mouse IgG(H+L) antibodies or Alexa Fluor 555 goat 120572-rabbit IgG(H+L) or Alexa Fluor 594 F (ab1015840)2 fragment of rabbit 120572-goatIgG (H+L) (A-11070 A-11020 or A-21428 A-21223 1 2000Invitrogen) and DAPI (1 120583gmL) counterstain were used forvisualization Fluorescence images were obtained using flu-orescence microscopy (Evos Amgmicro Life TechnologiesCarlsbad CA USA) and confocal laser scanning microscopy(Fluoview100 Olympus Tokyo Japan)

27 RT-PCR Total RNA from cells of in vitro differentiationwas prepared using Trizol Reagent (Invitrogen) followed bytreatment with DNase I (Ambion Austin TX USA) Two120583g of total RNA was reverse-transcribed into cDNA usingoligo (dT) primers according to the SuperScript III ReverseTranscriptase Kit (Invitrogen) The cDNA was then ana-lyzed by semiquantitative RT-PCR using the neuronal geneprimers Relative expression of mRNAs was assessed by nor-malizing levels of cDNA to the signal from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA RT-PCR reac-tions were carried out on an ABI2720 Thermal Cycler(Applied Biosystems Foster City CA USA) in final volumesof 30 120583L containing 1x reaction buffer (50mM KCl 2mMMgCl

2 and 25mM TAPS pH 93) 02mM dNTP 05 120583M

of each primer and 125 units of TaKaRa Ex Taq (TaKaRaBiotechnology Co Ltd Shiga Japan) Primer sequencesannealing temperatures cycle numbers and amplicon size

The Scientific World Journal 3

Vimentin DAPI DAPI

DAPI DAPI

VimentinDAPI Nestin NestinDAPI

Sox1 Sox1DAPI Tuj1 Tuj1DAPI

(a)

Vimentin NestinSox1 Tuj1

100

80

60

40

20

0

Posit

ive

DA

PI (

)

ND

lowast

(b)

Figure 1 Isolation characterization and conversion into neural lineage ofMEF cells ((a) (b))MEF cells did not express the neuronalmarkersbut highly expressed the fibroblast marker Magnification times200 scale bar is 50 120583m Single asterisk is 119875 lt 001

are shown in Supplementary Table 1 available online athttpdxdoiorg1011552014957548

28 Statistical Analysis Data was analyzed by using SPSS 120software All results were evaluated by a two-tailed Studentrsquospaired 119905-test and are shown between group mean plusmn SD from3 independent experiments 119875 lt 001 (lowast) was consideredstatistically significant

3 Results

31 Reprogramming of MEF Cells into Neuronal and GlialCells by Ascl1 and Nurr1 For the direct conversion of somaticcells into neuronal lineage cells we first prepared mouseembryonic fibroblasts (MEFs) by removing spinal cord partsfrom themouse fetus on embryonic day 145 (E145)Then wecultured the MEF in a Petri dish and checked the cells with

immunostaining using anti-vimentin antibody as a fibroblastmarker or anti-Nestin anti-Sox1 and anti-Tuj1 antibodiesas neural and pan-neuronal markers respectively We con-firmed that our cultured MEF cells were uniformly positiveagainst anti-vimentin but were negative against anti-Nestin -Sox1 and -Tuj1 antibodies (Figures 1(a) and 1(b)) Next MEFcells were infected with retroviral vectors containing Ascl1and Nurr1 and then cultured for 25 to 30 days in neuronalmedium (NM) which contained DMEMF12 culture mediasupplemented with insulintransferrinselenium (ITS) N2B27 and ascorbic acid (AA)

32 Induction of Neural Precursor (NP) Cells from FibroblastsSince the reprogramming potentials of MEF cells to neuronaland glial cells were demonstrated in our experiments weexamined whether MEF cells could be converted into neuralprecursor (NP) cells Isolated MEF cells were maintained


ANSplit

minus3 minus1 0NP induction

12NP expansion

17

Days

NM + bFGFEGF

(a)

0 DIV 7 DIV 10 DIV 14 DIV 17 DIV

(b)

DAPISox1 Sox1DAPI

(c)

DAPINestin NestinDAPI

(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

25

20

15

10

5

0

25

20

15

10

5

0

NestinSox1

lowast

lowast

NDND

(e)

NI AN

Nestin

Sox1

Msi1

Gapdh

(f)

Figure 2 Direct conversion of MEF cells into neural precursor (NP) cells (a) A schematic diagram for the generation of MEF-derivedNP cells (b) Representative phase-contrast images Magnification times200 scale bar is 100 120583m ((c) (d)) Representative immunofluorescenceimage of differentiated MEF cells stained with Nestin and Sox1 antibodies Yellow arrows on the above micrograph indicate Sox1-stainedcells Magnification times200 scale bar is 50 120583m (e) Reprogramming efficiency Sox1-positive (red bar) and Nestin-positive (green bar) cells perDAPI-positive cells Single asterisk is 119875 lt 001 (f) mRNA expression of neural precursor marker genes Nestin Sox1 and Msi1 by RT-PCR

in serum-containing medium and were infected with theretroviral vectors containing Ascl1 and Nurr1 Three daysafter infection infected cells were transferred onto a coateddish for 1 day and then cultured in NM containing 20 ngmLbFGF and 20 ngmL EGF for more than 17 days (Figure 2(a))Interestingly morphological changes began to appear at 7days after induction and the morphology of NP cells wasclear by approximately 14 to 17 days after induction with cellsshowing a large nucleus narrow cytoplasm and round shape(Figure 2(b)) At 21 days after induction potential NP cellswere analyzed for neural precursormarkers Sox1 Nestin andMusasi1 NP-like cells were clearly immunoreactive againstSox1 and Nestin antibodies in the nucleus and cytoplasmrespectively (Figures 2(c) and 2(d)) Sox1- andNestin-positivecells accounted for approximately 1682 plusmn 317 and 1968 plusmn351out of total cells respectively (Figure 2(e)) In additionRT-PCR experiments showed that NP-like cells showeddistinct expression of the neural precursor marker genes

Nestin Sox1 and Musasi1 (Figure 2(f)) Next we examinedwhether NP-like cells could directly differentiate into neu-ronal and glial cells For differentiation NP-like cells werecultured for an additional 14 days inNMmediawithout bFGFand EGF However NP-like cells were not differentiated intoeither neurons or glial cells These results indicate that theAscl1 and Nurr1 factors directly reprogram the somatic cellsinto neuronal cells without a NP-like cell stage while NP-likecells appeared to be maintained for a period of time

33 Direct Conversion of Fibroblasts to Pan-Neuronal CellsSince we confirmed that MEF cells were converted intoneuronal cells in our culture system as occurred in Wernigrsquosgroup [18] we examined whether MEFs could be directlyconverted into neurons MEF cells that were transducedwith retroviral vectors containing Ascl1 and Nurr1 wereinduced to neuronal cells in NM containing 20 ngmL bFGF


ANNMSplit

minus3 minus1 0 NP induction 12 Differentiation 28

Days

NM + bFGFEGF

(a)


(b)

Tuj1 DAPI Tuj1 DAPI

(c)

MAP2 DAPI MAP2 DAPI

(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

50

10

20

30

40

60

0

lowast lowast

MAP2Tuj1

50

10

20

30

40

60

0ND ND

(e)

Map2

Tuj1

Gapdh

NI AN

(f)

Figure 3 Direct conversion ofMEF cells into neurons (a) A schematic diagramof the generation ofMEF-derived neurons (b) Representativephase-contrast images of neuron-like cells showing bipolar projections Magnification times200 scale bar is 100 120583m ((c) (d)) Representativeimmunofluorescence image of differentiated MEF cells stained with Tuj1 and MAP2 antibodies Magnification times200 scale bar is 50 120583m (e)Reprogramming efficiency of theMEF cells into neuronal cells Tuj1-positive (green bar) andMAP2-positive (red bar) cells per DAPI-positivecells Single asterisk is 119875 lt 001 (f) mRNA expression of neuronal cell marker genes Tuj1 and Map2 by RT-PCR

and 20 ngmL EGF for 12 days Next differentiation oftransduced cells was induced by withdrawing bFGF and EGFfor a further 16 days or more (Figure 3(a)) The cells showeda neuronal cell type morphology exhibiting neurite-likeoutgrowth Neurites were extensively grown and branchedout to form neural connections with other cells as shown ina typical neuronal cell type at days 25 to 27 (Figure 3(b))Reprogrammed cells were stained with the pan-neuronalmarker Tuj1 and the mature neuronal marker MAP2 Tuj1-positive cells were approximately 51 plusmn 302 and MAP2-positive cells were approximately 46 plusmn 401 of total cellsby our immunofluorescence assay (Figures 3(c) and 3(d))In addition the neuronal genes Tuj1 and MAP2 were clearlyexpressed in the reprogrammed cells as shown in our RT-PCR experiments (Figure 3(e)) These results indicate thatthe MEF cells were successfully and efficiently converted toneuronal cell type in our culture systems using Ascl1 andNurr1 factors

34 Direct Conversion of Fibroblasts to DA Neurons We nextexamined whether MEF cells could be directly convertedto DA neurons by Ascl1 and Nurr1 MEF cells were trans-duced with retroviral vectors containing Ascl1 and Nurr1Transduced cells were cultured in NM containing 20 ngmLbFGF and 20 ngmL EGF for 3 days At 3 days after neuralinduction 200 ngmL Shh and 100 ngmL FGF8b were addedto NM for 9 days and the cells were cultured in NMcontaining 200 ngmL Shh and 100 ngmL FGF8b in theabsence of bFGF and EGF DA neurons were induced for9 days by withdrawing Shh and FGF8b (Figure 4(a)) Cellswere morphologically changed to neuron-like cells whichwere positive against tyrosine hydroxylase (TH) and Tuj1antibodies (Figure 4(b)) The efficiency of TH+ cells outof Tuj1+ cells was approximately 33 plusmn 062 (Figure 4(c))Expression of Lmx1a Aadc Vmat2 Th and Tuj1 genes wasclearly detected by our RT-PCR experiments (Figure 4(d))These results indicate that the conversion of MEF cells into


AN

NMSplit

minus3 minus1 0 Neural DA neuroninduction IIinduction IDA neuron

induction22123

Differentiation

NMNM

NMShhFGF8b

31

Days

+ bFGFEGF+ bFGFEGF

+ ShhFGF8b

(a)

MergeTuj1DAPI THDAPI

(b)

NI

TH

AN

Posit

ive

Tuj1

()

lowast

10

20

30

40

0ND

(c)

NI AN

Lmx1a

Aadc

Vmat2

Th

Tuj1

Gapdh

(d)

Figure 4 Direct conversion of MEF cells into dopaminergic (DA) neurons (a) Schematic diagram of the generation of MEF-derived DAneurons (b) Representative immunofluorescence of the DA neurons stained with Tuj1 and TH antibodies A yellow arrow indicates DAneurons Magnification times200 scale bar is 50 120583m (I II and III) Higher magnification insert of B (c) Reprogramming efficiency of the MEFcells into DA neuron-like cells Single asterisk is 119875 lt 001 (d) mRNA expression of various DA neuronal marker genes by RT-PCR

a DA neuronal cell type can be successfully achieved underour systematic cell culture conditions suggesting that MEFcells could be directly reprogrammed into DA neuronal cellsby Ascl1 and Nurr1

4 Discussion

Neuronal and glial cells including neural precursor cells area promising cell source of cell-based therapy for neurologicaldisorders [21] It is also possible to directly generate adesired cell type from either skin or other somatic cells [8ndash19 22 23] In this study we demonstrated that a definedset of transcription factors Ascl1 and Nurr1 can directlyconvert embryonic mouse fibroblasts into neuronal cells

including DA neurons using our established culture systemssupplemented with various neurotrophic factors

A major concern when using embryonic fibroblasts forconversion is that contamination of neural precursors orglial and neuronal cells may be present in the startingmaterials To exclude this possibility we confirmed thatthere were only fibroblasts in our cell cultures by using thefibroblastmarker vimentin and various other neuralmarkersincluding Sox1 Nestin and Tuj1 We detected no Nestin-or Sox1- or Tuj1-positive cells (Figure 1(a)) suggesting nocontamination of neural cells in our fibroblast populationWhen we first directly converted the fibroblasts into neuralprecursors by using Ascl1 and Nurr1 transcription factorsreprogrammed cells were positive against neural markersand clearly expressed neural marker genes compared to


fibroblasts as shown in Figure 2 However NP cells didnot proliferate during the cell expansion step These resultssuggest that NP cells might be directly generated from MEFcells but they seem to be maintained only for a shortperiod of time during induction of MEFs to NP cells Ourreprogramming protocol should be further optimized forclinical applications

In addition we also directly reprogrammed fibroblaststo Tuj1-positive neurons using Ascl1 and Nurr1 in our cellculture conditions supplemented with various cytokinesInterestingly we found that fibroblasts were directly con-verted into neurons with high reprogramming efficiencywhich was approximately 510 plusmn 302 suggesting thatour reprogramming protocols can be used to generate acell source of cell-based therapy for neurological disordersMoreover we directly reprogrammed fibroblasts into TH-positive DA neurons using two Ascl1 and Nurr1 factors inour cell culture systems supplemented with SHH and FGF8bThe reprogramming efficiency of TH+ cells was relativelyhigh which was approximately 33 plusmn 062 These resultssuggest that the reprogramming protocol for generatingreprogrammed DA neurons may be used as an alternativecell source of cell-based therapy for clinical application ofParkinsonrsquos disease in the future

Nurr1 is a member of the nuclear receptor family regulat-ing genes that are involved in the induction andmaintenanceof DA neurons [24ndash27] Recently Caiazzo et al convertedmouse and human fibroblasts into functional DA neuronsusing the 3 factors Ascl1 Nurr1 and Lmx1a with relativelyhigh efficiency (sim18) [28]They also showed that the factorsAscl1 and Nurr1 directly reprogrammed fibroblasts into DAneuronal cells with low reprogramming efficiency (sim8)In addition neurotrophic factors such as SHH and FGF8have been known to be critical for the specification anddevelopment of a neuronal subtype [29 30] Recently Castroet al reported that Ascl1 can directly control the specificationof neural progenitors as well as the later steps of neuronaldifferentiation andneurite outgrowth in the embryonic brainand in neural stem cell cultures [31] These reports supportthe fact that Ascl1 andNurr1 are sufficient for reprogrammingsomatic cells into neuronal cells under our culture condi-tions using various neurotrophic factors Importantly ourapproach prevents development of tumorigenicity of ESCsor iPSCs when they were transplanted in an undifferentiatedstate Therefore reprogramming of pan-neuronal and DAneuronal cells can be used for cell-based therapy to treatneurological disorders such as Parkinsonrsquos disease and mayalso be invaluable formechanistic studies and drug screening

5 Conclusions

This study demonstrates that mouse embryonic fibroblastswere directly reprogrammed into pan-neurons and DA neu-rons using a combination of theAscl1 andNurr1 transcriptionfactors and various neurotrophic factors under our systematiccell culture conditions In particular the reprogrammingefficiency of pan-neuronal cells (Tuj1+) and DA neurons(TH+) was approximately 510 plusmn 302 and 33 plusmn 062

respectively indicating relatively high However it should befurther studiedwhether the reprogrammed neuronal cells arefunctional

Conflict of Interests

All authors declare that they have no actual or potentialconflict of interests including financial personal or otherrelationships with other people or organizations that couldinappropriately influence or be perceived to influence thiswork

Acknowledgments

This work was supported by Grants from the Ministryof Science ICT and Future Planning (2012M3A9C605013120100023160 and 20100020349) and the Ministry of Healthand Welfare (A110606 and A120392) of the Korean govern-ment

References

[1] K Takahashi and S Yamanaka ldquoInduction of pluripotent stemcells from mouse embryonic and adult fibroblast cultures bydefined factorsrdquo Cell vol 126 no 4 pp 663ndash676 2006

[2] K Takahashi K Okita M Nakagawa and S YamanakaldquoInduction of pluripotent stem cells from fibroblast culturesrdquoNature Protocols vol 2 no 12 pp 3081ndash3089 2007

[3] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007

[4] I H Park R Zhao J AWest et al ldquoReprogramming of humansomatic cells to pluripotency with defined factorsrdquo Nature vol451 no 7175 pp 141ndash146 2008

[5] F Soldner D Hockemeyer C Beard et al ldquoParkinsonrsquos diseasepatient-derived induced pluripotent stem cells free of viralreprogramming factorsrdquo Cell vol 136 no 5 pp 964ndash977 2009

[6] F Jia K D Wilson N Sun et al ldquoA nonviral minicircle vectorfor deriving human iPS cellsrdquo Nature Methods vol 7 no 3 pp197ndash199 2010

[7] D Kim C H Kim J I Moon et al ldquoGeneration of humaninduced pluripotent stem cells by direct delivery of reprogram-ming proteinsrdquo Cell Stem Cell vol 4 no 6 pp 472ndash476 2009

[8] S Kajimura P Seale K Kubota et al ldquoInitiation of myoblastto brown fat switch by a PRDM16-CEBP-120573 transcriptionalcomplexrdquo Nature vol 460 no 7259 pp 1154ndash1158 2009

[9] M Ieda J Fu P Delgado-Olguin et al ldquoDirect reprogrammingof fibroblasts into functional cardiomyocytes by defined fac-torsrdquo Cell vol 142 no 3 pp 375ndash386 2010

[10] P Huang Z He S Ji et al ldquoInduction of functional hepatocyte-like cells frommouse fibroblasts by defined factorsrdquoNature vol475 no 7356 pp 386ndash389 2011

[11] S Sekiya andA Suzuki ldquoDirect conversion ofmouse fibroblaststo hepatocyte-like cells by defined factorsrdquo Nature vol 475 no7356 pp 390ndash393 2011

[12] E Szabo S Rampalli R M Risueno et al ldquoDirect conversionof humanfibroblasts tomultilineage blood progenitorsrdquoNaturevol 468 no 7323 pp 521ndash526 2010

[13] J Kim J A Efe S Zhu et al ldquoDirect reprogramming of mousefibroblasts to neural progenitorsrdquo Proceedings of the National


Academy of Sciences of the United States of America vol 108 no19 pp 7838ndash7843 2011

[14] DWHan N Tapia A Hermann et al ldquoDirect reprogrammingof fibroblasts into neural stem cells by defined factorsrdquoCell StemCell vol 10 no 4 pp 465ndash472 2012

[15] Y Xue K Ouyang J Huang et al ldquoDirect conversion of fibrob-lasts to neurons by reprogramming PTB-regulated MicroRNAcircuitsrdquo Cell vol 152 no 1-2 pp 82ndash96 2013

[16] A S Yoo A X Sun L Li et al ldquoMicroRNA-mediatedconversion of human fibroblasts to neuronsrdquo Nature vol 476no 7359 pp 228ndash231 2011

[17] E Y Son J K Ichida B J Wainger et al ldquoConversion of mouseand human fibroblasts into functional spinal motor neuronsrdquoCell Stem Cell vol 9 no 3 pp 205ndash218 2011

[18] T Vierbuchen A Ostermeier Z P Pang Y Kokubu T CSudhof and M Wernig ldquoDirect conversion of fibroblasts tofunctional neurons by defined factorsrdquo Nature vol 463 no7284 pp 1035ndash1041 2010

[19] U Pfisterer A Kirkeby O Torper et al ldquoDirect conversion ofhuman fibroblasts to dopaminergic neuronsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 25 pp 10343ndash10348 2011

[20] S Hong D Y Hwang S Yoon et al ldquoFunctional analysis ofvarious promoters in lentiviral vectors at different stages of invitro differentiation of mouse embryonic stem cellsrdquoMolecularTherapy vol 15 no 9 pp 1630ndash1639 2007

[21] O Lindvall and Z Kokaia ldquoStem cells in human neurode-generative disordersmdashtime for clinical translationrdquo Journal ofClinical Investigation vol 120 no 1 pp 29ndash40 2010

[22] E Lujan S Chanda H Ahlenius T C Sudhof andMWernigldquoDirect conversion of mouse fibroblasts to self-renewing tripo-tent neural precursor cellsrdquoProceedings of theNational Academyof Sciences of the United States of America vol 109 no 7 pp2527ndash2532 2012

[23] Z P Pang N Yang T Vierbuchen et al ldquoInduction of humanneuronal cells by defined transcription factorsrdquoNature vol 476no 7359 pp 220ndash223 2011

[24] J H Kim J M Auerbach J A Rodrıguez-Gomez et alldquoDopamine neurons derived from embryonic stem cells func-tion in an animalmodel of Parkinsonrsquos diseaserdquoNature vol 418no 6893 pp 50ndash56 2002

[25] D W Kim S Chung M Hwang et al ldquoStromal cell-derivedinducing activity Nurr1 and signalingmolecules synergisticallyinduce dopaminergic neurons from mouse embryonic stemcellsrdquo Stem Cells vol 24 no 3 pp 557ndash567 2006

[26] B Kadkhodaei T Ito E Joodmardi et al ldquoNurr1 is requiredfor maintenance of maturing and adult midbrain dopamineneuronsrdquo Journal of Neuroscience vol 29 no 50 pp 15923ndash15932 2009

[27] B Kadkhodaei A Alvarsson N Schintu et al ldquoTranscriptionfactor Nurr1 maintains fiber integrity and nuclear-encodedmitochondrial gene expression in dopamine neuronsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 110 no 6 pp 2360ndash2365 2013

[28] M Caiazzo M T DellrsquoAnno E Dvoretskova et al ldquoDirectgeneration of functional dopaminergic neurons from mouseand human fibroblastsrdquo Nature vol 476 no 7359 pp 224ndash2272011

[29] S H Lee N Lumelsky L Studer J M Auerbach and R DMcKay ldquoEfficient generation of midbrain and hindbrain neu-rons from mouse embryonic stem cellsrdquo Nature Biotechnologyvol 18 no 6 pp 675ndash679 2000

[30] N Narita M Kato M Tazoe K Miyazaki M Narita andN Okado ldquoIncreased monoamine concentration in the brainand blood of fetal thalidomide- and valproic acid-exposed ratputative animal models for autismrdquo Pediatric Research vol 52no 4 pp 576ndash579 2002

[31] D S Castro B Martynoga C Parras et al ldquoA novel function ofthe proneural factor Ascl1 in progenitor proliferation identifiedby genome-wide characterization of its targetsrdquo Genes andDevelopment vol 25 no 9 pp 930ndash945 2011

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therapy to treat neurological disorders such as Parkinsonrsquosdisease

2 Materials and Methods

21 Cell Culture MEFs were isolated and cultured asdescribed previously [18] from embryonic day (E) 145wild-type BALBc mice embryos Mouse experiments wereapproved by the Institutional Animal Care and Use Com-mittee of Korea University (KUIACUC-2012-111) and wereperformed in accordance with government and institutionalguideline and regulations Briefly MEFs were expanded upto passage 2 in an MEF medium consisting of DMEM con-taining 10 FBS 1 NEAA and 1 penicillinstreptomycin(all from Gibco Grand Island NY USA) at 37∘C 5 CO

2in

95 humidity At passage number 2 theMEF phenotype wasconfirmed by immunocytochemical analysis with a positivemarker (vimentin) and negative markers (Sox1 Nestin orTuj1)

22 Retroviral Vectors Construction Production and Titra-tion Human Nurr1 cDNAs were amplified with primers foreach gene using high-fidelity cloned Pfu DNA polymerase(Stratagene La Jolla CA USA) and subcloned into theEcoRV site of the vector pUC19 Retroviral vectors expressingNurr1 were constructed by inserting the respective cDNAderived from pUC19 into the monocistronic retroviral vectorpCL Retroviral vectors expressing Ascl1 were gifts fromCH Park (Department of Microbiology Hanyang UniversitySeoul Korea) Retroviral vectors were introduced into theretrovirus packaging cell line 293 GPG by transient trans-fection with Lipofectamine 2000 (Invitrogen Carlsbad CAUSA) Supernatants were respectively collected each dayfor two weeks and stored in a deep freezer at minus80∘C [20]Mouse embryonic fibroblasts (MEFs) (5 times 104 cellswell) weretransduced with viruses at multiplicity of infection (MOI) of10 in the presence of polybrene (2 120583gmL) for 18 h at passage3 At 2 days after transduction the transduced cells wereseeded onto Cellstart-coated (Invitrogen) 24-well (5 times 104cellswell) or 6-well (25 times 105 cellswell) plates and werecultured for 1 day in Dulbeccorsquos modified Eagle medium(DMEM) containing 10 fetal bovine serum (FBS) and1 penicillinstreptomycin The next day the medium wasreplaced with DMEMF12-based neuronal medium (NM)supplemented with 1 ITS 1 N2 supplement 1 B27 1ascorbic acid and 1 penicillinstreptomycin The mediumwas replaced every other day

23 Neuronal Precursor Cells To induce neuronal precursor(NP) cells the MEF medium was replaced with NM con-taining 20 ngmL bFGF and 20 ngmL EGF (RampD SystemsMinneapolis MN USA) at 3 days after transduction MEFcells were cultured for an additional 17 days and NP-like cellswere analyzed by immunofluorescence and semiquantitativereverse transcription (RT)-RCR

24 Neurons To induce neurons the medium was replacedand cultured with NM containing 20 ngmL bFGF and

20 ngmL EGF (RampD Systems) for 12 days at 3 days aftertransduction The NM was changed to bFGF-EGF-free NMfor a further 16 days Neuron-like cells were analyzed byimmunofluorescence and semiquantitative RT-PCR

25 DA Neurons To induce DA neurons the medium wasreplaced with NM containing 20 ngmL bFGF and 20 ngmLEGF at 3 days after transduction for 3 days Next 200 ngmLShh and 100 ngmL FGF8b (RampD Systems) were added to themedium and the cells were cultured for an additional 9 daysThe medium was changed with NM supplemented with onlyboth Shh (200 ngmL) and FGF8b (100 ngmL) and then cellswere cultured for an additional 10 days DA neuron-like cellswere induced bywithdrawing both Shh and FGF8b fromNMOver the next 25 days reprogrammed cells began to changeto neuron-like cell morphology DA neuron-like cells wereanalyzed by immunofluorescence and semiquantitative RT-PCR

26 Immunofluorescence Assay Cells were fixed in 4paraformaldehyde and were stained with the following pri-mary antibodies rabbit 120572-rat Nurr1 (sc-991 1 200 SantaCruz Biotechnology Santa Cruz CA USA) goat 120572-humanAsc1 (sc-13219 1 200 Santa Cruz Biotechnology) mouse 120572-human vimentin (V5255 1 200 Sigma St Louis MO USA)rabbit 120572-mouse Sox1 (ab22572 1 200 Abcam CambridgeUK) mouse 120572-human Nestin (MAB5326 1 200 MilliporeBillerica MA USA) rabbit 120572-rat Tuj1 (MRB-435p 1 1000Covance Princeton NJ USA) mouse 120572-human MAP2(M2320 1 200 Sigma St Louis MO USA) rabbit 120572-ratTH (AB152 1 200 Millipore) Appropriate Alexa Fluor 488or 594 F (ab1015840)2 fragment of goat 120572-rabbit or 120572-mouse IgG(H+L) antibodies or Alexa Fluor 555 goat 120572-rabbit IgG(H+L) or Alexa Fluor 594 F (ab1015840)2 fragment of rabbit 120572-goatIgG (H+L) (A-11070 A-11020 or A-21428 A-21223 1 2000Invitrogen) and DAPI (1 120583gmL) counterstain were used forvisualization Fluorescence images were obtained using flu-orescence microscopy (Evos Amgmicro Life TechnologiesCarlsbad CA USA) and confocal laser scanning microscopy(Fluoview100 Olympus Tokyo Japan)

27 RT-PCR Total RNA from cells of in vitro differentiationwas prepared using Trizol Reagent (Invitrogen) followed bytreatment with DNase I (Ambion Austin TX USA) Two120583g of total RNA was reverse-transcribed into cDNA usingoligo (dT) primers according to the SuperScript III ReverseTranscriptase Kit (Invitrogen) The cDNA was then ana-lyzed by semiquantitative RT-PCR using the neuronal geneprimers Relative expression of mRNAs was assessed by nor-malizing levels of cDNA to the signal from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA RT-PCR reac-tions were carried out on an ABI2720 Thermal Cycler(Applied Biosystems Foster City CA USA) in final volumesof 30 120583L containing 1x reaction buffer (50mM KCl 2mMMgCl

2 and 25mM TAPS pH 93) 02mM dNTP 05 120583M

of each primer and 125 units of TaKaRa Ex Taq (TaKaRaBiotechnology Co Ltd Shiga Japan) Primer sequencesannealing temperatures cycle numbers and amplicon size


Vimentin DAPI DAPI

DAPI DAPI



(a)


100

80

60

40

20

0

Posit

ive

DA

PI (

)

ND

lowast

(b)




3 Results





ANSplit


12NP expansion

17

Days

NM + bFGFEGF

(a)


(b)

DAPISox1 Sox1DAPI

(c)


(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

25

20

15

10

5

0

25

20

15

10

5

0

NestinSox1

lowast

lowast

NDND

(e)

NI AN

Nestin

Sox1

Msi1

Gapdh

(f)






ANNMSplit


Days

NM + bFGFEGF

(a)


(b)

Tuj1 DAPI Tuj1 DAPI

(c)

MAP2 DAPI MAP2 DAPI

(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

50

10

20

30

40

60

0

lowast lowast

MAP2Tuj1

50

10

20

30

40

60

0ND ND

(e)

Map2

Tuj1

Gapdh

NI AN

(f)





AN

NMSplit


induction22123

Differentiation

NMNM

NMShhFGF8b

31

Days

+ bFGFEGF+ bFGFEGF

+ ShhFGF8b

(a)


(b)

NI

TH

AN

Posit

ive

Tuj1

()

lowast

10

20

30

40

0ND

(c)

NI AN

Lmx1a

Aadc

Vmat2

Th

Tuj1

Gapdh

(d)



4 Discussion








5 Conclusions





Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Vimentin DAPI DAPI

DAPI DAPI



(a)


100

80

60

40

20

0

Posit

ive

DA

PI (

)

ND

lowast

(b)




3 Results





ANSplit


12NP expansion

17

Days

NM + bFGFEGF

(a)


(b)

DAPISox1 Sox1DAPI

(c)


(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

25

20

15

10

5

0

25

20

15

10

5

0

NestinSox1

lowast

lowast

NDND

(e)

NI AN

Nestin

Sox1

Msi1

Gapdh

(f)






ANNMSplit


Days

NM + bFGFEGF

(a)


(b)

Tuj1 DAPI Tuj1 DAPI

(c)

MAP2 DAPI MAP2 DAPI

(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

50

10

20

30

40

60

0

lowast lowast

MAP2Tuj1

50

10

20

30

40

60

0ND ND

(e)

Map2

Tuj1

Gapdh

NI AN

(f)





AN

NMSplit


induction22123

Differentiation

NMNM

NMShhFGF8b

31

Days

+ bFGFEGF+ bFGFEGF

+ ShhFGF8b

(a)


(b)

NI

TH

AN

Posit

ive

Tuj1

()

lowast

10

20

30

40

0ND

(c)

NI AN

Lmx1a

Aadc

Vmat2

Th

Tuj1

Gapdh

(d)



4 Discussion








5 Conclusions





Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ANSplit


12NP expansion

17

Days

NM + bFGFEGF

(a)


(b)

DAPISox1 Sox1DAPI

(c)


(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

25

20

15

10

5

0

25

20

15

10

5

0

NestinSox1

lowast

lowast

NDND

(e)

NI AN

Nestin

Sox1

Msi1

Gapdh

(f)






ANNMSplit


Days

NM + bFGFEGF

(a)


(b)

Tuj1 DAPI Tuj1 DAPI

(c)

MAP2 DAPI MAP2 DAPI

(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

50

10

20

30

40

60

0

lowast lowast

MAP2Tuj1

50

10

20

30

40

60

0ND ND

(e)

Map2

Tuj1

Gapdh

NI AN

(f)





AN

NMSplit


induction22123

Differentiation

NMNM

NMShhFGF8b

31

Days

+ bFGFEGF+ bFGFEGF

+ ShhFGF8b

(a)


(b)

NI

TH

AN

Posit

ive

Tuj1

()

lowast

10

20

30

40

0ND

(c)

NI AN

Lmx1a

Aadc

Vmat2

Th

Tuj1

Gapdh

(d)



4 Discussion








5 Conclusions





Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ANNMSplit


Days

NM + bFGFEGF

(a)


(b)

Tuj1 DAPI Tuj1 DAPI

(c)

MAP2 DAPI MAP2 DAPI

(d)

NI AN NI AN

Posit

ive

DA

PI (

)

Posit

ive

DA

PI (

)

50

10

20

30

40

60

0

lowast lowast

MAP2Tuj1

50

10

20

30

40

60

0ND ND

(e)

Map2

Tuj1

Gapdh

NI AN

(f)





AN

NMSplit


induction22123

Differentiation

NMNM

NMShhFGF8b

31

Days

+ bFGFEGF+ bFGFEGF

+ ShhFGF8b

(a)


(b)

NI

TH

AN

Posit

ive

Tuj1

()

lowast

10

20

30

40

0ND

(c)

NI AN

Lmx1a

Aadc

Vmat2

Th

Tuj1

Gapdh

(d)



4 Discussion








5 Conclusions





Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AN

NMSplit


induction22123

Differentiation

NMNM

NMShhFGF8b

31

Days

+ bFGFEGF+ bFGFEGF

+ ShhFGF8b

(a)


(b)

NI

TH

AN

Posit

ive

Tuj1

()

lowast

10

20

30

40

0ND

(c)

NI AN

Lmx1a

Aadc

Vmat2

Th

Tuj1

Gapdh

(d)



4 Discussion








5 Conclusions





Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusions





Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Efficient Reprogramming of Mouse ...downloads.hindawi.com/journals/tswj/2014/957548.pdf · Direct Conversion of Fibroblasts to Pan-Neuronal Cells. Since we conrmed