Research Article Modeling Neurological Disease by Rapid

Research ArticleModeling Neurological Disease by Rapid Conversion of HumanUrine Cells into Functional Neurons

Shu-Zhen Zhang12 Li-Xiang Ma3 Wen-Jing Qian4 Hong-Fu Li2

Zhong-Feng Wang4 Hong-Xia Wang1 and Zhi-Ying Wu12

1Department of Neurology Institute of Neurology Huashan Hospital Institutes of Brain Science andState Key Laboratory of Medical Neurobiology Shanghai Medical College Fudan University Shanghai 200040 China2Department of Neurology Research Center of Neurology in Second Affiliated Hospitaland the Collaborative Innovation Center for Brain Science Zhejiang University School of Medicine Hangzhou 310009 China3Department of Anatomy Histology amp Embryology Shanghai Medical College Fudan University Shanghai 200030 China4Institutes of Brain Science Institute of Neurobiology and State Key Laboratory of Medical Neurobiology Fudan UniversityShanghai 200030 China

Correspondence should be addressed to Zhi-Ying Wu zhiyingwuzjueducn

Received 11 April 2015 Revised 18 July 2015 Accepted 22 July 2015

Academic Editor Gary E Lyons

Copyright copy 2016 Shu-Zhen Zhang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Somatic cells can be directly converted into functional neurons by ectopic expression of defined factors andor microRNAs Sincethe first report of conversion mouse embryonic fibroblasts into functional neurons the postnatal mouse and human fibroblastsastroglia hepatocytes and pericyte-derived cells have been converted into functional dopaminergic and motor neurons both invitro and in vivo However it is invasive to get all these materials In the current study we provide a noninvasive approach toobtain directly reprogrammed functional neurons by overexpression of the transcription factors Ascl1 Brn2 NeuroD c-Myc andMyt1l in human urine cells These induced neuronal (iN) cells could express multiple neuron-specific proteins and generate actionpotentials Moreover urine cells fromWilsonrsquos disease (WD) patient could also be directly converted into neurons In conclusiongeneration of iN cells from nonneural lineages is a feasible and befitting approach for neurological disease modeling

1 Introduction

Reprogramming techniques have been used to generateinduced pluripotent stem cells (iPSCs) from human fibrob-lasts [1] Patient-derived iPSCs differentiating into maturecells could be useful tools for diseasemodeling and cell-basedtherapy [2] Patients with age-related macular degenerationhave benefited from the iPSCs-derived retinal pigmentedepithelium cell treatment [3] However the lengthy proce-dures of the iPSCs induction and target cells differentiation[4] in addition to the tumorigenic properties [1] restrictedthe use of iPSCs

Direct reprogramming is another option to generatepatient-specific cells Reprogrammed cells do not passthrough the pluripotent state and the entire conversion pro-cedure takes less than twomonthsTherefore reprogrammed

cells may serve as potential alternative tools for diseasemodeling and cell-based therapy An important milestone inthe field of cell lineage conversions was the discovery thatthe single factorMyoDwas sufficient to convert fibroblasts tomyoblasts [5] Somatic cells have been successfully convertedinto myoblasts [6] chondrocytes [7] functional cardiomy-ocytes [8] functional hepatocyte-like cells [9] hepatic stemcells [10] multilineage blood progenitors [11] neural stemcells [12] functional neurons [13ndash15] even more specificneurons such as motor neurons [16] and dopaminergicneurons [17 18]

Themost commonly used somatic cells for direct conver-sion are fibroblasts [16] The alternative materials are brownfat cells [6] placenta [7] hepatocytes [14] pericyte-derivedcells [15] and astrocytes [18] However it is invasive to getthese cells Recently several groups generated human iPSCs

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 2452985 8 pageshttpdxdoiorg10115520162452985

2 Stem Cells International

[19 20] and neuron stem cells [21] from exfoliated renalepithelial cells present in urine providing a noninvasiveapproach to obtain reprogramming cells The isolation ofurinary cells is simple cost-effective and universal Howeverthe direct conversion of urine cells to functional neurons hasnot been reported so far

In the present study we showed that human urine cellscould be directly reprogrammed into functional neuronsusing a combination of Ascl1 Brn2 NeuroD c-Myc andMyt1l and various neurotrophic factors These induced neu-ronal (iN) cells expressed multiple neuron-specific proteinsand generated action potentials Generation of iN cells fromnonneural lineages could have important implications forneurological disease modeling and regenerative medicine

2 Materials and Methods

21 Culture and Verification of Human Urinary Cells Theurinary cells were collected fromhealthy individuals andWDpatients after obtaining informed consents The exfoliatedcells were then isolated using a previously established pro-tocol with brief modifications [19] Donors were asked todrink 300ndash500mL water before urine collection The vulvawas wiped with iodine to keep sterile Approximately 200mLaseptic middle stream of the micturition was collected intoa sterilized glass bottle The urines were centrifuged at1000 rpm for 15min at room temperature immediately aftercollection The upper liquid was discarded and 2mL renalepithelial cell growth medium (REGM) with growth factors(Lonza cat number CC-4127) was added to the bottomsediment The cell-like organisms were seeded on one well of24-well plate (Corning) at a density of about 05 lowast 106cm2Half of the medium was discarded and replaced by 1mL ofnew REGM medium per day for 3 successive days Smallclones normally appeared at day 5 after planting Usually oneto five small colonies were observed per well Cells exhibitedshort spindle morphologies when they reached confluenceThese cells were subcultured or frozen for further usage Thestudy protocolwas approved by the research ethics committeeof Huashan Hospital

22 Reprogramming of Urinary Cells Five retroviruses(Lenti-EF1120572-EGFP-TRE-ASCL1 Lenti-EF1120572-EGFP-TRE-BRN2 Lenti-EF1120572-EGFP-TRE-NEUROD Lenti-EF1120572-EGFP-TRE-MYT1L and Lenti-EF1120572-EGFP-TRE-C-MYC)were produced for the ectopic expression of human Ascl1Brn2 NeuroD c-Myc and Myt1l respectively Retrovirusescarrying these five transcriptional factors were produced inthe 293T cell line Viral supernatant was concentrated byAmicon Ultra Centrifugal Filters (Millipore) and filteredthrough 022120583m cellulose acetate filter before being addedto urine cells Primary monolayer astrocyte cultures wereestablished as described previously [22]

Twenty-four hours after the recovery the five retroviruseswere mixed at 1 1 1 1 1 and were added to 1 lowast 105 primaryurinary cells at multiplicity of infection (MOI) of 75ndash10 Themedium was changed to 1 1 mixture of EPi and N2 medium(EPiN2 DMEMF2 N2 and NEAA [Invitrogen]) supplied

with 1 120583gmLDOX for 2 days Cells were harvested and platedonto astrocyte coated cover slips (1 times 104cm2) in N2mediumsupplemented with DOX Y21732 (10120583M Tocris) cAMP(1 120583M) Vit-C (200120583M) 05 FBS (Gibco) and neurotrophicfactors including NT-3 BDNF GDNF and IGF (10 ngmLeach RampD systems)The culture medium was changed everyother day until used

23 RNA Isolation and Relative Quantitative PCR Total RNAwas extracted usingMiniBESTUniversal RNAExtraction Kit(TaKaRa) The reverse-transcription reaction was conductedusing the PrimeScript RT Master Mix (TaKaRa) Primersused are the same as those reported previously [19] SYBRPremix ExTaq (TaKaRa)was used for the relative quantitativePCR reaction

24 Electrophysiology Theelectrophysiology experiment wasperformed according to the previously described protocolwith somemodifications [23] Cells selected for electrophysi-ological recordings had neuron-like shapes with fine branch-ing nerve projections Action potentials were recorded withcurrent-clamp whole cell configuration The bath solutioncontained the following (in mM) 125 NaCl 25 KCl 25NaHCO

3 125 NaH

2PO4 2 CaCl 1 MgCl

2 and 25 glucose

The pipette solution contained the following (in mM) 123 K-gluconate 10 KCl 1 MgCl

2 10 HEPES 1 EGTA 01 CaCl

2

1 K2ATP 02 Na

4GTP and 4 glucose pH adjusted to 72 with

KOHMembrane potentials were kept aroundminus65 tominus70mVand step currents were injected to elicit action potentials Forwhole cell voltage-dependent current recordings the sameinternal solution as aforementioned was used

25 Immunofluorescence Cells were fixed in 4 parafor-maldehyde for 10min and blocked by PBS solution con-taining 5 donkey serum and 02 Triton X-100 for1 hour at room temperature Primary antibodies wereapplied overnight and secondary antibodies were appliedfor 2 hours The following antibodies were used rabbitanti-Tuj1 (Covance 1 1000) mouse anti-Tuj1 (Covance1 1000) mouse anti-MAP2 (Sigma 1 500) rabbit anti-synaptophysin (Chemicon 1 1000) rabbit anti-v-Glu (Mil-lipore 1 2000) and rabbit-anti-GABA (DSHB 1 500)Alexa-488- Alexa-594- and Cy5-conjugated secondary anti-bodies were obtained from Invitrogen 410158406-Diamidino-2-phenylindole (DAPI) was from Sigma (1 10000)

26 Efficiency Calculation The Tuj1-positive cells with thinprocesses at least three times longer than their cell bodieswere counted We selected 3 cover slips and averaged thenumber of iN cells We then divided this number by thenumber of cells plated before infection to get the percentageof the starting population of cells that adopted neuron-likecharacteristics

3 Results

To investigate whether human urinary cells could be directlyconverted into neurons we collected urine samples from the

Stem Cells International 3

L1CAME-CadherinOccludinClaudin 1NR3C2

SLC2A1Twist 1

L1CA

M

E-Ca

dher

in

Occ

ludi

n

Clau

din

1

NR3

C2

SLC2

A1

Twist

1

0000

0001

0002

0003

01020304

Relat

ive e

xpre

ssio

n of

120573-a

ctin

Figure 1 Cultured urine cells express renal epithelial and epithelialmarkers The culture urine cells express renal epithelial cell markersNR3C2 L1CAM and SLC2A1 at high level Epithelial cell markersOccludin Claudin 1 and E-cadherin were also expressed in thosecells The fibroblast marker Twist 1 was expressed at a very low level119873 = 7

volunteers with the informed consents These cells expressedrenal epithelial cell markers NR3C2 L1CAM and SLC2A1 athigher levels and epithelial cell markers Occludin Claudin1 and E-cadherin at lower levels (Figure 1) The expressionof Twist 1 a marker of fibroblast cell was very low This dataindicated that in our culture systemsmost of the cells isolatedfrom the urines were more similar to renal proximal tubularepithelial cells

It was reported in 2011 that forced expression of neuronallineage specific transcriptional factors Ascl1 Brn2 and Neu-roD in combination with Myt1l could successfully converthuman fibroblast into iN neurons [24] So we tried these4 transcriptional factors in the urinary cells Unfortunatelythese four factors could only convert urinary cells intoneuron-like cells which died 4sim6 days later although wechanged induction and culture condition Then we triedthe protooncogene Myc which enhanced the efficiency ofiPSC generation [25] in combination with the aforemen-tioned 4 factors We also changed the culture medium fromN3 medium [24] to N2 medium which was used for theinduction and maintenance of neurons induced from EScells or iPSCs To facilitate the survival of the neurons Y-27632-dihydrochloride and FBS were added to the mediumFinally urine cells were successfully converted into matureneurons The conversion procedure was simplified as shownin Figure 2(a)

For the first 4 days the cells showed epithelial-likemorphology and sustained proliferation From day 4 cellsbegan to change their shape We analyzed the expressionof the transcriptional factors on day 4 by observing the

GFP protein expression We found that almost all the cellsexpressed GFP but we could not know which cells expressedthe 5 factors at the same time On day 5 about 30 ofthe cells elongated and became long spindle cells Somegrew dendrite-like structures Unfortunately only a smallpercentage of these cells could be converted into neuronsMost of these cells began to die at almost the same time Sowe changed the medium every day from day 6 to removethe dead cells and retain the survival of the healthy cellsCells which were successfully converted into neurons grewlong processes and exhibited neuron-like morphology Andthese cells could be labeled by neuron lineage marker Tuj1(Supplementary Figure 1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520162452985)

Two weeks after infection with the aforementioned fac-tors the induced neuron cells showed neuronal morpholo-gies and were labeled with neuronal antibodies Tuj1 andMAP2 (Figures 2(b) and 2(c)) in the DOX induced group butnot in the groupwithoutDOX induction (Figure 2(c)) About155plusmn 001 (two independent experiments) of the initiatedcells were converted into Tuj1-positive cells

Immunofluorescence analysis showed that the iNcell expressed the mature neuron marker synaptophysin(Figure 3(a)) and there were GABAergic (Figure 3(b)) andglutamatergic (Figure 3(c)) neurons in the iN cells on day 25

Though it expressed synaptic vesicle protein synap-tophysin (Figure 3(a)) and fast-activating and inactivatinginward Na+ currents as well as outward K+ currents couldbe induced (Figure 4(a)) on day 24 these cells remainedfunctionally immature as revealed by their inability to gen-erate action potentials (Figure 4(b) upper panel) The meanmembrane capacity is 1521 plusmn 273 pF (mean plusmn sem 119899 =33) The average resting membrane potential of iN cells wasminus4489 plusmn 245mV (mean plusmn sem 119899 = 9) After extendedculture periods to 5 weeks the average resting membranepotential of iN cells is minus4950 plusmn 237mV (mean plusmn sem119899 = 14) and we could detect induced action potentials whichcould be blocked by the TTX treatment (Figure 4(b) lowerpanel) in 214 (119899 = 14) of the iN cells

To further verify this approach of direct conversion wecollected urinary cells from WD patients Using the fivefactors mentioned above we converted urine cells from bothnormal individuals and WD patients into neurons by day 12(Figure 5)

4 Discussion

In this study we found that a defined set of transcriptionfactors (Ascl1 Brn2 NeuroD c-Myc and Myt1l) coulddirectly reprogramme human urine cells into neuronal cellsTo our knowledge this is the first report concerning the directconversion of human urine cells into functional neuronsThese obtained iN cells could express multiple neuron-specific proteins and generate action potentials

For decades the paradigmof drug discovery anddevelop-ment as well as disease modeling had relied on immortalizedcell lines and animal models of human diseases Howevercandidate compounds for treating central nervous systemdefects failed in clinical trials in over 90 of cases due to poor


0DayEPIN2

Virus

351 2 3 4 12 25

(a)

GFP Tuj1

MAP2 Merge

(b)

MergeMAP2 GFP

(c)

Figure 2 Generation of neurons from human urine cells (a) Schematic protocol for conversion of urinary cells into neurons (b) Twelve daysafter induction urine derived-iN cells showed neuronalmorphologies And the urine-iN cells expressed both Tuj1 andMAP2 (c)Without theinduction of DOX the urine cells could not change into neuron cells (upper panel) In the presence of DOX the DOX induced transcriptionalfactors (Ascl1 Brn2 NeuroD c-Myc and Myt1l) convert urine cells into neuron directly (lower panel) Scale bars 50120583m


GFP Synaptophysin MAP2 Merge

(a)

GFP Synaptophysin GABA Merge

(b)

GFP Synaptophysin V-Glu Merge

(c)

Figure 3The iN cells expressedmature neuronmarker synaptophysinGABA or v-Glu (a) Twenty-four days afterDOX treatment the urine-iN cells expressed synaptophysin (b) Immunofluorescent study revealed GABA positive iN cells (c) Immunofluorescent study revealed thatiN cells were positive for v-Glu Scale bars 50 120583m (a) and 20 120583m (b c)

targeting lack of efficacy and unacceptable side effects [26]The last 10 years has witnessed the breakthrough discovery inbioscience The generation of human iPSCs [1] has provideda platform for drug research and disease modeling forsafety and efficacy The direct reprogramming technique[13] has provided another in vitro drug testing platformfor compound screening and exploring of the mysteries ofvarious diseases Researchers now have the opportunity tostudy human diseases in living developing neural cells thatcarry the disease-specific genetic variants

Until now fibroblasts are the most commonly usedsomatic cells for direct reprogramming [8ndash13 16 17] Besidesbrown fat cells [6] placenta [7] hepatocytes [14] pericyte-derived cells [15] and astrocytes [18] are also used Theproblem is that it is invasive to get them Therefore it ishard to persuade patients to donate The renal epithelialcells present in urine are idealized materials The isolationof urinary cells is simple cost-effective and universal Themethod for the collection and culture of urinary cells has alsobeen established [20]

By forced expression of 5 transcription factors Ascl1Brn2 Myt1l NeuroD and c-Myc we could generate func-tional neurons from human urine cells The iN cells showedneuronal morphologies and were labeled with neuronalantibodies Tuj1 and MAP2 Most of the iN cells were gluta-matergic neurons which are the same as the data reportedpreviously [24] We could also observe the expression ofsynaptic vesicle protein synaptophysin and fast-activatingand inactivating inward Na+ currents as well as outward K+currents Our results showed that neurons could be success-fully reprogrammed from urine cells of healthy individuals

Then we tried this method in WD patientsrsquo urinary cellsAs shown in Figure 5 urinary cells from both healthy indi-viduals and WD patients could be converted into neuronsPrevious report has demonstrated that the direct reprogram-ming astrocytes from skin fibroblasts of amyotrophic lateralsclerosis showed similar toxicity towards motor neuronsas astrocytes from autopsies of patients [27] This meansthat this method could be used to establish cell models ofneurological diseases such as WD


+TTX

+TTX

500pA

500pA

100mS

5mS(a)

+TTX

+TTX

minus50mV

minus65mV

50mV

100mS

50mV

100mS(b)

Figure 4 Membrane properties of the urine-iN cells Whole cell recording was conducted on urine-iN cells identified by differentialinterference contrast microscopy (a) Representative traces of membrane currents Fast-activating and inactivating Na+ currents wereprominent in all the iN cells The Na+ currents could be blocked by tetrodotoxin (TTX) (b) Representative traces of action potentials inresponse to step current injections 35 days after induction Membrane potential was maintained at approximately minus52mV And the actionpotentials could be blocked by TTX treatment


Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m

Figure 5 Urine cells from patients could be converted into neuron Twelve days after doxycycline treatment the urine from both normalindividuals and WD patients could be converted into neurons as shown by the expression of Tuj1 and MAP2 Scale bars 50 120583m

In 2012 Kim et al reviewed the common feature of thedirect conversion first the conversion process seems to bevery rapid second the functional maturation of the iN cellsseems to take several weeks third the efficiency of hiN cellsgeneration is much lower in adult human cells than mouseadult cells last human iN cells derived from embryonicor neonatal human cells functionally and physiologicallymature much faster than adult cell derived human iNs [28]The reprogramming procedure of the urinary cells fits allthe above features It takes 5 days to see the cells whichhave neuronal morphology Only about more than 1 of theurinary cells became neuron cells And it takes 5 weeks toinduce action potential in about only 21 of the iN cells Themost significant progress of the procedure is that the urinarycells are in endless supply although the protooncogene c-Mycwas used in this process

5 Conclusion

This study demonstrates that exfoliated cells in urine could beefficiently converted into functional neurons Andwe providea new way for preclinical studies and disease modeling ofneurological diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Shu-Zhen Zhang and Li-XiangMa contributed equally to thiswork

Acknowledgments

The authors sincerely thank the healthy individuals andWD patients for their help and willingness to participate inthis study This work was supported by a grant from theNational Natural Science Foundation of China to Zhi-YingWu (81330025 and 81125009)

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] 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

[3] S D Schwartz J-P Hubschman G Heilwell et al ldquoEmbryonicstem cell trials for macular degeneration a preliminary reportrdquoThe Lancet vol 379 no 9817 pp 713ndash720 2012

[4] X-Q Zhang and S-C Zhang ldquoDifferentiation of neural precur-sors and dopaminergic neurons from human embryonic stemcellsrdquoMethods inMolecular Biology vol 584 pp 355ndash366 2010

[5] R L Davis H Weintraub and A B Lassar ldquoExpression of asingle transfected cDNA converts fibroblasts tomyoblastsrdquoCellvol 51 no 6 pp 987ndash1000 1987

[6] 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

[7] R Ishii D Kami M Toyoda et al ldquoPlacenta to cartilagedirect conversion of human placenta to chondrocytes withtransformation by defined factorsrdquoMolecular Biology of the Cellvol 23 no 18 pp 3511ndash3521 2012

[8] M Ieda J-D Fu P Delgado-Olguin et al ldquoDirect reprogram-ming of fibroblasts into functional cardiomyocytes by definedfactorsrdquo Cell vol 142 no 3 pp 375ndash386 2010


[9] 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

[10] B Yu Z-Y He P You et al ldquoReprogramming fibroblasts intobipotential hepatic stem cells by defined factorsrdquo Cell Stem Cellvol 13 no 3 pp 328ndash340 2013

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

[12] 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

[13] 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

[14] S Marro Z P Pang N Yang et al ldquoDirect lineage conversionof terminally differentiated hepatocytes to functional neuronsrdquoCell Stem Cell vol 9 no 4 pp 374ndash382 2011

[15] M Karow R Sanchez C Schichor et al ldquoReprogramming ofpericyte-derived cells of the adult human brain into inducedneuronal cellsrdquo Cell Stem Cell vol 11 no 4 pp 471ndash476 2012

[16] 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

[17] J Kim S C Su H Wang et al ldquoFunctional integration ofdopaminergic neurons directly converted from mouse fibrob-lastsrdquo Cell Stem Cell vol 9 no 5 pp 413ndash419 2011

[18] R C Addis F-C Hsu R L Wright M A Dichter D ACoulter and J D Gearhart ldquoEfficient conversion of astrocytesto functional midbrain dopaminergic neurons using a singlepolycistronic vectorrdquoPLoSONE vol 6 no 12 Article ID e287192011

[19] T Zhou C Benda S Dunzinger et al ldquoGeneration of humaninduced pluripotent stem cells from urine samplesrdquo NatureProtocols vol 7 no 12 pp 2080ndash2089 2012

[20] T Zhou C Benda S Duzinger et al ldquoGeneration of inducedpluripotent stem cells from urinerdquo Journal of the AmericanSociety of Nephrology vol 22 no 7 pp 1221ndash1228 2011

[21] L Wang L Wang W Huang et al ldquoGeneration of integration-free neural progenitor cells from cells in human urinerdquo NatureMethods vol 10 no 1 pp 84ndash89 2013

[22] AMaximov Z P Pang D G R Tervo and T C Sudhof ldquoMon-itoring synaptic transmission in primary neuronal culturesusing local extracellular stimulationrdquo Journal of NeuroscienceMethods vol 161 no 1 pp 75ndash87 2007

[23] W-J Zhao M Zhang Y Miao X-L Yang and Z WangldquoMelatonin potentiates glycine currents through a PLCPKCsignalling pathway in rat retinal ganglion cellsrdquo Journal ofPhysiology vol 588 no 14 pp 2605ndash2619 2010

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

[25] M Nakagawa M Koyanagi K Tanabe et al ldquoGeneration ofinduced pluripotent stem cells without Myc from mouse andhuman fibroblastsrdquoNature Biotechnology vol 26 no 1 pp 101ndash106 2008

[26] I Kola and J Landis ldquoCan the pharmaceutical industry reduceattrition ratesrdquoNature Reviews Drug Discovery vol 3 no 8 pp711ndash715 2004

[27] K Meyer L Ferraiuolo C J Miranda et al ldquoDirect conversionof patient fibroblasts demonstrates non-cell autonomous toxic-ity of astrocytes tomotor neurons in familial and sporadic ALSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 2 pp 829ndash832 2014

[28] J Kim R Ambasudhan and S Ding ldquoDirect lineage repro-gramming to neural cellsrdquoCurrentOpinion inNeurobiology vol22 no 5 pp 778ndash784 2012

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Stem CellsInternational



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Microbiology


[19 20] and neuron stem cells [21] from exfoliated renalepithelial cells present in urine providing a noninvasiveapproach to obtain reprogramming cells The isolation ofurinary cells is simple cost-effective and universal Howeverthe direct conversion of urine cells to functional neurons hasnot been reported so far

In the present study we showed that human urine cellscould be directly reprogrammed into functional neuronsusing a combination of Ascl1 Brn2 NeuroD c-Myc andMyt1l and various neurotrophic factors These induced neu-ronal (iN) cells expressed multiple neuron-specific proteinsand generated action potentials Generation of iN cells fromnonneural lineages could have important implications forneurological disease modeling and regenerative medicine

2 Materials and Methods

21 Culture and Verification of Human Urinary Cells Theurinary cells were collected fromhealthy individuals andWDpatients after obtaining informed consents The exfoliatedcells were then isolated using a previously established pro-tocol with brief modifications [19] Donors were asked todrink 300ndash500mL water before urine collection The vulvawas wiped with iodine to keep sterile Approximately 200mLaseptic middle stream of the micturition was collected intoa sterilized glass bottle The urines were centrifuged at1000 rpm for 15min at room temperature immediately aftercollection The upper liquid was discarded and 2mL renalepithelial cell growth medium (REGM) with growth factors(Lonza cat number CC-4127) was added to the bottomsediment The cell-like organisms were seeded on one well of24-well plate (Corning) at a density of about 05 lowast 106cm2Half of the medium was discarded and replaced by 1mL ofnew REGM medium per day for 3 successive days Smallclones normally appeared at day 5 after planting Usually oneto five small colonies were observed per well Cells exhibitedshort spindle morphologies when they reached confluenceThese cells were subcultured or frozen for further usage Thestudy protocolwas approved by the research ethics committeeof Huashan Hospital

22 Reprogramming of Urinary Cells Five retroviruses(Lenti-EF1120572-EGFP-TRE-ASCL1 Lenti-EF1120572-EGFP-TRE-BRN2 Lenti-EF1120572-EGFP-TRE-NEUROD Lenti-EF1120572-EGFP-TRE-MYT1L and Lenti-EF1120572-EGFP-TRE-C-MYC)were produced for the ectopic expression of human Ascl1Brn2 NeuroD c-Myc and Myt1l respectively Retrovirusescarrying these five transcriptional factors were produced inthe 293T cell line Viral supernatant was concentrated byAmicon Ultra Centrifugal Filters (Millipore) and filteredthrough 022120583m cellulose acetate filter before being addedto urine cells Primary monolayer astrocyte cultures wereestablished as described previously [22]

Twenty-four hours after the recovery the five retroviruseswere mixed at 1 1 1 1 1 and were added to 1 lowast 105 primaryurinary cells at multiplicity of infection (MOI) of 75ndash10 Themedium was changed to 1 1 mixture of EPi and N2 medium(EPiN2 DMEMF2 N2 and NEAA [Invitrogen]) supplied

with 1 120583gmLDOX for 2 days Cells were harvested and platedonto astrocyte coated cover slips (1 times 104cm2) in N2mediumsupplemented with DOX Y21732 (10120583M Tocris) cAMP(1 120583M) Vit-C (200120583M) 05 FBS (Gibco) and neurotrophicfactors including NT-3 BDNF GDNF and IGF (10 ngmLeach RampD systems)The culture medium was changed everyother day until used

23 RNA Isolation and Relative Quantitative PCR Total RNAwas extracted usingMiniBESTUniversal RNAExtraction Kit(TaKaRa) The reverse-transcription reaction was conductedusing the PrimeScript RT Master Mix (TaKaRa) Primersused are the same as those reported previously [19] SYBRPremix ExTaq (TaKaRa)was used for the relative quantitativePCR reaction

24 Electrophysiology Theelectrophysiology experiment wasperformed according to the previously described protocolwith somemodifications [23] Cells selected for electrophysi-ological recordings had neuron-like shapes with fine branch-ing nerve projections Action potentials were recorded withcurrent-clamp whole cell configuration The bath solutioncontained the following (in mM) 125 NaCl 25 KCl 25NaHCO

3 125 NaH

2PO4 2 CaCl 1 MgCl

2 and 25 glucose

The pipette solution contained the following (in mM) 123 K-gluconate 10 KCl 1 MgCl

2 10 HEPES 1 EGTA 01 CaCl

2

1 K2ATP 02 Na

4GTP and 4 glucose pH adjusted to 72 with

KOHMembrane potentials were kept aroundminus65 tominus70mVand step currents were injected to elicit action potentials Forwhole cell voltage-dependent current recordings the sameinternal solution as aforementioned was used

25 Immunofluorescence Cells were fixed in 4 parafor-maldehyde for 10min and blocked by PBS solution con-taining 5 donkey serum and 02 Triton X-100 for1 hour at room temperature Primary antibodies wereapplied overnight and secondary antibodies were appliedfor 2 hours The following antibodies were used rabbitanti-Tuj1 (Covance 1 1000) mouse anti-Tuj1 (Covance1 1000) mouse anti-MAP2 (Sigma 1 500) rabbit anti-synaptophysin (Chemicon 1 1000) rabbit anti-v-Glu (Mil-lipore 1 2000) and rabbit-anti-GABA (DSHB 1 500)Alexa-488- Alexa-594- and Cy5-conjugated secondary anti-bodies were obtained from Invitrogen 410158406-Diamidino-2-phenylindole (DAPI) was from Sigma (1 10000)

26 Efficiency Calculation The Tuj1-positive cells with thinprocesses at least three times longer than their cell bodieswere counted We selected 3 cover slips and averaged thenumber of iN cells We then divided this number by thenumber of cells plated before infection to get the percentageof the starting population of cells that adopted neuron-likecharacteristics

3 Results

To investigate whether human urinary cells could be directlyconverted into neurons we collected urine samples from the



SLC2A1Twist 1

L1CA

M

E-Ca

dher

in

Occ

ludi

n

Clau

din

1

NR3

C2

SLC2

A1

Twist

1

0000

0001

0002

0003

01020304

Relat

ive e

xpre

ssio

n of

120573-a

ctin










4 Discussion




0DayEPIN2

Virus

351 2 3 4 12 25

(a)

GFP Tuj1

MAP2 Merge

(b)

MergeMAP2 GFP

(c)




(a)


(b)


(c)







+TTX

+TTX

500pA

500pA

100mS

5mS(a)

+TTX

+TTX

minus50mV

minus65mV

50mV

100mS

50mV

100mS(b)



Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m



5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



SLC2A1Twist 1

L1CA

M

E-Ca

dher

in

Occ

ludi

n

Clau

din

1

NR3

C2

SLC2

A1

Twist

1

0000

0001

0002

0003

01020304

Relat

ive e

xpre

ssio

n of

120573-a

ctin










4 Discussion




0DayEPIN2

Virus

351 2 3 4 12 25

(a)

GFP Tuj1

MAP2 Merge

(b)

MergeMAP2 GFP

(c)




(a)


(b)


(c)







+TTX

+TTX

500pA

500pA

100mS

5mS(a)

+TTX

+TTX

minus50mV

minus65mV

50mV

100mS

50mV

100mS(b)



Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m



5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0DayEPIN2

Virus

351 2 3 4 12 25

(a)

GFP Tuj1

MAP2 Merge

(b)

MergeMAP2 GFP

(c)




(a)


(b)


(c)







+TTX

+TTX

500pA

500pA

100mS

5mS(a)

+TTX

+TTX

minus50mV

minus65mV

50mV

100mS

50mV

100mS(b)



Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m



5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)


(b)


(c)







+TTX

+TTX

500pA

500pA

100mS

5mS(a)

+TTX

+TTX

minus50mV

minus65mV

50mV

100mS

50mV

100mS(b)



Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m



5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


+TTX

+TTX

500pA

500pA

100mS

5mS(a)

+TTX

+TTX

minus50mV

minus65mV

50mV

100mS

50mV

100mS(b)



Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m



5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Nor

mal

per

son

Tuj1 MAP2 Merge

Wils

onrsquos

dise

ase p

atie

nt

50120583m



5 Conclusion






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

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