RESEARCH OUTPUTS / RÉSULTATS DE RECHERCHE
Author(s) - Auteur(s) :
Publication date - Date de publication :
Permanent link - Permalien :
Rights / License - Licence de droit d’auteur :
Bibliothèque Universitaire Moretus Plantin
Institutional Repository - Research Portal
Dépôt Institutionnel - Portail de la Recherche
researchportal.unamur.be
University of Namur
Mitochondrial remodeling in hepatic differentiation and dedifferentiation
Wanet, Anaïs; Remacle, Noémie; Najar, Mehdi; Sokal, Etienne; Arnould, Thierry; Najimi,
Mustapha; Renard, Patricia
Published in:
The international journal of Biochemistry & Cell biology
DOI:
10.1016/j.biocel.2014.07.015
Publication date:
2014
Document Version
Early version, also known as pre-print
Link to publication
Citation for pulished version (HARVARD):
Wanet, A, Remacle, N, Najar, M, Sokal, E, Arnould, T, Najimi, M & Renard, P 2014, 'Mitochondrial remodeling in
hepatic differentiation and dedifferentiation', The international journal of Biochemistry & Cell biology, vol. 54C,
pp. 174-185. https://doi.org/10.1016/j.biocel.2014.07.015
General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain
• You may freely distribute the URL identifying the publication in the public portal ?
Take down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
ContentslistsavailableatScienceDirect
The
International
Journal
of
Biochemistry
&
Cell
Biology
j ourna l h o m e pa ge :w w w . e l s e v i e r . c o m / l o c a t e / b i o c e l
Mitochondrial
remodeling
in
hepatic
differentiation
and
dedifferentiation
Anaïs
Wanet
a,
Noémie
Remacle
a,
Mehdi
Najar
b,
Etienne
Sokal
c,
Thierry
Arnould
a,
Mustapha
Najimi
c,
Patricia
Renard
a,∗aLaboratoryofBiochemistryandCellBiology(URBC),NAmurResearchInstituteforLIfeSciences(NARILIS),UniversityofNamur(UNamur),61ruede
Bruxelles,5000Namur,Belgium
bLaboratoryofClinicalCellTherapy,InstitutJulesBordet,UniversitéLibredeBruxelles(ULB),Brussels,Belgium
cUniversitéCatholiquedeLouvain,InstitutdeRechercheCliniqueetExpérimentale(IREC),LaboratoryofPediatricHepatologyandCellTherapy,Brussels,
Belgium
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received23January2014
Receivedinrevisedform20June2014 Accepted22July2014
Availableonline30July2014 Keywords:
Oxidativephosphorylation Hepatocytes
Mesenchymalstromalcells Mitochondrialturnover Cell(de)differentiation
a
b
s
t
r
a
c
t
Mitochondrialbiogenesisandmetabolismhaverecentlyemergedasimportantactorsofstemnessand differentiation.Ontheonehand,thedifferentiationofstemcellsisassociatedwithaninductionof mitochondrialbiogenesisandashiftfromglycolysistowardoxidativephosphorylations(OXPHOS).In addition,interferingwithmitochondrialbiogenesisorfunctionimpactsstemcelldifferentiation.Onthe otherhand,someinversechangesinmitochondrialabundanceandfunctionareobservedduringthe reprogrammingofsomaticcellsintoinducedpluripotentstemcells(iPSCs).Yetalthoughgreatpromises incelltherapymightgeneratebetterknowledgeofthemechanismsregulatingthestemnessand differ-entiationofsomaticstemcells(SSCs)—whicharepreferredoverembryonicstemcells(ESCs)andiPSCs becauseofethicalandsafetyconsiderations—littleinterestwasgiventothestudyoftheirmitochondria. Thisstudyprovidesadetailedcharacterizationofthemitochondrialbiogenesisoccurringduringthe hep-atogenicdifferentiationofbonemarrow-mesenchymalstemcells(BM-MSCs).Duringthehepatogenic differentiationofBM-MSCs,anincreasedabundanceofmitochondrialDNA(mtDNA)isobserved,aswell asanincreasedexpressionofseveralmitochondrialproteinsandbiogenesisregulators,concomitant withincreasedOXPHOSactivity,capacity,andefficiency.Inaddition,oppositechangesinmitochondrial morphologyandintheabundanceofseveralOXPHOSsubunitswerefoundduringthespontaneous ded-ifferentiationofprimaryhepatocytes.Thesedatasupportreversemitochondrialchangesinadifferent contextfromgenetically-engineeredreprogramming.Theyargueinfavorofamitochondrialinvolvement inhepaticdifferentiationanddedifferentiation.
©2014ElsevierLtd.Allrightsreserved.
Abbreviations:␣1AT,␣-1-antitrypsin;BM-MSCs,bone-marrowmesenchymalstemcells;COX1,subunitIofthecytochromecoxidase;COX2,subunitIIofthecytochromec oxidase;COX4,subunitIVofthecytochromecoxidase;DRP1,dynamin-relatedprotein1;ECAR,extracellularacidificationrate;ERR,estrogen-relatedreceptors;ESCs, embry-onicstemcells;FCCP,carbonylcyanide4-(trifluoromethoxy)phenylhydrazone;G6PC,glucose-6-phosphatasecatalyticsubunit;iPSCs,inducedpluripotentstemcells;mtDNA, mitochondrialDNA;ND2,NADHdehydrogenasesubunit2;NRF-1and-2,nuclearrespiratoryfactor1and2;OCR,oxygenconsumptionrate;OTC,ornithinetranscarbamylase; nDNA,nuclearDNA;OXPHOS,oxidativephosphorylation;PEPCK1,phosphoenolpyruvatecarboxykinase1;PGC-1,PPARgammacoactivator1;PPAR,peroxisome proliferator-activatedreceptor;PPIE,peptidyl-prolylcis-transisomeraseE;PRC,PGC-1-relatedcoactivator;SSCs,somaticstemcells;TAT,tyrosineaminotransferase;TDO2,tryptophan 2,3-dioxygenase;TFs,transcriptionfactors;TFAM,mitochondrialtranscriptionfactorA.
∗ Correspondingauthor.LaboratoryofBiochemistryandCellBiology(URBC),NAmurResearchInstituteforLIfeSciences(NARILIS),UniversityofNamur(UNamur),61rue deBruxelles,5000Namur,Belgium,Tel.:+32081724128;fax:+32081724135.
E-mailaddresses:[email protected](A.Wanet),[email protected](N.Remacle),[email protected](M.Najar),[email protected](E.Sokal),
[email protected](T.Arnould),[email protected](M.Najimi),[email protected](P.Renard).
http://dx.doi.org/10.1016/j.biocel.2014.07.015
1. Introduction
Dependingonphysiologicandcellularcues,mitochondriacan display differences in their abundance, morphology and func-tions. Mitochondrial biogenesis is a complex process involving lipidmembraneformation,mitochondrialDNA(mtDNA) replica-tionandtranscription,andcoordinatedsynthesisofmitochondrial proteinsencodedbybothnuclearandmitochondrialgenomes.The coordinationbetweenthenuclearandmitochondrialgenomesis achievedthroughtheexpressionofnucleus-encoded mitochon-drialproteinswhichcontrolmtDNAreplicationandtranscription, suchasthemitochondrial transcriptionfactor A(TFAM). Inthe nucleus,theexpressionof mitochondrialgenes iscontrolledby alimitednumber oftranscriptionfactors(TFs),includingNRF-1 and-2(nuclearrespiratory factors1and2),PPARs(peroxisome proliferator-activatedreceptors␣,/␦,and␥)andERRs (estrogen-relatedreceptors␣,,and␥).(Interestedreaderscanreferto(Hock andKralli,2009)foradetailedreviewoftheinvolvementofthese TFsinmitochondrialbiogenesis).TheactivityoftheseTFsisitself controlledby thePGC-1(PPARgammacoactivator 1)family of coactivators:PGC-1␣;PGC-1;andPRC(PGC-1-related coactiva-tor)(HockandKralli,2009).Amongthese,thebestdescribedis PGC-1␣,aproteinconsideredtobethemasterregulatorof mito-chondrialbiogenesisandfunction(Fernandez-MarcosandAuwerx, 2011).
Anumberofrecentstudiesevidencedanenhanced mitochon-drialbiogenesisinvariousstemcelldifferentiationmodels(Chen etal.,2012,Xuetal.,2013).InESCs,themitochondrialphenotype hasbeendescribedas“immature,”consistingoffewmitochondria, containingpoorlydevelopedcristae,anddisplayingaperinuclear location(Choetal.,2006,Lonerganetal.,2007,StJohnetal.,2005). Whilethesecells mainlyrelyonglycolysisfortheirenergy pro-duction,differentiatingcellsdisplay anincreasedmitochondrial massandmtDNAabundance,amoredevelopedmitochondrial net-work,andashifttowardOXPHOStomeettheirenergydemands (Chungetal.,2010,Facucho-Oliveiraetal.,2007,Lonergan,Bavister, 2007,Prigioneetal.,2010,Suhretal.,2010).Inaddition,theuse ofmoleculespromotingorinhibitingmitochondrialbiogenesisor function,orinterferingwiththeexpressionofmitochondrial bio-genesisregulatorsorproteinsinvolvedinmitochondrialfunction, hasbeendemonstrated to impactstemness and cell differenti-ation (Huang et al.,2011, Tormoset al.,2011, Xuetal., 2013). Forinstance,theattenuationofmitochondrialfunctionusing car-bonyl cyanide m-chlorophenylhydrazone (CCCP)in ESCsresults in increased transcriptional levels of the pluripotency markers Sox2,Oct4,and Nanog,and intherepression of transcriptional programsassociated withlineagedifferentiation (Mandal etal., 2011).Conversely,thereprogrammingofsomaticcellsintoiPSCs isaccompaniedbyinversemodifications,inaprocesscalled “mito-chondrialrejuvenation”(Folmesetal.,2011,Prigione,Fauler,2010,
Suhr,Chang,2010,Varumetal.,2011).Thesedatahaveledtothe hypothesisthattransitionsinmitochondrialmetabolismregulate, orareregulatedby,differentiationandreprogrammingevents.
Inordertobetterappreciatetheinvolvementofmitochondriain differentiationprocesses,twomainpointsremaintobeaddressed. Ontheonehand,duetothelackofanextensivecharacterization ofthemitochondriainthedifferentstemcelldifferentiation mod-elspreviouslystudied,itiscurrentlyunclearifthemitochondrial biogenesisprocessalwaysinvolvesanincreaseinallorsomeof theconstituentsofmitochondria(membrane,proteinandmtDNA) andifitsystematicallyresultsin increasedmitochondrial activ-ity.Inaddition,thekineticsofthemitochondrialbiogenesishas notbeenaddressedinmoststudies,anditremainsunclearwhen, and for how long, themitochondrial biogenesis is induced. On theotherhand,whilemoststudiesofmitochondrial biogenesis instemnessanddifferentiationhavebeenperformedonESCsand
iPSCs,few studiesreportanenhanced mitochondrialbiogenesis duringthedifferentiationofSSCssuchasmesenchymalstemcells (MSCs)(Chenetal.,2008,Palomakietal.,2013,Pietilaetal.,2012,
Tormos,Anso,2011).Ifthemitochondrialbiogenesisrepresentsa hallmarkofalltypesofstemcelldifferentiationprocessestherefore remainstobedemonstrated.AlthoughnotaspluripotentasESCs, SSCsofferseveraladvantagesoverESCs/iPSCsincelltherapy,such asbettersafetyandfewerethicalconcerns.AmongSSCs,MSCscan differentiateintomultiplecelltypes,includinghepatocytes(Banas etal.,2007,Mosnaetal.,2010).Interestingly,hepatocytesdisplaya muchhigherexpressionofseveralmitochondrialOXPHOSsubunits (Suppl.Fig.1)thanBM-MSCs,suggestingthatastrong mitochon-drialbiogenesismightbeassociatedwiththeirdifferentiation.
Primaryhepatocytes,however,cannotbemaintainedinculture inafullydifferentiatedandhighlymetabolicstate.Hepatocytesare subjecttoarapiddedifferentiationprocessduringinvitroculture. Thisprocessisinitiatedduringtheirisolation,andischaracterized by:rapid lossof hepaticgeneexpression,polarity,andactivity; re-entryintothecellcycle;andupregulationofcytoskeletonand mesenchymalproteinssuchasvimentin(Elautetal.,2006,Meyer et al., 2013). Improving our understandingof the mechanisms involvedinhepatocytededifferentiation,andeventually counter-actingthem,couldthusleadtomanyimportantapplicationssuchas pharmacologicaltesting.Thedemonstrationofaninvolvementof mitochondriainhepaticdifferentiationanddedifferentiation pro-cessesmightthereforebeusedasastartingpointforstudiesaimed toimprove/limithepatocytedifferentiation/dedifferentiation.
Inthisstudy,weprovideakineticanddetailedcharacterization ofthemitochondrialbiogenesisinitiatedduringthehepatogenic differentiationofBM-MSCs.Weprovideevidenceforinverse mito-chondrial changes during the spontaneous dedifferentiation of hepatocytes.Weinitiallyfoundevidenceforaninductionof mito-chondrialandmtDNAabundanceandanincreasedexpressionof severalmitochondrialproteinsandmitochondrialbiogenesis reg-ulators,concomitantwithanincreasedoxidativeactivity,during theearlyhepatogenicdifferentiationofBM-MSCs.Incontrast, dur-ing primary hepatocytededifferentiation wefound a reduction ofseveralmitochondrial proteinsinvolvedin OXPHOS. Further-more,whileafissionofthemitochondrialnetworkwasobserved inthedifferentiationmodel,atrendtowardanincreasedfusion was observed during hepatocyte dedifferentiation. These data demonstratetheexistenceofoppositechangesinmitochondrial morphologyandfunctionbetweenthehepaticdifferentiationand hepatocyte dedifferentiation models. They further support the involvementofmitochondriainstemnessandcelldifferentiation.
2. Materialandmethods
2.1. Ethicalguidelines
Thisstudywasapprovedbythelocalethicscommitteeofthe Jules Bordet Institute(Belgium) for obtaining humanBM-MSCs afterinformedconsentfromdonors.Theuseofhumanlivertissue forresearchwasapprovedbytheethicalcommitteeofSt-Luc Hos-pitalandoftheUniversitéCatholiquedeLouvain,Belgium.Primary hepatocyteswereobtainedfromtheHepatocyteandLiverStemcell TissueBankofCliniquesStLuc,actingundertheagreementofthe BelgianMinistryofHealth.
2.2. Cellcultureanddifferentiation
Culturesof humanBM-MSCs wereestablished aspreviously described(Najaretal.,2013)from4differenthealthydonors(aged 2–22years old). BM-MSCs wereexpanded in Dulbecco’s Modi-fiedEagleMediumlowglucose(DMEM-LG)supplementedby1%
penicillinstreptomycinmixture(LifeTechnologies,Carlsbad,CA, USA)and 10%fetal bovineserum(FBS,PAA/GEHealthcare, Pis-cataway,NJ,USA).Priortothehepatogenicinduction,BM-MSCs wereseeded ontype-1 collagen(BD Biosciences, SanJose, CA) coateddishesandexpandedtill95%confluency(«day0»). Hepato-genicinductionwascarriedoutbyincubatingBM-MSCsinIscove’s ModifiedDulbecco’sMedium(IMDM,LifeTechnologies) contain-ing1%penicillin streptomycinmixture and supplementedwith 20ng/mlEGF(PeproTech,RockyHill,NJ,USA)and10ng/ml FGF-2(PeproTech) for 48hand then with10ng/ml FGF-2,20ng/ml HGF(PeproTech),0,61mg/mlnicotinamide(Sigma–Aldrich, Saint-Louis,MO,USA)andinsulin-transferrin-selenium(ITS)mixture1X (LifeTechnologies) for 10 days.Cells werethen treated with a hepatogenicmaturationcocktailcontaining20ng/mloncostatinM (OSM)(PeproTech),1Mdexamethasone(Sigma–Aldrich)andITS for10days.UndifferentiatedBM-MSCsweremaintainedinIMDM supplementedwith1%FBS. Allexperimentswereperformedat passage5–6.
Primary human hepatocytes were isolated from 3 healthy cadaveric donors (4 days, 27 and 48 years old) using a clas-sictwo-stepperfusiontechniqueaspreviouslydescribed(Najimi etal.,2007).Hepatocyteswereseededontype-1collagencoated dishes and maintained in Williams’E medium (Life Technolo-gies)supplementedwith1%penicillin-streptomycin,10%FBS(Life Technologies), 1.275M dexamethasone (Aacidexam) and 1.66 10−5U/mlinsulin(LillyBeneluxSA,Brussels,Belgium)foran8-day dedifferentiationperiod.
Allcellsweremaintainedat37◦Cinahumidifiedatmosphere containing5%CO2.
2.3. PeriodicacidSchiffstaining
Cellswereseededoncoverslips,fixedwith4% paraformalde-hydefor 20minand then stainedusingtheperiodicacid-schiff stainingsystem(Sigma–Aldrich).Briefly,cellswereincubatedwith 1%periodicacidfor10min,rinsedseveraltimeswithdeionized water,incubatedwithSchiff’sreagentfor15min,rinsedseveral timeswithtapwaterandthenobservedunderacontrast-phase lightmicroscope.
2.4. Nucleoidsandmitochondrialnetworkvisualizationand analysis
Toanalyzethemitochondrialnetworkmorphology,expanded, differentiated and undifferentiated BM-MSCs seeded on cover-slipswerestainedwith100nMMitotracker®GreenFM(Molecular Probes,LifeTechnologies)for30minat37◦CandwithCellMaskTM
Orange(MolecularProbes,LifeTechnologies)atadilution1:4000 during the last 5min. Live cells were observed using confocal microscopy(TCSSP5II,LeicaMicrosystems,Wetzlar,Germany). Dedifferentiating hepatocytes seeded on coverslips were fixed with4% paraformaldehyde for 20min, permeabilized for 5min withPBS-1%TritonX-100,andthenincubatedfor2hwith anti-TOM20antibody(BDBioscience#612278)dilutedinPBS-1%BSA (1:100).Cellswerethenincubatedfor1hwithanAlexa-labeled secondaryantibody(MolecularProbes)(1:1000)andobservedby confocalmicroscopy.Thelengthandbranchingstatusofthe mito-chondrialnetworkwasdeterminedbycalculatingtheaspectratio (AR)andendpoints/branchpointsratioofmitochondrialparticles in the entire cell sections using the Image J software accord-ingtoDeVosandSheetz(DeVosandSheetz,2007).Toanalyze nucleoidsaswellasthepercentageofthecytoplasmicarea occu-piedbymitochondria,cellswereseededoncoverslips,fixedwith 4%paraformaldehydefor20min,permeabilizedfor5minwith PBS-1%TritonX-100,andthenincubatedfor2hwithananti-TOM20 antibody(SC-11415, 1:200) (for mitochondriastaining) and an
anti-DNAantibody(ProgenAC-30-10,1:10)(fornucleoidstaining) orananti--cateninantibody(BD610153,1:100)(forcytoplasm staining) dilutedinPBS -1%BSA. Cellswerethen incubatedfor 1hwithAlexa-labeled secondaryantibodies (MolecularProbes) (1:1000) and observed by confocalmicroscopy. The number of nucleoidspercellsection,aswellastheareaoccupiedby mito-chondrialandnucleoidorcytoplasmicstainingswerecalculated usingtheImageJsoftware.
2.5. DeterminationoftherelativeabundanceofmtDNA
TotalDNAwasisolatedusingtheWizardGenomicDNA purifi-cationsystem(Promega,Fitchburg,MI,USA).TherelativemtDNA copynumberwasestimatedbyquantitativereal-timePCR(qPCR) usingprimersforthemitochondrialgeneNADHdehydrogenase subunit2 (ND2) andnormalized tothegenomic DNA byusing primersforthebeclingene(TableA1).Reactionswereperformed usingSYBRGreen PCRMastermix(Roche)andanABI7900HT FastRealTimePCRSystem(AppliedBiosystems,LifeTechnologies). ResultsareexpressedrelativetoexpandingBM-MSCs.
2.6. Real-timeRT-qPCRanalysis
TotalRNAwasisolatedusingtheRNeasyMinikitandQiacube (Qiagen,Venlo,TheNetherlands)andreverse-transcribedusingthe cDNAfirststrandsynthesiskit(RocheAppliedScience,Pensberg, Germany).RT-qPCRwasperformedusingSYBRGreenPCR Mas-termix(Roche),theprimerslistedinTableA1andanABI7900 HTFastRealTimePCRSystem.ThemRNAabundanceof glucose-6-phosphatase catalytic subunit (G6PC), phosphoenolpyruvate carboxykinase1(PEPCK1),ornithinetranscarbamylase(OTC)and tyrosineaminotransferase(TAT)wasdeterminedusingaPCRarray from Qiagen according to the manufacturer’s instructions. All resultswerenormalizedtothemRNAabundanceofthe peptidyl-prolylcis-transisomeraseE(PPIE),andareexpressedrelativeto expandingBM-MSCsorhepatocytesat«0h».
2.7. Westernblotanalysis
Totalcell protein lysates were obtainedusing a buffer con-taining 7M urea, 2M thiourea, 1% CHAPS, 1% ASB14, 1% SDS, 30mMTris,pH8.5supplementedwithCompleteProteaseInhibitor Cocktail (Roche)and 4% phosphatase inhibitor cocktail (25mM Na3VO4,250mM4-nitrophenylphosphate,250mM
glycerophos-phate,125mMNaF).Anamountof25gofproteinswasresolved on12%(PPAR␥)or4–15%(otherproteins)Mini-PROTEANTGX pre-castgels(Biorad,Hercules,CA,USA).Westernblottinganalysiswas performedusingtheprimaryantibodieslistedin TableA2, sec-ondaryantibodiescoupledtoInfrareddyes[(1:5000(anti-goatIgG) or1:10000(anti-mouseoranti-rabbitIgG),Li-CorBiosciences, Lin-coln,NE,USA)]and detectionbyinfraredfluorescence(Odyssey scanner,Li-CorBiosciences).Thefluorescencesignalintensityof thedetectedbandswascalculatedusingtheOdysseyV3.0 applica-tionsoftware,normalizedtotheoneofHistone3(usedasloading control).ResultsareexpressedrelativetoexpandingBM-MSCsor todedifferentiatinghepatocytesat18h.
2.8. Respirationassays
TheextracellularfluxanalyzerXF96(SeahorseBiosience,North Billerica,MA,USA)wasusedtomeasuretheoxygenconsumption rate(OCR)andextracellularacidificationrate(ECAR)attheendof thehepatogenicinductionstep(day12).Thenecessitytoperform theanalysisonthesamedayexcludedthepossibilitytoanalyzethe respirationofcorrespondingexpandingBM-MSCs.Thedaypriorto theassay,cellswereseededatadensityof20,000cells/well.AXF
TableA1
SequencesofprimersusedinqPCR.
Genes Forwardprimer(5→3) Reverseprimer(5→3)
Albumin TGTTGCATGAGAAAACGCCA GTCGCCTGTTCACCAAGGAT
ATP5A1(ATPsynthase␣subunit1) TCGGTTCTGACCTCGATGCT ACCCGCATAGATAACAGCCAC
Beclin(nucleargene) CCCTCATCACAGGGCTCTCTCCA GGGACTGTAGGCTGGGAACTATGC
COX1(cytochromecoxidase,subunit1) CCTGACTGGCATTGTATTAGC TTTTGGCGTAGGTTTGGTCT COX2(cytochromecoxidase,subunit2) AGATGCAATTCCCGGACGT CATGAAACTGTGGTTTGCTCC COX4i1(cytochromecoxidase,subunit4isoform1) ACCGCGCTCGTTATCATGTG CATGTCCAGCATCCTCTTGGT mtHSP70(mitochondrialheatshockprotein70) CTGAAGAAGACCGGCGAAAGA AGCTTGTTGCACTCATCAGCA ND2(NADHdehydrogenase,subunit2(mitochondrialgene)) TGTTGGTTATACCCTTCCCGTACTA CCTGCAAAGATGGTAGAGTAGATGA
OTC(ornithinetranscarbamoylase) GCGAAATTCGGAATGCACCTT GCTCTGCCAACTTGGTTACAC
PGC-1␣(peroxisomeproliferator-activatedreceptorgamma,coactivator1alpha) TCCTTTCTCTCGCCCAACACGATCT GCATCCGACAGGACAAACAGTGGA PGC-1(peroxisomeproliferator-activatedreceptorgamma,coactivator1beta) AGCATTGGCTTGGAGCATCT AGGGCTCGTGTTGGTTTCAA PPAR␥(peroxisomeproliferator-activatedreceptorgamma) GATGACAGCGACTTGGCAAT AGGGCTTGTAGCAGGTTGTC PPIE(Peptidyl-prolylcis-transisomeraseE) CCGCTCTTGACCCTGCATAT TCCAAGCAGACCCTGAGGAA
Slug ATTCGGACCCACACATTACCT CTGTTGCAGTGAGGGCAAGAA
Sox9 CACACAGCTCACTCGACCTTG TTCTTCGGTTATTTTTAGGATCATCTC
TDO2(tryptophan2,3-dioxygenase) GAGGAACAGGTGGCTGAATTT GCTCCCTGAAGTGCTCTGTA
Vimentin GAGGCTGCCAACCGGAACAATG TCCATTTCACGCATCTGGCGTTC
␣1AT(alpha-1antitrypsin) GGGTCAACTGGGCATCACTAA CCCTTTCTCGTCGATGGTCA
cellmitostresstest(SeahorseBioscience)wasperformedaccording tothemanufacturer’sinstructionsandusingthefollowing chem-ical concentrations:1M oligomycin,0.5M carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone(FCCP), 1MantimycinA and 1M rotenone. Once OCR and ECAR measurements were performed,cellswerelysedin0.5NNaOHandOCRandECAR mea-surementswerenormalizedtotheproteincontentofthewell.Basal respirationwascalculatedbysubtractingthenon-mitochondrial oxygenconsumption(OCRfollowingtheadditionofbothantimycin Aandrotenone)tothebasalOCR.ATPcouplingefficiencyis calcu-latedbysubtractingthenon-mitochondrialoxygenconsumption fromtheOCRfollowingoligomycinadditionandisexpressedas apercentageofthebasalrespiration.Sparerespiratorycapacityis calculatedbysubtractingthenon-mitochondrialoxygen consump-tionfromtheOCRfollowingFCCPadditionandisexpressedasa percentageofthebasalrespiration.Foreachconditionandeach donor,4–12technicalreplicateswereperformed.
2.9. Statisticalanalyses
Dependingonthedatatocompare,one-tailedT-test(Suppl.Fig. 1(A)),onewayanalysisofvariance(Fig.7(B))oroneway(Fig.1(B),
Fig.2(B),Fig.4(B),Fig.6,Fig.7(C))/twoways(Fig.3(A)–(C),Fig.4(A),
Fig.5(A),Fig.5(B),Suppl.Fig.2,Suppl.Fig.3)repeatedmeasure ANOVAtestswereperformed,assumingdatanormaldistribution. Whenequalvariancetestfailed,Friedmanrepeatedmeasures anal-ysisofvarianceonrankswasperformedinstead(Fig.4(A)forCOX4 proteinabundance).Toidentifythesignificantlydifferentgroups, pairwisemultiplecomparisonsusingtheStudent-Newman-Keuls methodwereperformed(*or#,p<0.05;**or##,p<0.01;***or ###,p<0.001)(*,comparisonsbetweendifferenttimepoints;#, comparisonsbetweenDiffandUndiffataparticulartimepoint). AllstatisticalanalysesweredoneusingtheSigmaStat3.2software (Systat,SanJose,CA,USA).
3. Results
3.1. TheinvitrohepatogenicdifferentiationofBM-MSCs
BM-MSCsweresubmittedtoahepatogenicdifferentiation pro-tocolthatconsistsofasequentialincubationwithgrowthfactors, cytokinesandcofactors(Fig.1(A)).Thecellsobtainedduringor at theend of this process,defined asdifferentiated cells (Diff), werecomparedwithundifferentiatedcells(Undiff)culturedover thesameperiodinacontrolmedium,aswellaswithexpanding BM-MSCs(Exp)fromthecorrespondingpassage.Thesetwo con-trolconditionswereanalyzedtoobservethechangesduetocell differentiationperseortotheprolongedinvitrocultureperiod.
TheefficiencyofhepatogenicdifferentiationofBM-MSCswas evaluatedatthemorphologic,geneticandfunctionallevels.Atthe endofthelastmaturationstep,anincreasedmRNAabundanceof severalhepaticmarkerssuchasalbumin,␣-1-antitrypsin(␣1AT)or tryptophan2,3-dioxygenase(TDO2)(p=0.053)wasnoticed.Atthe sametime,theabundanceofthepluripotentmarkerSox9mRNA wassuppressed(Fig.1(B)).
Whilerelativelyheterogeneous,differentiatedcellsalsodevelop an increasedability tostoreglycogen asevidenced byperiodic acid–Schiff(PAS)staining(Fig.1(C)).Onthesemicrographs,one canobservetheacquisitionofthetypicalhepatocyte-likepolygonal shapeofthedifferentiatedcells.
3.2. ThemitochondrialnetworkofBM-MSCsundergoes morphologicalchangesduringhepatogenicdifferentiation
Although the globular shape and perinuclear localization of mitochondriain ESCsand iPSCs hasbeenregarded asa poten-tialmarkerof pluripotency (Lonergan,Bavister, 2007), it is not clearly establishedifthemorphologyof themitochondrial net-workispluripotency-dependentorstemcellspecific.Stainingof
TableA2
AntibodiesusedforWestern-blottingapplications.
Target Manufacturer Reference Dilutionfactor
Histone3 CellSignalingTechnology(Danvers,MA,USA) #4499 1:5000
COX1(Cytochromecoxidase,subunit1) Abcam(Cambridge,UK) ab14705 1:2000
COX2(Cytochromecoxidase,subunit2) Abcam(Cambridge,UK) ab110258 1:2000
COX4(Cytochromecoxidase,subunit3) LifeTechnologies(Carlsbad,CA,USA) A21348 1:500
ATPsynthase␣ LifeTechnologies(Carlsbad,CA,USA) A21350 1:1000
mtHSP70(mitochondrialheatshockprotein70) EnzoLifeSciences(Farmingdale,NY,USA) ALX-804-077 1:1000 PPAR␥(peroxisomeproliferator-activatedreceptorgamma) SantaCruzBiotechnology(Dallas,TX,USA) SC-7196 1:500 PGC-1␣(peroxisomeproliferator-activatedreceptorgamma,
coactivator1alpha)
Fig.1.ThehepatogenicdifferentiationofBM-MSCsresultsintheacquisitionofhepaticfeatures.(A):Representationofthehepatogenicdifferentiationprotocoland exper-imentalconditions.(B):mRNAabundanceofseveralpluripotency(Sox9)andhepaticmarkers(Albumin,␣-1-antitrypsin(␣1AT),tryptophan2,3-dioxygenase(TDO2))in expanding,differentiatedandundifferentiatedBM-MSCsdeterminedusingRT-qPCR.Dataarepresentedasmean±SEM(n=4independentdonorcells).(C):Periodic-acid Schiffstainingillustratingtheabilityofsomedifferentiatedcellstostoreglycogen.
Fig.2. ThehepatogenicdifferentiationofBM-MSCsisaccompaniedbychangesinthemitochondrialnetworkmorphology.(A):Livingexpanding,differentiatedand undif-ferentiatedBM-MSCswerestainedwithmitotrackergreen®FM(green)andcellMaskTMOrange(red)andobservedusingconfocalmicroscopy.(B):Twoparametersofthe mitochondrialnetworkmorphology,theaspectratioandendpoints/branchpointsratiowereanalyzedfor10cellsforeachconditionandfor4differentdonors.Datawere firstaveragedforthe10cellsobtainedforeachdonorineachconditionandarepresentedasmean±SEMofthedataobtainedforthe4independentdonors.
Fig.3.AnincreaseinmitochondriaandmtDNAabundanceisobservedduringthehepatogenicdifferentiationofBM-MSCs.(A):Thepercentageofthecytoplasmicarea (visualizedby-cateninimmunostaining,seeFig.3(D))occupiedbymitochondria(visualizedbyTOM20immunostaining,seeFig.3(D))wasdeterminedfor10cellsper donorforeachconditionandfor4differentdonors.Datawerefirstaveragedforthe10cellsobtainedforeachdonorineachconditionandarepresentedasmean±SEMof thedataobtainedforthe4independentdonors.(B):RelativeabundanceofthemtDNAcontentdeterminedbyquantitativePCR.Dataarepresentedasmean±SEM(n=4 independentdonorcells).(C):Thenumberofnucleoidspercellsection(determinedbynucleoidimmunostaining,seeFig.3(E))wascalculatedfor10cellsperdonorforeach conditionandfor4differentdonors.Datawerefirstaveragedforthe10cellsobtainedforeachdonorineachconditionandarepresentedasmean±SEMofthedataobtained forthe4independentdonors.(D):Immunofluorescencestainingofmitochondria(usingananti-TOM20antibody,ingreen)andcytoplasm(usingananti--cateninantibody, inred)visualizedbyconfocalmicroscopy.(E):Immunofluorescencestainingofnucleoids(usingananti-DNAantibody,ingreen)andmitochondria(usingananti-TOM20 antibody,inred)visualizedbyconfocalmicroscopy.
themitochondrialnetworkofBM-MSCswithMitoTracker®Green (aprobethatstainsmitochondriaregardlessofmembrane poten-tial)(Zhang,1994)andCellMaskTMOrange(forstainingtheplasma
membrane)revealedthatexpandingBM-MSCsdisplayadeveloped networkcomposedofthread-likemitochondriaspreadthroughout thecytoplasm(Fig.2(A)).Undifferentiatedcellsalsodisplaya mito-chondrialpopulationorganizedinabranched/reticulatednetwork, yetwefoundamorefragmentedmorphology,mostlycomposedof rod-likeparticles,indifferentiatedcells.Quantitativeanalysisof mitochondrialmorphologywiththeImageJsoftware(DeVosand Sheetz,2007)demonstrated,indifferentiatedcells,adecreasein
theaspectratio,whichmeasurestheelongationofmitochondrial particles,andanincreasedratioofendpointstobranchpoints, whichisinverselyproportionaltothemitochondrialnetworking (Fig.2(B)).
3.3. HepatogenicdifferentiationofBM-MSCsisaccompaniedbya mitochondrialbiogenesisresultinginenhancedmitochondrial respiration
BM-MSCsappeartodisplayamoreelongatedandmore devel-oped mitochondrial network than the one reported in ESCs.
Fig.4.AnincreaseinseveralmitochondrialproteinabundanceandrespirationisobservedduringthehepatogenicdifferentiationofBM-MSCs.(A):Relativeabundance ofseveralmitochondrialproteinswasanalyzedbyWestern-Blot.Signalintensitywasquantifiedandnormalizedtotheabundanceofhistone3(loadingcontrol).Dataare presentedasmean±SEM(n=4independentdonorcells).(B):OCRchangesinresponsetooligomycin,FCCPandantimycinAincombinationwithrotenonewereusedto calculatethebasalrespiration,respirationcouplingefficiencyandsparerespiratorycapacityofdifferentiatedandundifferentiatedcellsatday12.ThebasalECARwasalso assessed.Dataarepresentedasmean±SEM(n=4independentdonorcells).
Nevertheless,wehypothesizedthatbothquantitativeand quali-tativechangesin mitochondrialcomposition,perhapsdrivenby mitochondrial biogenesisregulators and leading toa metabolic shifttowardincreasedOXPHOS,mayaccompanythehepatogenic differentiationofBM-MSCs.Wethusfirstanalyzedon immuno-fluorescencesectionsobservedbyconfocalmicroscopythefraction of cytoplasmic area occupied by mitochondria (Fig. 3 (A) and (D)).Interestingly,weevidencedasignificantincreaseinthe per-centageofthecytoplasmicareaoccupiedbymitochondria,rising from11.23±0.71%inexpandingBM-MSCs,to15.93±1.25%and 16.32±0.96%attheendofthehepaticinductionandmaturation stepsrespectively, while slightly decreasingin undifferentiated cells. We next analyzed the evolution of mtDNA copy number duringthecelldifferentiationprocess.Weobservedasignificant inductionof mtDNA copy number in differentiatingcells when comparedwithexpandingBM-MSCs, reaching2.13±0.14 folds attheendoftheinductionstep(day12)and3.87±0.25foldsat theendofthematurationstep(day22).ThemtDNAcopy num-berremainedstableinundifferentiatedcells(Fig.3(B)).Although nochangewasfoundattheendofthehepaticinductionstep,we evidencedanincreaseintherelativenumberofnucleoidspercell attheend ofthehepaticmaturationstepindifferentiatedcells
(Fig. 3 (C) and (E)).Nevertheless, no significantdifference was evidencedwhenconsideringtheareadensityofnucleoidsrelative tomitochondria,whichcouldbeexplainedbyadecreasedsizeof nucleoidsindifferentiatingcellswhencomparedtoexpandingcells (Suppl.Fig.2(A)and(B)).
Giventhatmitochondrialbiogenesisalsorequiresthe synthe-sisandimportofmanymitochondrialproteins,wenextanalyzed theabundanceofseveralmitochondrialproteinsthroughoutthe differentiationprocess.TheabundanceofmtHSP70,a mitochon-drial matrix chaperone required for the import and folding of mostmitochondrialinnermembraneandmatrixproteins(Deocaris etal.,2006),althoughnotsignificantlymodifiedatthemRNAlevel (Suppl.Fig.3)wasfoundtobesignificantlyinducedattheprotein levelindifferentiatingcells(Fig.4(A)).Inaddition,theabundance ofseveralproteinsinvolvedintheOXPHOSchainincreased dur-ingthehepatogenicdifferentiationofBM-MSCs,bothatthemRNA (Suppl.Fig.3)andproteinlevels(Fig.4(A)andSuppl.Fig.4).The proteinabundanceofthesubunitIIofthecytochromecoxidase (COX2),whichisencodedbymtDNA,wasfoundtobesignificantly increasedindifferentiatedcells(vs.expandingorundifferentiated BM-MSCs),withamaximumabundancereachedattheendofthe hepatogenicinductionstep(day12).Acomparableinductionof
Fig.5. AnincreasedexpressionofseveralmitochondrialbiogenesisregulatorsisobservedduringthehepatogenicdifferentiationofBM-MSCs.(A)PGC-1␣,PGC-1and PPAR␥mRNAabundancewasassessedbyRT-qPCR.Dataarepresentedasmean±SEM(n=4independentdonorcells).(B):Relativeproteinabundancewasanalyzedby Western-Blot.Signalintensitywasquantifiedandnormalizedtotheabundanceofhistone3(loadingcontrol).Dataarepresentedasmean±SEM(n=4independentdonor cells).
thesubunitIV(COX4,encodedbynDNA)occurredduringthe hep-atogenicinductionstep,whiletheabundanceofthesubunit␣of theATPsynthaseincreasedcontinuouslyduringthehepatogenic inductionand maturationsteps.While anincreased abundance ofthesubunitIofthecytochromecoxidase(COX1)(encodedby mtDNA)wasalsofoundindifferentiatingcells,thedifferencewas notsignificantinourexperimentalconditions.
TheincreasedabundanceofseveralOXPHOScomplexsubunits promptedustoanalyzethemitochondrialrespirationattheendof thehepatogenicinductionstep.TheOCRwasthusmeasuredina basalconditionaftertheadditionofoligomycinA(anATPsynthase inhibitor,inordertoevaluatethecouplingefficiencyofthe respi-ratorychain),FCCP(anuncouplingprotonophore,toevaluatethe sparerespiratorycapacityofcells),andacombinationofantimycin Aandrotenone(mitochondrialcomplexIIIandIinhibitors, respec-tively).Theseconditionswerenecessaryinordertocalculateand subtractthenon-mitochondrialoxygenconsumptionfromother measurements(BrandandNicholls,2011)(Fig.4(B)).Wefound thatthebasalrespirationwassignificantlyincreasedin differenti-atedvs.undifferentiatedcells,whichsuggeststhatdifferentiated cellsrely moreonOXPHOSfor theirATPproduction. Moreover, thecouplingefficiency,representingthefractionofthebasal res-pirationthatisusedforATPproduction,wassignificantlyhigher indifferentiatedcells.Thesparerespiratorycapacity,which repre-sentsthecell’sabilitytoincreasesubstratesandelectronfluxacross theOXPHOSchaintorespondtoanincreasedenergydemand,was alsohigherindifferentiatedcells.Together,thesedatademonstrate that,inabasalcondition,differentiatedcellshaveamoreefficient OXPHOSsystemandrelymoreonOXPHOS.Also,differentiatedcells canincrease theirrelianceonOXPHOSduringincreasedenergy demand.InadditiontomeasuringtheOCR,wealsoanalyzedthe ECARinabasalcondition.WeobservedaslightlylowerECARin differentiatedvs.undifferentiatedcells,butthedifferencewasnot statisticallysignificant.Altogether,thesedatastronglysupporta metabolicshifttowardincreasedOXPHOSduringthehepatogenic inductionofBM-MSCs.
3.4. Severalmitochondrialbiogenesisregulatorsareincreased duringthehepatogenicdifferentiationofBM-MSCs
Inordertoinvestigatethepotentialmechanismsregulatingthe mitochondrialbiogenesisindifferentiatingBM-MSCs,weanalyzed therelativemRNAabundanceofmostofthewell-knownregulators of mitochondrial biogenesis.These regulators includedPGC-1␣, PGC-1,PPAR␣,-␥and-␦,Nrf1andNrf2␣,ERR␣,POLG,POLG2, PolRmt,andTFAMatdifferenttime pointsofthedifferentiation process.Althoughnochangewasobservedin theexpressionof mostofthesecandidates(datanotshown),weevidencedatrend towardaninductionofthemRNAabundanceofPPAR␥(withno isoformdiscrimination)andoftheglobalco-regulatorsPGC-1␣and PGC-1(Fig.5(A)).Attheproteinlevel,weobservedasignificant inductionofPPAR␥1attheendofthedifferentiation,andofboth PPAR␥2andPGC-1␣attheendoftheinductionandmaturation steps(Fig.5(B)).Togetherwiththepreviousresults,thesedata fur-thersupportthatanenhancedmitochondrialbiogenesisprocess occursduring thehepatogenicdifferentiationof BM-MSCs. This processinvolvesatleasttwowell-knownregulatorsof mitochon-drialbiogenesisandresultsinthereplicationofmtDNA,synthesis ofmitochondrialproteinsandincreasedmitochondrialrespiration. 3.5. Mitochondrialmodificationsoccurduringtheinvitro
dedifferentiationofhumanprimaryhepatocytes
ThereprogrammingofsomaticcellsintoiPSCscorrelateswitha reductionofmitochondrialabundanceandfunction,aremodeling ofwhichisrequiredfortheinductionofpluripotencyiniPSCs(Xu etal.,2013).Wethusaimedtodetermineifsimilarstructuralor functionalremodelingofmitochondriaoccursduringthe sponta-neousinvitrodedifferentiationofhepatocytes,inordertoevidence reversemitochondrial changescorrelatingwithhepatocyte (de-)differentiation.
A drastic loss of multiple hepatic gene markers, includ-ingalbumin(p=0.055),glucose-6-phosphatase(G6PC)(p=0.054),
Fig.6. Primaryhumanhepatocytededifferentiationisassociatedwiththerapidlossofhepatic(albumin,G6PC,PEPCK1,OTC,TAT)andprogressiveacquisitionofmesenchymal markers(vimentin,snail).mRNAabundancewasassessedbyRT-qPCR.Dataarepresentedasmean±SEM(n=3independentdonorcells).
phosphoenolpyruvatecarboxykinase1(PEPCK1),ornithine tran-scarbamylase (OTC) and tyrosine amino-transferase (TAT) was noted within 18h post-isolation at the mRNA level (Fig. 6). Regardingmesenchymalmarkers(whichareinducedduringthe spontaneous in vitro dedifferentiation of hepatocytes (Meyer, Dzieran, 2013), a statistically significantinduction of vimentin, alongwitha moderateincreasein theabundanceof slug,were observedatthemRNAlevel(Fig.6).
Interestingly, the analysis of the mitochondrial morphology throughoutthededifferentiationprocessexhibitedchangestothe network structure that weretheopposite of those observed in thedifferentiationmodel (Fig.7(A)).Indeed, themitochondrial aspectratioincreased,whiletheratioofendpointstobranchpoints decreasedduringthededifferentiationprocess(Fig.7(B)),which supportsanelongationandincreasednetworkingofmitochondria. Inaddition, wefounda repressionof theabundanceofseveral OXPHOScomplexsubunitsheretoforedemonstratedtobeinduced inthedifferentiationmodel:COX1,COX2,COX4,andthesubunit␣ oftheATPsynthase(Fig.7(C)andSuppl.Fig.5).Thesedatasuggest thatoppositechangesinthemitochondrialnetworkmorphology, andintheabundanceofproteinsinvolvedinmitochondrialactivity, occurduringthedifferentiationofBM-MSCsintohepatocyte-like cellsandinthededifferentiationofprimaryhepatocytes.
4. Discussion
Recent discoveries have demonstrated a strong interplay between mitochondrial biogenesis/function and stem-ness/differentiation programs in ESCs and iPSCs. However, evidenceofasimilarphenomenoninMSCsis(toourknowledge) veryscarce,andlimitedtothecontextsofosteogenic(Chen,Shih, 2008,Palomaki,Pietila,2013,Pietilaetal.,2010,Pietila,Palomaki, 2012)andadipogenic(Hofmannetal.,2012,Huang,Chen,2011,
Tormos, Anso, 2011) differentiation. It is still unclear whether thisphenomenon isahallmark ofcelldifferentiationprograms, ordisplaysspecificitiesdependingonthestemcelltypesor lin-eagesintowhichtheyaredifferentiated.Investigationofmultiple modelsisthereforerequired.Inthisstudyweprovide,forthefirst time, a detailed characterization of the mitochondrial changes occurring during the hepatogenic differentiation of BM-MSCs. Wefirstevidencedanincreasedmitochondriatocytoplasmratio, suggestingthatthecytoplasmofdifferentiatingcellsisenriched in mitochondria compared to expanding or undifferentiating cells.TheinductionofmtDNAabundanceduringthehepatogenic
differentiation (3.87 folds) appeared stronger than during the osteogenic differentiation ofBM-MSCs (1.37folds) (Chen, Shih, 2008) and resulted at the end of the hepatic maturation step in anincreased number of nucleoids.Nevertheless, we didnot findanysignificantchangeintheareadensityofnucleoidswhen normalizedtothemitochondrialcontent(Suppl.Fig.2(A)),which canbeexplained both byan increase inmitochondrial content aswellasbyadecreasein nucleoidsize(Suppl.Fig.2(B)).This decreaseinnucleoidsizebothindifferentiatedand undifferenti-atedcellsversusexpandingcellsmightbeexplainedbytheserum withdrawal in those conditions, as the serum deprivation has beenassociatedwithmorecondensednucleoids(Prachar,2010). Obviously this nucleoid compaction does not impact mtDNA-encodedgene expression,asboth COX1andCOX2aresimilarly expressed in expanding and undifferentiatedBM-MSCs (Suppl.
Fig.3).
InadditiontotheincreaseinmtDNA,weobserved(asalready demonstratedduringtheosteogenic(Chen,Shih,2008)and adi-pogenic(Hofmann, Beyer,2012)differentiationofBM-MSCs)an increasedexpressionforseveralmitochondrial proteins, includ-ing OXPHOS subunits. Although the mitochondrial chaperone mtHSP70isupregulatedatthepost-transcriptionallevelonly,we evidencedanincreasedmRNAabundanceforCOX1,COX2,COX4i1 andATP5A1.Interestingly,themRNAlevelsofnDNA-encoded sub-units(COX4i1andATP5A1)wereinducedtoalesserextentthan theproteinlevels,whilesimilarmRNAandproteininductionfolds werefoundformtDNA-encodedsubunits.Thesedatasuggestthat nDNA-encodedandmtDNA-encodedsubunitexpressionis upre-gulatedbydifferentmechanismstofinallyreachanincreasedand balancedproductionofOXPHOScomplexes.
AlthoughwefoundacontinuousincreaseinthemtDNA abun-dance and a still significantly increased abundance in several mitochondrial proteinsand biogenesis regulators at the end of thehepatogenicmaturationstep(day 22),wedidnotice apeak intheabundanceofseveralmitochondrialproteinsattheendof theinductionstep(day12).ThesedatasuggestthatthemtDNA copynumberisnotnecessarilyassociatedwithOXPHOSsubunit expression,whichisinagreementwithdatashowingthatmtDNA isnotcorrelatedwiththecytochromecoxidaseactivityindifferent celltypes(VandenBogertetal.,1993).Thedifferentconstituent of mitochondria (lipids, mtDNA and proteins) might therefore bedifferentiallyregulated.Interestingly,studiesofadipogenicor osteogenicdifferentiationofBM-MSCsalsoevidencedanincrease inmitochondrial biogenesisandfunctionduringtheearlysteps
Fig.7.OppositechangesinthemitochondrialnetworkmorphologyandOXPHOSproteinabundanceareobservedindedifferentiatingprimaryhepatocytes.(A)The mito-chondrialnetworkmorphologywasvisualizedbyperformingaTOM20immunofluorescencecombinedwithconfocalmicroscopyvisualization.(B):Theaspectratioand endpoints/branchpointsratiowereanalyzedfor12cellsforeachtime-pointofthededifferentiationprocess.Dataarepresentedasmean±standarddeviation.(C).Relative abundanceofseveralmitochondrialproteinswasanalyzedbyWestern-Blot.Signalintensitywasquantifiedandnormalizedtotheabundanceofhistone3(loadingcontrol). Dataarepresentedasmean±SEM(n=3independentdonorcells)andexpressedrelativetothe18htimepoint.
ofthedifferentiationprocess(Chen,Shih,2008,Pietila,Palomaki, 2012,Tormos,Anso,2011).Itremainstobedeterminedwhether thisincreasedmitochondrialabundanceandactivity,which pre-cedesthehepatogenicmaturationinourmodel,isimportantfor thecommitmentandearlyinductionofdifferentiation,and/orif thecontinuousincreaseinmtDNAandmitochondrialabundance arealsorequiredforthelatedifferentiationstages.
Atthefunctionallevel,wedemonstratedanincreased oxida-tiveactivity(basalrespiration),capacity(respiratoryreserve),and efficiency(couplingefficiency)ofdifferentiatingcells.Therefore, bothquantitativeandqualitativemodificationsofthe mitochon-drialnetworkoccurredduringthehepatogenicdifferentiationof BM-MSCs, resulting in enhanced mitochondrial respiration. We nextevidencedanincreasedexpressionoftheco-activatorPGC-1␣ (Fig.5),consideredakeyregulatorofmitochondrialbiogenesisand oxidativemetabolism(Scarpulla,2011).Wealsofoundaninduction ofPPAR␥bothduringthestepsofhepatogenicinduction(isoform2) andmaturation(bothisoforms).Interestingly,thesedataare con-sistentwiththepreviouslydescribedPPAR␥-dependentinduction ofPGC-1␣and-expression(Dengetal.,2011,Hondaresetal., 2006).Theseco-regulatorsmayinturndrivethemitochondrial bio-genesisprocessandoxidativeswitchbyco-activatingthemanyTFs thatregulatemitochondrialgeneexpression.
Theinterestdevotedtomitochondrialabundanceandactivityin stemnessanddifferentiationresultsfromtherelevanceconferred byobservationsmadeinmultiplemodels(diversestemcellsand differentiationtypes(Chen,Hsu,2012,Xuetal.,2013)).Yetthere isalsotheintriguingfactthatoppositemodificationsoccur dur-ingthereprogrammingofsomaticcellsintoiPSCs(Xuetal.,2013). Thisprocesshasbeen,sofar,onlydemonstratedintheartificial contextofgenetically-engineeredreprogramming.Inthisstudy,we tookadvantageofthespontaneousdedifferentiationprocess occur-ringduringprimaryhepatocytesinvitroculturetotestwhether oppositechangesareobservedduringhepaticdifferentiationand dedifferentiation.
Interestingly,weevidencedadecreasedabundanceinseveral OXPHOSproteinsubunits(Fig.7(C)),allofwhichwereinducedin theBM-MSCshepatogenicdifferentiationmodel(Fig.4(A)). Impor-tantly,thespontaneousdedifferentiationprocessofhepatocytes doesnotleadtothere-establishmentofpluripotencylikeiniPSC reprogramming.Our datathereforesuggestthatthediminished mitochondrialOXPHOSsubunitabundanceisobservedassoonas cellsareloosingtheirfullydifferentiatedphenotype,anddonot requirethe re-acquisitionof pluripotency. Althoughthis would requirevalidationinothermodels,thesedatamightsuggestthat mitochondriaareremodeledthroughoutthekineticsof differentia-tion/dedifferentiationprogramsandnotinvolvedinatime-limited switchbetweenpluripotencyandcommitmenttoadifferentiation program.
BM-MSCsdisplaytubularinterconnectedmitochondriaspread throughoutthecytoplasm.WhileESCsaregenerallyreportedto displaygranularmitochondria(althoughsomediscrepanciesexist, dependingonthestudies)(Lonerganetal.,2006,Mandal,Lindgren, 2011,Sathananthanetal.,2002,StJohn,Ramalho-Santos,2005), weobserved inhumanumbilicalcord-derivedMSCs, aswellas inadulthumanlivermesenchymalstem/progenitorcells,amore comparablemitochondrialmorphologythaninBM-MSCs (unpub-lisheddata).Thisdevelopedtubularmitochondrialnetworkmay thusbetypicalofmesenchymalstem/progenitorcells.During hep-atogenicdifferentiation,themitochondrialnetworkofBM-MSCs undergoesafragmentation,leadingtosmallermitochondria.The reasons for these modifications in the mitochondrial network morphologyduringhepatogenicdifferentiationarestillunknown. However,severalhypothesesmaypartlyexplainthesechanges. First,an increasingnumber ofstudiespoint towardan involve-mentofmitochondrialfusion/fissionprocessesincellproliferation anddifferentiationprograms.AsrecentlyreviewedbyMitra(Mitra, 2013),multipleevidencesupportsthehypothesisthatareduced expressionoractivityofdynamin-relatedprotein1(Drp1,involved in the mitochondrial fission machinery) results in unopposed
mitochondrialfusionandpromotescellproliferationthroughthe build-upofcyclinE,whichregulatestheentryintheS-phaseof thecellcycle.Thehighlyfusedmitochondrialnetworkof expand-ingBM-MSCs(Fig.2)maythereforepartlyreflecttheproliferation status of these cells. In addition, we observed the progressive acquisitionofa slightlymorefusedmitochondrialnetwork dur-ingthededifferentiationofhepatocytes(Fig.7(A)and(B)).Since isolated hepatocytes re-enterproliferation during isolation and invitroculture(Elaut,Henkens,2006),this hypothesismayalso partlyexplaintheincreasedbranchingandelongationof mitochon-driaduringearlydedifferentiation.Secondly,ithasbeensuggested thatmitochondrialdynamicsinfluenceOXPHOScapacity(Benard etal.,2010).Forinstance,ithasbeendemonstratedthatinhibiting mitochondrialfission,throughdown-regulatingDrp1expression, results in decreased mtDNA abundance and OXPHOS activity (Paroneetal.,2008).Conversely,OXPHOSactivityisalsoreportedto influencemitochondrialdynamics(Benard,Bellance,2010). There-fore,themodificationsofthemitochondrialnetworkmorphology ofdifferentiatingBM-MSCsmayrepresentaprerequisite(ora con-sequence)oftheobservedincreasedOXPHOSactivity.Thirdly,the oppositemodificationsinmitochondrialnetworkmorphologymay resultfromchangesincytoskeletonstructureduringthehepatic differentiationanddedifferentiationprocesses.Indeed,wefound animportantdecreasein-actinand␣-tubulinabundanceduring thedifferentiationofBM-MSCs,whileastronginductionofthese proteinswasobservedduringthededifferentiationofhepatocytes (datanotshown).Interestingly,mitochondriallocation,shape,and tubularstatusareknowntoberegulatedthroughtheirinteraction with,and modificationof, shapeproteinsand cytoskeleton ele-ments(suchasthemicrotubules,microfilaments,andintermediary filamentfamilies),resultinginchangesinmitochondrial morphol-ogy(Kuznetsovetal.,2009).
5. Conclusion
Whileweobservedafragmentationofthemitochondrial net-workmorphologyand aninductionofmitochondrial biogenesis andactivityduringthehepatogenicdifferentiationofBM-MSCs, wereportedseveraloppositechangesduringthespontaneous ded-ifferentiationofprimaryhepatocytes.Theseobservationssupport thehypothesisofastronginvolvementofmitochondriaand mito-chondrialmetabolisminregulatingthedifferentiationofstemcells anddedifferentiationorreprogrammingevents.Furtherresearchis stillrequiredtounravelthemechanismsinvolvedintheinterplay betweenthesetwoprocesses.Understandinghowtheseprocesses areregulated,anddeterminingiftheyregulateeachother,isof greatinterestfortheimprovementofdirecteddifferentiationof stemcellsintospecificcelltypes.Eventhoughthereiscontinuous improvementoftheirefficiency,currentdifferentiationprotocols do not enableone toreach a terminal differentiationstatus as observedinprimarysomaticcells.Thisisparticularlytrueinthe contextof hepatic differentiation,in which even themost effi-cientprotocolsdonotallowforreachingsimilarlevelsofhepatic functions,orinducingallhepaticactivitiessimultaneously(Zhang etal.,2013,Zhouetal.,2012).Ontheotherhand,understanding howoppositemitochondrialmodificationsoccurduringprimary hepatocytededifferentiationmay,inturn,helptopreservethem inamoredifferentiatedstatus.Understandingtheroleplayedby mitochondriainhepaticdifferentiationanddedifferentiationmay thereforebeusefulinregenerativemedicineandpharmacological testing.
Conflictsofinterest
Theauthorsdeclarenoconflictofinterest
Acknowledgments
Anaïs Wanet is a recipient of the FNRS fellowship (Fonds NationaldelaRechercheScientifique,Belgium).Theauthorsthank Pr.SonveauxgroupandparticularlyPaoloPorporato(FATH,IREC, UniversitéCatholiquedeLouvain,Brussels,Belgium)fortheirhelp with respiration analyses, as well as Antoine Fattaccioli, Aude Sauvage,CatherineDemazyandNoëlleNinane(URBC,University ofNamur,Belgium)fortechnicalsupport.
AppendixA. Supplementarydata
Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.biocel. 2014.07.015.
References
BanasA,YamamotoY,TerataniT,OchiyaT.Stemcellplasticity:learningfrom hepatogenicdifferentiationstrategies.DevelopmentalDynamics:AnOfficial PublicationoftheAmericanAssociationofAnatomists2007;236:3228–41.
BenardG,BellanceN,JoseC,MelserS,Nouette-GaulainK,RossignolR.Multi-site controlandregulationofmitochondrialenergyproduction.Biochimicaet Bio-physicaActa2010;1797:698–709.
BrandMD,NichollsDG.Assessingmitochondrialdysfunctionincells.The Biochem-icalJournal2011;435:297–312.
ChenCT,HsuSH,WeiYH.Mitochondrialbioenergeticfunctionandmetabolic plas-ticityinstemcelldifferentiationandcellularreprogramming.Biochimicaet BiophysicaActa2012;1820:571–6.
ChenCT,ShihYR,KuoTK,LeeOK,WeiYH.Coordinatedchangesofmitochondrial biogenesisandantioxidantenzymesduringosteogenicdifferentiationofhuman mesenchymalstemcells.StemCells2008;26:960–8.
ChoYM,KwonS,PakYK,SeolHW,ChoiYM,ParkdoJ,etal.Dynamicchanges inmitochondrialbiogenesisandantioxidantenzymesduringthespontaneous differentiationofhumanembryonicstemcells.BiochemicalandBiophysical ResearchCommunications2006;348:1472–8.
ChungS,ArrellDK,FaustinoRS,TerzicA,DzejaPP.Glycolyticnetworkrestructuring integraltotheenergeticsofembryonicstemcellcardiacdifferentiation.Journal ofMolecularandCellularCardiology2010;48:725–34.
DeVosKJ,SheetzMP.Visualizationandquantificationofmitochondrialdynamics inlivinganimalcells.MethodsinCellBiology2007;80:627–82.
DengT,SieglaffDH,ZhangA,LyonCJ,AyersSD,CvoroA,etal.Aperoxisome proliferator-activatedreceptorgamma(PPARgamma)/PPARgammacoactivator 1betaautoregulatoryloopinadipocytemitochondrialfunction.TheJournalof BiologicalChemistry2011;286:30723–31.
Deocaris CC, Kaul SC,Wadhwa R. On the brotherhoodof the mitochondrial chaperonesmortalinand heatshockprotein60.CellStress&Chaperones 2006;11:116–28.
ElautG,HenkensT,PapeleuP,SnykersS,VinkenM,VanhaeckeT,etal.Molecular mechanismsunderlyingthededifferentiationprocessofisolatedhepatocytes andtheircultures.CurrentDrugMetabolism2006;7:629–60.
Facucho-OliveiraJM,AldersonJ,SpikingsEC,EggintonS.StJohnJC.Mitochondrial DNAreplicationduringdifferentiationofmurineembryonicstemcells.Journal ofCellScience2007;120:4025–34.
Fernandez-Marcos PJ, Auwerx J. Regulation of PGC-1alpha, a nodal regula-torofmitochondrialbiogenesis. TheAmericanJournalofClinicalNutrition 2011;93:884S–90S.
FolmesCD,NelsonTJ,Martinez-FernandezA,ArrellDK,LindorJZ,DzejaPP,etal.
Somaticoxidativebioenergeticstransitionsintopluripotency-dependent gly-colysistofacilitatenuclearreprogramming.CellMetabolism2011;14:264–71.
HockMB,KralliA.Transcriptionalcontrolofmitochondrialbiogenesisandfunction. AnnualReviewofPhysiology2009;71:177–203.
HofmannAD,BeyerM,Krause-BuchholzU,WobusM,BornhauserM,RodelG.
OXPHOSsupercomplexesasahallmarkofthemitochondrialphenotypeof adi-pogenicdifferentiatedhumanMSCs.PloSOne2012;7:e35160.
Hondares E, Mora O, Yubero P, Rodriguez de la Concepcion M, Iglesias R, Giralt M, et al. Thiazolidinediones and rexinoids induce peroxisome proliferator-activatedreceptor-coactivator(PGC)-1alphagenetranscription:an autoregulatoryloopcontrolsPGC-1alphaexpressioninadipocytesvia per-oxisomeproliferator-activatedreceptor-gammacoactivation.Endocrinology 2006;147:2829–38.
HuangPI,ChenYC,ChenLH,JuanCC,KuHH,WangST,etal.PGC-1alphamediates differentiationofmesenchymalstemcellstobrownadiposecells.Journalof AtherosclerosisandThrombosis2011;18:966–80.
KuznetsovAV,HermannM,SaksV,HengsterP,MargreiterR.Thecell-typespecificity ofmitochondrialdynamics.TheInternationalJournalofBiochemistry&Cell Biology2009;41:1928–39.
LonerganT,BavisterB,BrennerC.Mitochondria instemcells.Mitochondrion 2007;7:289–96.
LonerganT,BrennerC,BavisterB.Differentiation-relatedchangesinmitochondrial propertiesasindicatorsofstemcellcompetence.JournalofCellularPhysiology 2006;208:149–53.
MandalS,LindgrenAG,SrivastavaAS,ClarkAT,BanerjeeU.Mitochondrialfunction controlsproliferationandearlydifferentiationpotentialofembryonicstemcells. StemCells2011;29:486–95.
MeyerC,DzieranJ,LiuY,SchindlerF,MunkerS,MullerA,etal.Distinct dedifferentia-tionprocessesaffectcaveolin-1expressioninhepatocytes.CellCommunication andSignaling:CCS2013;11:6.
MitraK.Mitochondrialfission-fusionasanemergingkeyregulatorofcell prolifer-ationanddifferentiation.BioEssays:NewsandReviewsinMolecular,Cellular andDevelopmentalBiology2013;35:955–64.
Mosna F, Sensebe L, Krampera M. Human bone marrow and adipose tis-suemesenchymalstemcells:auser’sguide.Stem CellsandDevelopment 2010;19:1449–70.
NajarM,RaicevicG,Fayyad-KazanH,DeBruynC,BronD,ToungouzM,etal.Impact ofdifferentmesenchymalstromalcelltypesonT-cellactivation,proliferation andmigration.InternationalImmunopharmacology2013;15:693–702.
NajimiM,KhuuDN,LysyPA,JazouliN,AbarcaJ,SempouxC,etal.Adult-derived humanlivermesenchymal-likecellsasapotentialprogenitorreservoirof hepa-tocytes.CellTransplantation2007;16:717–28.
PalomakiS,PietilaM,LaitinenS,PesalaJ,SormunenR,LehenkariP,etal.HIF-1alpha isupregulatedinhumanmesenchymalstemcells.StemCells2013;31:1902–9.
ParonePA,DaCruzS,TonderaD,MattenbergerY,JamesDI,MaechlerP,etal. Pre-ventingmitochondrialfissionimpairsmitochondrialfunctionandleadstoloss ofmitochondrialDNA.PloSOne2008;3:e3257.
PietilaM,LehtonenS,NarhiM,HassinenIE,LeskelaHV,ArankoK,etal. Mito-chondrialfunctiondeterminestheviabilityandosteogenicpotencyofhuman mesenchymalstemcells.TissueEngineeringPartC,Methods2010;16:435–45.
PietilaM,PalomakiS,LehtonenS,RitamoI,ValmuL,NystedtJ,etal.Mitochondrial functionandenergymetabolisminumbilicalcordblood-andbone marrow-derivedmesenchymalstemcells.StemCellsandDevelopment2012;21:575–88.
PracharJ.Mouseandhumanmitochondrialnucleoid–detailedstructureinrelation tofunction.GeneralPhysiologyandBiophysics2010;29:160–74.
PrigioneA,FaulerB,LurzR,LehrachH,AdjayeJ.Thesenescence-related mitochon-drial/oxidativestresspathwayisrepressedinhumaninducedpluripotentstem cells.StemCells2010;28:721–33.
SathananthanH,PeraM,TrounsonA.Thefinestructureofhumanembryonicstem cells.ReproductiveBiomedicineOnline2002;4:56–61.
ScarpullaRC.MetaboliccontrolofmitochondrialbiogenesisthroughthePGC-1 fam-ilyregulatorynetwork.BiochimicaetBiophysicaActa2011;1813:1269–78.
StJohnJC,Ramalho-SantosJ,GrayHL,PetroskoP,RaweVY,NavaraCS,etal.The expressionofmitochondrialDNAtranscriptionfactorsduringearly cardiomy-ocyteinvitrodifferentiationfromhumanembryonicstemcells.Cloningand StemCells2005;7:141–53.
SuhrST,ChangEA,TjongJ,AlcasidN,PerkinsGA,GoissisMD,etal.Mitochondrial rejuvenationafterinducedpluripotency.PloSOne2010;5:e14095.
TormosKV,AnsoE,HamanakaRB,EisenbartJ,JosephJ,KalyanaramanB,etal. Mito-chondrialcomplexIIIROSregulateadipocytedifferentiation.CellMetabolism 2011;14:537–44.
VandenBogertC,DeVriesH,HoltropM,MuusP,DekkerHL,VanGalenMJ,etal.
Regulationoftheexpressionofmitochondrialproteins:relationshipbetween mtDNAcopynumberandcytochrome-coxidaseactivityinhumancellsand tissues.BiochimicaetBiophysicaActa1993;1144:177–83.
VarumS,RodriguesAS,MouraMB,MomcilovicO,AtEasleyC,Ramalho-SantosJ,etal.
Energymetabolisminhumanpluripotentstemcellsandtheirdifferentiated counterparts.PloSOne2011;6:e20914.
XuX,DuanS,YiF,OcampoA,LiuGH.IzpisuaBelmonteJC.Mitochondrialregulation inpluripotentstemcells.CellMetabolism2013;18:325–32.
ZhangYZ.Novelfluorescentacidicorganelle-selectivedyesand Mitochondrion-selectivedyesthatarewellretainedduringcellfixationandpermeabilization. MolecularBiologyoftheCell1994;5:113–653.
ZhangZ,LiuJ,LiuY,LiZ,GaoWQ,HeZ.Generation,characterizationandpotential therapeuticapplicationsofmatureandfunctionalhepatocytesfromstemcells. JournalofCellularPhysiology2013;228:298–305.
ZhouWL,MedineCN,ZhuL,HayDC.Stemcelldifferentiationandhumanliver disease.WorldJournalofGastroenterology:WJG2012;18:2018–25.
