Research Article TBX3 Knockdown Decreases Reprogramming ...downloads.hindawi.com/journals/sci/2016/6759343.pdfResearch Article TBX3 Knockdown Decreases Reprogramming Efficiency of

Research ArticleTBX3 Knockdown Decreases Reprogramming Efficiency ofHuman Cells

Moritz Klingenstein1 Stefanie Raab1 Kevin Achberger1

Alexander Kleger2 Stefan Liebau1 and Leonhard Linta1

1 Institute of Neuroanatomy Eberhard Karls University Tubingen 72074 Tubingen Germany2Department of Internal Medicine I Ulm University 89081 Ulm Germany

Correspondence should be addressed to Alexander Kleger alexanderklegeruni-ulmdeStefan Liebau stefanliebauuni-tuebingende and Leonhard Linta leonhardlintauni-tuebingende

Received 24 April 2015 Accepted 2 July 2015

Academic Editor Su-Chun Zhang

Copyright copy 2016 Moritz Klingenstein 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

TBX3 is a member of the T-box transcription factor family and is involved in the core pluripotency network Despite this role in thepluripotency network its contribution to the reprogramming process during the generation of human induced pluripotent stemcells remains elusive In this respect we performed reprogramming experiments applying TBX3 knockdown in human fibroblastsand keratinocytes Knockdown of TBX3 in both somatic cell types decreased the reprogramming efficiencies in comparison tocontrol cells but with unchanged reprogramming kinetics The resulting iPSCs were indistinguishable from control cells anddisplayed a normal in vitro differentiation capacity by generating cells of all three germ layers comparable to the controls

1 Introduction

Pluripotent embryonic stem cells (ESCs) are isolated fromthe inner cell mass of early embryos Pluripotency is definedby a high and indefinite proliferative potential and thecapacity to differentiate into cells of the three germ layerssubsequently able to generate all cell types and tissues ofan organism The possibility of culturing and investigatingESCs has led to a good understanding of the pluripotencynetwork but also of various signaling pathways involved incell differentiation events [1 2] The pluripotency network iscomprised of numerous transcription factors interconnectedwith each other by regulatory feedback loops Basically itkeeps the expression of stem cell genes active and repressesgenes involved in differentiation [3 4] These transcriptionfactors have such a dominant role in cell function that theoverexpression of a few factors is able to force a somatic cellback into a pluripotent state In this way somatic cells arereprogrammed into induced pluripotent stem cells (iPSCs)by overexpression of OCT4 SOX2 KLF4 or other relatedfactors [5 6]The discovery of this reprogramming technique

has not just widened the understanding of the pluripotencynetwork but is also an invaluable tool to generate donor andpatient specific stem cells for disease modeling and for futuretherapeutic strategies [7ndash12]

The T-box transcription factor family is involved in avariety of processes including differentiation and pluripo-tency In the pluripotency circuitry TBX3 has been shown tointeract with the core pluripotency factors NANOG OCT4and SOX2 to maintain the stem cell state and to inhibitdifferentiation Depletion of Tbx3 leads to differentiation ofmurine pluripotent stem cells [13] Moreover Tbx3 upholdspluripotency by acting as a downstream activator of WNTsignaling and plays a role in the reprogramming processby direct binding and activation of the Oct4 promoterDuring early differentiation processes Tbx3 is involved inmesendodermal differentiation heart development and limbformation [14] Additionally Tbx3 is highly expressed indefinite endoderm progenitors and together with Jmjd3 andEomes promotes the formation of the endoderm [15] Inthat respect Tbx3 was classified as one of the mesendodermpluripotency transcription factors

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 6759343 7 pageshttpdxdoiorg10115520166759343

2 Stem Cells International

Based on these findings we applied a knockdownofTBX3in human somatic cells to investigate the role of TBX3 in thereprogramming process

2 Materials and Methods

21 Keratinocyte and Fibroblast Cultivation The cultivationof keratinocytes from plucked human hair was mainlyperformed according to [16] In brief keratinocytes werecultured on 20 120583gmL collagen IV (Sigma-Aldrich) coateddishes in EpiLifemediumwithHKGS supplement (both fromGibco) until they reached sim70 confluency

Human foreskin fibroblasts (HFFs) (System Biosciences)were cultivated in Dulbeccorsquos modified Eaglersquos medium(DMEM) supplemented with 10 fetal bovine serum (FBS)and 1Glutamax 1 nonessential amino acids (NEAA) and1 antibiotic-antimycotic (all from Life Technologies)

22 Lentivirus Production Infection and Selection 4 times 106Lenti-X 293T cells (Clontech) were transfected with 8120583gDNA of plasmid pRRLPPTSFhOKSMcoidTompre FRT[17] or TRIPZ Human TBX3 shRNA (V2THS 135043 GEHealthcare) togetherwith 2120583g pMD2G and 55 120583g of psPAX2vector DNA (Addgene 12259 and 12260 from Didier Trono)using 1120583gmL polyethylenimine (PEI) Lenti-XConcentratorKit (Clontech) was used to concentrate the viral super-natant collected after two and four days Virus pellets wereresuspended in EpiLife with HKGS supplement Spinfectionwith 1000 g for 30min was used to transduce TRIPZ TBX3lentivirus into the cells on two consecutive days with amedium change after 4 h respectively Infected cells wereselected by puromycin (1 120583gmL) treatment for two days

23 Feeder Cells Reprogramming of Keratinocytes and Fibrob-lasts Rat embryonic fibroblasts (REFs) from embryonicday 14 were generated according to the protocol previouslydescribed in [18] and were cultured in DMEM containing10 fetal bovine serum (FBS) 1 Glutamax 1 NEAA and1 antibiotic-antimycotic (all from Life Technologies) REFswere treated with 75 120583gmL mitomycin C (Biomol) for 25hours for mitotic inactivation

Keratinocytes and fibroblasts were infected as mentionedabove over two days with 5 times 108 lentiviral particles in theappropriate medium containing 8 120583gmL polybrene (Sigma-Aldrich) 1 120583gmL doxycycline was added to the keratinocytesand fibroblasts depending on the condition from the first dayof infection

Two days after infection keratinocytes and fibroblastswere detached using TrypLE Express (Life Technologies)and were seeded on 15 times 105 mitotic inactive REF cells iniPSCmedium (DMEMF12 supplementedwith 20knockoutserum replacement 1 antibiotic-antimycotic 1 NEAA1 Glutamax 100 120583M 120573-mercaptoethanol 10120583M Y-2763250120583gmL ascorbic acid and 10 ngmL FGF2) Incubationconditions for reprogramming cells were 37∘C 5 CO

2

and 5 O2 Reprogramming cells were cultured on feeder

cells until iPSC colonies were clearly visible and were thenmechanically picked and transferred onto Matrigel-coated(Corning) cell culture dishes for feeder-free culture

24 Human iPSC Culture Germ Layer Differentiation iPSCswere cultivated under feeder-free conditions in FTDAmedium according to [19] For further passaging and germlayer differentiation iPSCs were detached using ReLeSR(Stemcell Technologies) according to the manufacturerrsquosmanual To form embryoid bodies detached cell clumpswere cultured in suspension using ultralow attachment flasks(Corning) for 7 days After seeding on Matrigel-coateddishes the embryoid bodies were cultured two additionalweeks adherently iPSC medium was used for germ layerdifferentiation

25 Immunocytochemistry and Alkaline Phosphatase StainingCells were fixed for 15 minutes with 4 paraformaldehyde(PFA) and 10 sucrose Pluripotency staining was performedusing StemLight Pluripotency Antibody Kit according tothemanufacturerrsquos recommended conditions (Cell Signaling)with Alexa secondary antibodies (all from Abcam) For germlayer differentiation immunocytochemistry stainings fixedcells were permeabilized with 02 Triton-X for 5 minutesfollowed by blocking with 5 bovine serum albumin (BSA)for 60 minutes Antibodies were used as described belowmouse anti-DESMIN (DAKO 1 500) goat anti-SOX17 (RampDSystems 1 500) rabbit anti-TUBB3 (Covance 1 2000) andAlexa secondary antibodies (all from Abcam) Cells weremounted with ProLong Gold Antifade Reagent with 410158406-diamidino-2-phenylindole (DAPI) (Life Technologies)

Cells for alkaline phosphatase (AP) staining were fixedin 4 PFA for two minutes Nitroblue tetrazolium5-bromo-4-chloro-3-indolyl phosphate (NBTBCIP) solution was pre-pared according to the manufacturerrsquos protocol (Sigma-Aldrich)The cells were incubated with the substrate solutionfor 20 minutes and the reaction was stopped with phosphate-buffered saline (PBS) Pictures were taken under artificiallight with a normal camera with particular caution on thesame acquisition settings and exposure time Colony num-bers were counted manually or quantified with the ImageJsoftware bymeasuring the total intensity in the well area aftersubtraction of the background value

26 Quantitative Reverse Transcription Polymerase ChainReaction Total RNA was isolated from cell lysates usingRNeasy MiniKit (Qiagen) QuantiTect primer assays wereused for all experiments (Qiagen Supplementary Table 1 inSupplementary Material available online at httpdxdoiorg10115520166759343)

PCR was performed on a StepOne instrument (AppliedBiosciences) using QuantiFast SYBR Green RT-PCR Kit(Qiagen) according to the manufacturerrsquos protocol Relativegene expression was calculated as a ratio of target gene con-centration to the housekeeping gene hydroxymethylbilanesynthase (HMBS) concentration

3 Results

31 Reprogramming of Human TBX3 Knockdown Cells Torule out cell type specific effects in our experimental setupwe chose somatic cells which originate from different germlayers mesodermal human foreskin fibroblasts (HFFs) and

Stem Cells International 3

Fibroblasts Keratinocytes

Lentivirus

TRE

shTBX3 Puro

Control (minusDOX)Knockdown (+DOX)


(a)

Infection

Seeding on REFs

qRT-PCRAP staining

iPSC transfer to Matrigel

sim50000 fibroblasts

Day 0

Day 17

Day 25

Day 4Day 2

Day 0

Day 22

Day 5Day 2

sim10000 keratinocytes



Day 22

Day minus2Day minus2

(b)

ControlKnockdown

15

10

05

00

d25d17d4d2d0

lowastlowastlowast lowastlowast

Rela

tiveT

BX3

expr

essio

nno

rmal

ized

with

HMBS

(c)

ControlKnockdown

20

15

10

05

00

d22d5d2d0

lowast lowast

Rela

tiveT

BX3

expr

essio

nno

rmal

ized

with

HMBS

(d)

Figure 1 Reprogramming fibroblasts and keratinocytes with a TBX3 knockdown (a) Infection of fibroblasts and keratinocytes with a TRIPZHumanTBX3 shRNA lentivirus inducible via doxycycline addition (b) Reprogramming time schemewith fibroblasts (left) and keratinocytes(right) along with starting cell numbers and experiment time points (c) TBX3 knockdown during reprogramming of fibroblasts at day 0(seeding infected cells on feeder layer) day 2 day 4 day 17 and day 25 (last day on feeder cells before transfer to feeder-free system onMatrigel) Control is not treated with doxycycline (d) TBX3 knockdown during reprogramming of keratinocytes at day 0 (seeding infectedcells on feeder layer) day 2 day 5 and day 22 (last day on feeder cells before transfer to feeder-free system onMatrigel) Control is not treatedwith doxycycline


lowastlowastlowast

KnockdownControl

Relative intensity0 05 1 15

(a)

KnockdownControl

Number of colonies0 5 10 15 20

(b)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion

d0 d2 d4 d17 d25 iPSC d0 d2 d4 d17 d25 iPSC d0 d2 d4 d17 d25 iPSC

15

10

05

00

minus05

20

15

10

05

00

minus05

minus10

25

20

15

10

05

00

minus05

lowast

lowast lowast

(c)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion

d0 d2 d5 d22 d0 d2 d5 d22 d0 d2 d5 d22

60

40

20

0

500

400

300

200

10015

10

5

0

15

10

5

0lowast

(d)

Figure 2 TBX3 knockdown in fibroblasts and keratinocytes during reprogramming causes decreased reprogramming efficiency (a) Alkalinephosphatase (AP) staining at day 17 of fibroblast reprogramming with a highly significant reduction (23) of iPSC colonies (b) Alkalinephosphatase (AP) staining at day 22 of keratinocyte reprogramming with a reduction (91) of iPSC colonies (c) Relative expression ofOCT4 SOX2 and NANOG during fibroblast reprogramming (d) Relative expression of OCT4 SOX2 and NANOG during keratinocytereprogramming

ectodermal human hair follicle keratinocytes First weinfected both cell types with a lentiviral vector containing apuromycin resistance for selection of infected cells as well asa doxycycline (DOX) inducible shRNAdirected againstTBX3(Figure 1(a)) After successful puromycin selection cells wereeither treated with doxycycline (+DOX) or left untreated asa control (minusDOX) Subsequently a defined number of allcell types were infected with a reprogramming lentiviruscontaining OCT4 SOX2 KLF4 and C-MYC After two daysinfected cells were transferred onto mitotically inactivated

feeder cells (rat embryonic fibroblasts) At indicated timepoints RNA samples were collected and alkaline phosphatase(AP) stainings were performed (Figure 1(b)) TBX3 knock-down efficiency was traced throughout the reprogrammingprocess by qRT-PCR For HFFs the knockdown was sim50(Figure 1(c)) while keratinocytes displayed an initial knock-down of approximately 30 showing an increase at later timepoints (Figure 1(d)) However it must be taken into accountthat all measured cell populations contained a considerableamount of rat feeder cells Due to the highly conserved


Control KnockdownOCT4 DAPI

SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

OCT4 DAPI

SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

(a)

Control KnockdownTUBB3 DAPI

DESMIN DAPI

SOX17 DAPI

TUBB3 DAPI

DESMIN DAPI

SOX17 DAPI

(b)

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC

TUBB3 PAX6 T MYH6 AFP FOXA2TUBB3 PAX6 T MYH6 AFP FOXA2

Control Knockdown

(c)

Figure 3 Reprogrammed fibroblasts under TBX3 knockdown have the same stem cell properties as control iPSCs (a) Pluripotency stainingof iPSCs generated from fibroblasts is positive for the pluripotency markers OCT4 SOX2 and NANOG as well as for the surface markersTRA-1-60 TRA-1-81 and SSEA4 (b) Immunocytochemistry of germ layer differentiation shows positive staining for ectoderm (TUBB3)mesoderm (DESMIN) and endoderm (SOX17) for TBX3 knockdown and control respectively (c) Expression of ectodermal genes (TUBB3PAX6) mesodermal genes (T MYH6) and endodermal genes (AFP FOXA2) in germ layer differentiation shows pluripotent differentiationpotential of the generated iPSCs in both control and TBX3 knockdown cells Scale bar 100120583m

sequence in both species the feeder cells partly contribute tothe measured TBX3 levels Therefore knockdown efficiencyin the reprogramming cells might be considerably higher

32 TBX3 Knockdown Reduces Reprogramming EfficiencyAt the end of the reprogramming process when large iPScolonies were clearly visible AP stainings were performedand the stained areas were measured In both cell types thereduced levels of TBX3 led to a reduced number of colonies

23 in HFF (highly significant) and 91 in keratinocytes(Figures 2(a) and 2(b)) Of note only a very low numberof keratinocytes were infected and reprogrammed (due toslow proliferation after infection) leading to a highly reducednumber of iPS colonies compared to HFFs which dividecontinuously and fast during the reprogramming processNeither colony size nor colony morphology was noticeablyaltered in knockdown cultures The reduced reprogrammingefficiency was confirmed by qRT-PCR The RNA levels of


OCT4 SOX2 and NANOG were additionally reduced inboth TBX3 knockdown cell types most probably due tothe lower number of reprogrammed cells (Figures 2(c) and2(d)) In later passage iPSCs RNA levels for OCT4 SOX2and NANOG levels were comparable to control culturesAdditionally knockdown cells at later passages displayed acomparable growth speed (not shown) and colony morphol-ogy To generally test the full pluripotent capacity of theknockdown iPSCs we analyzed later passage HFF derivediPSCs in more detail in terms of pluripotency factor expres-sion and differentiation potential iPSCs were positive forthe transcription factors OCT4 SOX2 and NANOG as wellas the surface antigens TRA-1-60 TRA-1-81 and SSEA4 inimmunofluorescence staining (Figure 3(a)) comparable tothe control cells In an in vitro differentiation experimentknockdown iPSCs generated cells positive for TUBB3 as anectodermal marker DESMIN as a mesodermal marker andSOX17 as an endodermal marker as shown by immunoflu-orescence staining (Figure 3(b)) This ability to differentiateinto all three germ layers could be underlined by qRT-PCR (Figure 3(c)) Comparable expression levels for differentdifferentiationmarkers between knockdown and control cellsshowed that the knockdown does not lead to an alteredgerm layer preference or diminished differentiation potentialTaken together the TBX3 knockdown significantly reducedthe reprogramming efficiency but the still arising iPSCs werecomparable to control iPSCs

4 Discussion

TBX3 is a core member of the pluripotency network oftranscription factors which keeps ESCs and iPSCs in thepluripotent state Therefore we were interested if a knock-down of TBX3 expression would affect the reprogrammingprocess or alter the resulting stem cells in their charac-teristics For this we used two human cell types fibrob-lasts and keratinocytes containing an inducible shRNAdirected against TBX3 Reprogramming of these cells didnot show altered reprogramming kinetics but a reducedreprogramming efficiency compared to control cells Thisresult was similar for both tested cell types The numberof arising colonies was significantly smaller than in controlAP stainings RNA levels of pluripotency factors were alsoreduced during the reprogramming process indicating lowerpluripotent cell numbers Fully reprogrammed cells of laterpassages however did not exhibit altered RNA levels ofpluripotency factors They were also comparable to controlsin stem cell marker immunofluorescence stainings Afterinduction of differentiation they expressed markers of thethree germ layers in a ratio comparable to control cellswithout altered germ layer preference This stays in contrastto a previous study indicating that TBX3 knockdown inhibitsneural rosette formation [20] However since we solelyinvestigated RNA levels and not the appearance of neuralrosettes a disturbed rosette morphology cannot be excludedInterestingly we found that although the knockdownofTBX3was very constant throughout the reprogramming processin later passage iPSCs it was not visible any more Herewe suppose that the shRNA does not reduce the very low

expression levels any further or that iPSCs may be able tosilence the shRNA expression after several passages

In the future studies could investigate if the reductionof reprogramming efficiency as observed with TBX3 knock-down cells will also be present in TBX3 depleted cells or ifthese cells even fail to be reprogrammed at all If stable iPSCscan be obtained in this way their differentiation behaviorshould be studied in detail since TBX3 is well known toalso have important contributions in certain differentiationpathways

Conflict of Interests

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

Authorsrsquo Contribution

Stefanie Raab andMoritz Klingenstein contributed equally tothis paper

Acknowledgments

The authors would like to thank Clara Misbah for thetechnical assistance This study was funded by the DeutscheForschungsgemeinschaft (Stefan Liebau BO17184-1) theForschungskern SyStaR to Alexander Kleger BIU (BohringerIngelheim Ulm to Alexander Kleger) and the Else-Kroner-Fresenius-Stiftung (2011 A200 to Alexander Kleger and Ste-fan Liebau) Alexander Kleger is indebted to the Baden-Wurttemberg Stiftung for the financial support of thisresearch project by the Eliteprogramme for Postdocs Alexan-der Kleger is also an Else-Kroner-FreseniusMemorial FellowLeonhard Linta is supported by a fortune junior grant of theFaculty of Medicine Tubingen (2248-0-0)

References

[1] Y Tanaka E Hysolli J Su et al ldquoTranscriptome signature andregulation in human somatic cell reprogrammingrdquo Stem CellReports vol 4 no 6 pp 1125ndash1139 2015

[2] E A Mason J C Mar A L Laslett et al ldquoGene expressionvariability as a unifying element of the pluripotency networkrdquoStem Cell Reports vol 3 no 2 pp 365ndash377 2014

[3] GMartello and A Smith ldquoThe nature of embryonic stem cellsrdquoAnnual Review of Cell and Developmental Biology vol 30 no 1pp 647ndash675 2014

[4] V Karwacki-Neisius J Goke R Osorno et al ldquoReducedOct4 expression directs a robust pluripotent state with distinctsignaling activity and increased enhancer occupancy by Oct4and Nanogrdquo Cell Stem Cell vol 12 no 5 pp 531ndash545 2013

[5] 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

[6] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[7] Q Feng N Shabrani J N Thon et al ldquoScalable generation ofuniversal platelets from human induced pluripotent stem cellsrdquoStem Cell Reports vol 3 no 5 pp 817ndash831 2014


[8] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[9] L Linta M Stockmann T M Boeckers A Kleger and SLiebau ldquoThe potential of iPS cells in synucleinopathy researchrdquoStem Cells International vol 2012 Article ID 629230 6 pages2012

[10] M Rao ldquoIPSC-based cell therapy an important step forwardrdquoStem Cell Reports vol 1 no 4 pp 281ndash282 2013

[11] F A Soares M Sheldon M Rao C Mummery and L Val-lier ldquoInternational coordination of large-scale human inducedpluripotent stem cell initiatives wellcome Trust and ISSCRworkshops white paperrdquo Stem Cell Reports vol 3 no 6 pp 931ndash939 2014

[12] L Ye C Swingen and J Zhang ldquoInduced pluripotent stemcells and their potential for basic and clinical sciencesrdquo CurrentCardiology Reviews vol 9 no 1 pp 63ndash72 2013

[13] J Han P Yuan H Yang et al ldquoTbx3 improves the germ-linecompetency of induced pluripotent stem cellsrdquoNature vol 463no 7284 pp 1096ndash1100 2010

[14] C E Weidgang R Russell P R Tata et al ldquoTBX3 directs cell-fate decision toward mesendodermrdquo Stem Cell Reports vol 1no 3 pp 248ndash265 2013

[15] A E R Kartikasari J X Zhou M S Kanji et al ldquoThe histonedemethylase Jmjd3 sequentially associates with the transcrip-tion factors Tbx3 andEomes to drive endodermdifferentiationrdquoThe EMBO Journal vol 32 no 10 pp 1393ndash1408 2013

[16] T Aasen A Raya M J Barrero et al ldquoEfficient and rapidgeneration of induced pluripotent stem cells from humankeratinocytesrdquo Nature Biotechnology vol 26 no 11 pp 1276ndash1284 2008

[17] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011

[18] L Linta M Stockmann K N Kleinhans et al ldquoRat embryonicfibroblasts improve reprogramming of human keratinocytesinto induced pluripotent stem cellsrdquo Stem Cells and Develop-ment vol 21 no 6 pp 965ndash976 2012

[19] S Frank M Zhang H R Scholer and B Greber ldquoSmallmolecule-assisted line-independent maintenance of humanpluripotent stem cells in defined conditionsrdquo PLoS ONE vol 7no 7 Article ID e41958 2012

[20] T Esmailpour and T Huang ldquoTBX3 promotes human embry-onic stem cell proliferation and neuroepithelial differentiationin a differentiation stage-dependentmannerrdquo StemCells vol 30no 10 pp 2152ndash2163 2012

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Enzyme Research



Microbiology


Based on these findings we applied a knockdownofTBX3in human somatic cells to investigate the role of TBX3 in thereprogramming process

2 Materials and Methods

21 Keratinocyte and Fibroblast Cultivation The cultivationof keratinocytes from plucked human hair was mainlyperformed according to [16] In brief keratinocytes werecultured on 20 120583gmL collagen IV (Sigma-Aldrich) coateddishes in EpiLifemediumwithHKGS supplement (both fromGibco) until they reached sim70 confluency

Human foreskin fibroblasts (HFFs) (System Biosciences)were cultivated in Dulbeccorsquos modified Eaglersquos medium(DMEM) supplemented with 10 fetal bovine serum (FBS)and 1Glutamax 1 nonessential amino acids (NEAA) and1 antibiotic-antimycotic (all from Life Technologies)

22 Lentivirus Production Infection and Selection 4 times 106Lenti-X 293T cells (Clontech) were transfected with 8120583gDNA of plasmid pRRLPPTSFhOKSMcoidTompre FRT[17] or TRIPZ Human TBX3 shRNA (V2THS 135043 GEHealthcare) togetherwith 2120583g pMD2G and 55 120583g of psPAX2vector DNA (Addgene 12259 and 12260 from Didier Trono)using 1120583gmL polyethylenimine (PEI) Lenti-XConcentratorKit (Clontech) was used to concentrate the viral super-natant collected after two and four days Virus pellets wereresuspended in EpiLife with HKGS supplement Spinfectionwith 1000 g for 30min was used to transduce TRIPZ TBX3lentivirus into the cells on two consecutive days with amedium change after 4 h respectively Infected cells wereselected by puromycin (1 120583gmL) treatment for two days

23 Feeder Cells Reprogramming of Keratinocytes and Fibrob-lasts Rat embryonic fibroblasts (REFs) from embryonicday 14 were generated according to the protocol previouslydescribed in [18] and were cultured in DMEM containing10 fetal bovine serum (FBS) 1 Glutamax 1 NEAA and1 antibiotic-antimycotic (all from Life Technologies) REFswere treated with 75 120583gmL mitomycin C (Biomol) for 25hours for mitotic inactivation

Keratinocytes and fibroblasts were infected as mentionedabove over two days with 5 times 108 lentiviral particles in theappropriate medium containing 8 120583gmL polybrene (Sigma-Aldrich) 1 120583gmL doxycycline was added to the keratinocytesand fibroblasts depending on the condition from the first dayof infection

Two days after infection keratinocytes and fibroblastswere detached using TrypLE Express (Life Technologies)and were seeded on 15 times 105 mitotic inactive REF cells iniPSCmedium (DMEMF12 supplementedwith 20knockoutserum replacement 1 antibiotic-antimycotic 1 NEAA1 Glutamax 100 120583M 120573-mercaptoethanol 10120583M Y-2763250120583gmL ascorbic acid and 10 ngmL FGF2) Incubationconditions for reprogramming cells were 37∘C 5 CO

2

and 5 O2 Reprogramming cells were cultured on feeder

cells until iPSC colonies were clearly visible and were thenmechanically picked and transferred onto Matrigel-coated(Corning) cell culture dishes for feeder-free culture

24 Human iPSC Culture Germ Layer Differentiation iPSCswere cultivated under feeder-free conditions in FTDAmedium according to [19] For further passaging and germlayer differentiation iPSCs were detached using ReLeSR(Stemcell Technologies) according to the manufacturerrsquosmanual To form embryoid bodies detached cell clumpswere cultured in suspension using ultralow attachment flasks(Corning) for 7 days After seeding on Matrigel-coateddishes the embryoid bodies were cultured two additionalweeks adherently iPSC medium was used for germ layerdifferentiation

25 Immunocytochemistry and Alkaline Phosphatase StainingCells were fixed for 15 minutes with 4 paraformaldehyde(PFA) and 10 sucrose Pluripotency staining was performedusing StemLight Pluripotency Antibody Kit according tothemanufacturerrsquos recommended conditions (Cell Signaling)with Alexa secondary antibodies (all from Abcam) For germlayer differentiation immunocytochemistry stainings fixedcells were permeabilized with 02 Triton-X for 5 minutesfollowed by blocking with 5 bovine serum albumin (BSA)for 60 minutes Antibodies were used as described belowmouse anti-DESMIN (DAKO 1 500) goat anti-SOX17 (RampDSystems 1 500) rabbit anti-TUBB3 (Covance 1 2000) andAlexa secondary antibodies (all from Abcam) Cells weremounted with ProLong Gold Antifade Reagent with 410158406-diamidino-2-phenylindole (DAPI) (Life Technologies)

Cells for alkaline phosphatase (AP) staining were fixedin 4 PFA for two minutes Nitroblue tetrazolium5-bromo-4-chloro-3-indolyl phosphate (NBTBCIP) solution was pre-pared according to the manufacturerrsquos protocol (Sigma-Aldrich)The cells were incubated with the substrate solutionfor 20 minutes and the reaction was stopped with phosphate-buffered saline (PBS) Pictures were taken under artificiallight with a normal camera with particular caution on thesame acquisition settings and exposure time Colony num-bers were counted manually or quantified with the ImageJsoftware bymeasuring the total intensity in the well area aftersubtraction of the background value

26 Quantitative Reverse Transcription Polymerase ChainReaction Total RNA was isolated from cell lysates usingRNeasy MiniKit (Qiagen) QuantiTect primer assays wereused for all experiments (Qiagen Supplementary Table 1 inSupplementary Material available online at httpdxdoiorg10115520166759343)

PCR was performed on a StepOne instrument (AppliedBiosciences) using QuantiFast SYBR Green RT-PCR Kit(Qiagen) according to the manufacturerrsquos protocol Relativegene expression was calculated as a ratio of target gene con-centration to the housekeeping gene hydroxymethylbilanesynthase (HMBS) concentration

3 Results

31 Reprogramming of Human TBX3 Knockdown Cells Torule out cell type specific effects in our experimental setupwe chose somatic cells which originate from different germlayers mesodermal human foreskin fibroblasts (HFFs) and



Lentivirus

TRE

shTBX3 Puro



(a)

Infection

Seeding on REFs

qRT-PCRAP staining



Day 0

Day 17

Day 25

Day 4Day 2

Day 0

Day 22

Day 5Day 2




Day 22


(b)

ControlKnockdown

15

10

05

00

d25d17d4d2d0


Rela

tiveT

BX3

expr

essio

nno

rmal

ized

with

HMBS

(c)

ControlKnockdown

20

15

10

05

00

d22d5d2d0

lowast lowast

Rela

tiveT

BX3

expr

essio

nno

rmal

ized

with

HMBS

(d)



lowastlowastlowast

KnockdownControl


(a)

KnockdownControl


(b)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion


15

10

05

00

minus05

20

15

10

05

00

minus05

minus10

25

20

15

10

05

00

minus05

lowast

lowast lowast

(c)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion

d0 d2 d5 d22 d0 d2 d5 d22 d0 d2 d5 d22

60

40

20

0

500

400

300

200

10015

10

5

0

15

10

5

0lowast

(d)






SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

OCT4 DAPI

SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

(a)


DESMIN DAPI

SOX17 DAPI

TUBB3 DAPI

DESMIN DAPI

SOX17 DAPI

(b)

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC


Control Knockdown

(c)







4 Discussion








Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Lentivirus

TRE

shTBX3 Puro



(a)

Infection

Seeding on REFs

qRT-PCRAP staining



Day 0

Day 17

Day 25

Day 4Day 2

Day 0

Day 22

Day 5Day 2




Day 22


(b)

ControlKnockdown

15

10

05

00

d25d17d4d2d0


Rela

tiveT

BX3

expr

essio

nno

rmal

ized

with

HMBS

(c)

ControlKnockdown

20

15

10

05

00

d22d5d2d0

lowast lowast

Rela

tiveT

BX3

expr

essio

nno

rmal

ized

with

HMBS

(d)



lowastlowastlowast

KnockdownControl


(a)

KnockdownControl


(b)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion


15

10

05

00

minus05

20

15

10

05

00

minus05

minus10

25

20

15

10

05

00

minus05

lowast

lowast lowast

(c)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion

d0 d2 d5 d22 d0 d2 d5 d22 d0 d2 d5 d22

60

40

20

0

500

400

300

200

10015

10

5

0

15

10

5

0lowast

(d)






SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

OCT4 DAPI

SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

(a)


DESMIN DAPI

SOX17 DAPI

TUBB3 DAPI

DESMIN DAPI

SOX17 DAPI

(b)

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC


Control Knockdown

(c)







4 Discussion








Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowastlowastlowast

KnockdownControl


(a)

KnockdownControl


(b)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion


15

10

05

00

minus05

20

15

10

05

00

minus05

minus10

25

20

15

10

05

00

minus05

lowast

lowast lowast

(c)

KnockdownControl

KnockdownControl

KnockdownControl

OCT4 SOX2 NANOG

vers

us iP

SCRe

lativ

e exp

ress

ion

d0 d2 d5 d22 d0 d2 d5 d22 d0 d2 d5 d22

60

40

20

0

500

400

300

200

10015

10

5

0

15

10

5

0lowast

(d)






SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

OCT4 DAPI

SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

(a)


DESMIN DAPI

SOX17 DAPI

TUBB3 DAPI

DESMIN DAPI

SOX17 DAPI

(b)

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC


Control Knockdown

(c)







4 Discussion








Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

OCT4 DAPI

SOX2 DAPI

NANOG DAPI

TRA-1-60 DAPI

TRA-1-81 DAPI

SSEA4 DAPI

(a)


DESMIN DAPI

SOX17 DAPI

TUBB3 DAPI

DESMIN DAPI

SOX17 DAPI

(b)

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC

3000000

2000000

1000000

350

250

150

50

5

4

3

2

1

0

Fold

indu

ctio

n co

mpa

red

to u

ndiff

eren

tiate

d iP

SC


Control Knockdown

(c)







4 Discussion








Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion








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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