Journal of Cell Science eut rvd nqeisgtit h ope rcs fplrzto uighptct ifrnito,idctn e ee and key small indicating using differentiation, genes hepatocyte inactivating during By polarization RT-PCR. of process real-time differentiation. complex using hepatocyte the confirmed governing into molecules was insight signaling functional genes unique into clustered these provide and that of networks results demonstrated to several we assigned were RNA, of genes interfering candidate expression the The interactions, -protein categories. predicted and known on Based mroeei.Drn eaoyedfeetain pcfcroutes specific differentiation, hepatocyte hepatocyte During embryogenesis. However, diseases. liver understood. poorly biology of still cell is pathogenesis polarization liver is both the polarization to many and significance hepatocyte considerable of in of hallmark therefore involved a the mechanisms is of understanding polarity molecular complete al., A of et cholestasis. e.g. Decaens loss diseases, 2004; hepatic or Boyer, between and Perturbation border (Wang 2008). the poles polarized lateral create In and (TJs) salts. apical junctions biliary tight recycled the of is hepatocytes, uptake and the circulation the for into responsible components various with secretes contact in in blood, is the is which secreted. (sinusoidal), is which domain bile membrane (BC), which basal into The canaliculi environment, bile external the of with contact network membrane continuous a plasma hepatocytes adjacent form and basolateral front-facing of poles and apical The domains. apical hepatocyte distinct of of formation the maintenance functionally involves hepatocytes and be of establishment polarization The to the polarity. polarized liver by the of be function ensured correct must the is is, hepatocytes That liver. adult cells, the in epithelial functional all for As Introduction words: Key filament-binding that encode as identified that such were those pathways, genes signaling include of of genes number components These a and polarity. stage, molecules, hepatocyte mature- activity differentiation and kinase maintaining the tyrosine immature-HLCs and on protein hFHPCs, establishing based protein, of compared for samples was essential triplicate expression cells be on hepatocyte-like might performed polarized differential progenitor was functional the profiling generate hepatic When to expression fetal protocol HLCs. gene human differentiation Global two-stage of hFHPCs. a mechanisms differentiation from used the we (HLCs) The study, However, understood. this polarity. powerful In completely a development. hepatocyte not provides hepatocyte of hepatocytes are functional maintenance polarity into and (hFHPCs) hepatocyte establishment cells of the generation requires the hepatocytes regulating of functioning correct The Summary 10006 Beijing, University, Medical Zhang Capital Haiyan work Regeneration, this and of to Regulation Lu equally ` contributed and Xin authors Protection *These Liu, Liver for Baoqing Laboratory Li, Municipal China Xueyang Biology, Cell Li, of Weihong Department Zhang*, Weitao Hua*, Mingxi hepatocyte-like functional phenotype a into progenitor differentiation hepatic cell human during polarity of hepatocyte establishment the regulating mechanisms Molecular 5800 o:10.1242/jcs.110551 doi: 5800–5810 125, Science Cell of Journal 2012 August 6 Accepted uhrfrcrepnec ( correspondence for Author 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. h salsmn fhptct oaiybgn uigliver during begins polarity hepatocyte of establishment The eai rgntrcls ifrnito,Hptct oaiy eeexpression Gene polarity, Hepatocyte Differentiation, cells, progenitor Hepatic [email protected] ) PTK7 and PARD3 rmt eai oaiyfrainadafc -ci raiain These organization. F-actin affect and formation polarity hepatic promote nvitro in bre uigtefrttietr(age l,20) They 2008). al., to able et are alpha- were (CK19), (Wang 19 that on trimester hepatic (ALB), fetus albumin first based AFP, fetal from express the isolated expression during Human promoter aborted were markers. (AFP) (hFHPCs) lineage fetoprotein purified cells on specific progenitor performed of with be establishment should the cells insight analyses driving specific polarity, events most molecular hepatocyte the the gain into To possible models limitations human differentiation. 2009; represent have hepatocyte they systems al., of origins, respective these their et Although adipose to 2010). inherent human (Bonora-Centelles al., 2008; or liver et cells 2008) Saulnier al., HepaRG induced Beretta, stromal 2011), and et and al., 2011) tissue-derived (Parent et al., (Chiao cells (Jozefczuk et progenitor cells cells Jozefczuk stem 2010; stem pluripotent differentiated al., that et embryonic cells Synnergren human of within population from identified heterogeneous was total, expression the gene these potential liver-specific several In of (HLCs). the pathways cells analysis, reports, and hepatocyte-like generating genes microarray for key importance the Using examined the have 2009). Zaret, to studies and Lemaigre, Lemaigre of 2004; Boyer, delivery 2004; and the (Wang for membrane defined plasma are mechanisms and oe ytmfrsuyn h oeua ehnssgoverning mechanisms molecular the studying for system model PTK7 , PARD3 , eerhArticle Research PRKCI ` and CDC42 9, . Journal of Cell Science eai oaiywsfrhrepoe yiatvtn their inactivating by Results explored technology. (siRNA) RNA further interfering small using was expression polarity hepatic eea fteegnswscnimduigra-ieRT-PCR. real-time using ( genes confirmed of two of was expression function genes The The these polarization. of core hepatocyte a several regulating be may in were CDC42 that factor differentiation. genes hFHPCs as during polarity, cell regulated of maintenance or establishment PARD3 eeaefntoa n oaie Lsfo FPsand hFHPCs from HLCs polarized and functional generate hepatocyte of process system the model regulate useful that differentiation. a mechanisms and the source study potential to a are hFHPCs Thus, HLCs functional into differentiate long-term proliferate td,w sdatosaedfeetainpooo odifferentiate this to protocol In (Sall4) differentiation S1A). two-stage 4 a Fig. protein used material we sal-like (supplementary study, and 2009) al., 2010), Delta-like et al., 2008), (Oikawa et al., (Yanai et (Dlk) Inada 2006; protein molecule al., adhesion cells/progenitor et cell stem (Schmelzer epithelial hepatic (EpCAM) CK19, ALB, the (Wang AFP, express marker protocol hFHPCs cells this hepatic 2008). using al., fetal hFHPCs human et produced from isolate expression and promoter to fetus, AFP aborted protocol on based a cells progenitor established previously We hepatocyte-like functional cells into hFHPCs of Differentiation identified We of polarity. network interactive hepatocyte the with in associated roles genes their their and for of investigated functions further molecular were stages morphogenesis regulators cell as in different serve genes involved the that Subsequently, identified. of genes were differentiation hFHPCs of of pool profiles comparing common a expression By differentiation, mature-HLCs. gene undifferentiated and differential on immature-HLCs, profiling expression hFHPCs, gene global performed nti td,w sdatosaedfeetainpooo to protocol differentiation two-stage a used we study, this In , PRKCI and CDC42 nvitro in PTK7 nw opa e oe nthe in roles key play to known , Wn ta. 08 n can and 2008) al., et (Wang and nvitro in PARD3 Zage l,2012). al., et (Zhang ntefrainof formation the in ) PTK7 , ifrnitdclsa a 0ehbtdCPB/ activity, CYP2B1/2 exhibited 10 day at cells differentiated eut ugs htti ifrnito rtclgvsrs to rise gives protocol differentiation These this 1E). HLCs. (Fig. functional that 5 day suggest at cells the results greater in were activities 10 equivalent day the at cells than the in activities CYP1A2 and CYP1A1 10 to compared ota tdy0(i.1) sexpected, As 1C). (Fig. 0 day at compared upregulated significantly that were 10 day to and 5 from day rate HGF- secretion at the ALB cells the and staining, cells PAS to progenitor Similar the cells. at induced in cells visible no the whereas was (PAS), all staining acid-Schiff nearly positive periodic 1B, for at positive Fig. stained cells in 10 in day shown As detected points. were 1A; time activity (Fig. different enzyme CYP450 5 and secretion day over of increased levels resembling two S1B). the significantly Fig. but material or CK8 supplementary structures AFP, express and one not intercellular ALB did with They and canaliculi. nuclei hepatic nucleoli round distinctly at prominent cells polygonal differentiated containing a The higher with 0. morphology shape, expressed hepatocyte day typical at and exhibited than 10 AFP, CK8 day and of ALB binucleated of levels with levels differentiated morphology lower OSM epithelial the expressed an and of showed centers, majority DEX 5 The day HGF, days. at of cells 5 were HGF combination another cells of for hepatic the days treatment immature by 5 the matured by stage, further second cells the hepatic In were immature treatment. hFHPCs become stage, first to the In induced HLCs. functional into hFHPCs of oaiaino Jascae rti O1adFatnwere F-actin and The ZO-1 points. time protein different TJ-associated at analyzed of was process, localization TJs differentiation of this assembly in whether cell the established determine is undergo To polarity polarity. hFHPCs hepatocyte acquire differentiation, may and functional morphogenesis to parallel In hepatocyte-like polarized cells into hFHPCs of Differentiation ofrhrcaatrz h el,gyoe trg,ALB storage, glycogen cells, the characterize further To 6 eaoyeplrt omto 5801 formation polarity Hepatocyte .%o h el tdy5(i.1) The 1D). (Fig. 5 day at cells the of 4.3% neslbldotherwise. labeled unless .Tedt r rsne sthe as presented mean are data The 5. epciey * MROD, respectively. and EROD by measured points. time ( different at assay by PROD evaluated was hFHPCs differentiated ( erto a nlzdb ELISA. * by analyzed was secretion ( at points. staining time PAS different by analyzed was hFHPCs ( time points. different at microscopy contrast ( progeny. their and of hFHPCs properties Functional 1. Fig. E D A P ciiyo Y11adCPA was CYP1A2 and CYP1A1 of Activity ) Y212ezm ciiyi the in activity enzyme CYP2B1/2 ) opooyo el ne phase- under cells of Morphology ) , .5cmae ihdy0. day with compared 0.05 6 B ..( s.d. nrclua lcgni the in glycogen Intracellular ) n P 5 , ) cl as 50 bars: Scale 3). .5cmae ihday with compared 0.05 , 90 C Albumin ) 6 .%o the of 2.5% m m Journal of Cell Science -ci rwantoktruhtecls In cells. the through network a drew F-actin ICdxrni h FPs * hFHPCs. the in FITC-dextran the and plane hwn Jbtenteclsa a 0 ,nces J ih ucin E,ruhedpamcrtclm ,Gli t iohnra ( mitochondria. Mt, Golgi; G, reticulum; endoplasmic rough rER, junction; * tight times. indicated TJ, the nucleus; at N, hFHPCs 10. differentiated the day in at TER cells as the measured between TJ a showing progeny. their and hFHPCs in functions and structures TJ-associated 2. Fig. at concentrated strongly in was and F-actin an boundaries, days, along cell-cell HGF five After observed arrow). for were panel, left stimulation ZO-1 2A, of (Fig. border spots cell-cell small individual cells, the through both in cultures 10 day we and 2A, 5 Fig. day the 0, in day shown from and As sections phalloidin examined microscopy. FITC-conjugated confocal with and visualized anti-ZO-1 using detected 5802 n -ci.( F-actin. and J,tetaspteileetia eitne(E)adthe and (TER) resistance electrical transepithelial of function the barrier the evaluated TJs, the we TJs, between functional of form acquisition TJs that ultra-thin differentiation. possibility Moreover, during the HLCs 10. hFHPC-derived 2C). raise day (Fig. HLCs results hFHPC-derived at in These belt cells the TJ at the adjacent displayed expressed sections two predominantly was of JAM-A later. border determined (JAM-A) that was TJ- shows 2008), A al., 2B another et contact Fig. molecule Paris of 2007; cell-cell adhesion al., localization et (Braiterman junctional The of arrow). protein, sites panel, associated at right 2A, increased (Fig. spots in linear time, double-staining a same size; in F-actin the borders and At cell-cell thick abundance pattern. along 10, in concentrated increased day was protein were At ZO-1 cell cells arrow). the the panel, throughout around contact middle bundles cell-cell 2A, of (Fig. sites layer at spots punctate as ots hte hs opoeei etrsrpeetthe represent features morphogenetic these whether test To x-z and ora fCl cec 2 (23) 125 Science Cell of Journal x-y B x-z el tdy1 tie o A- eeiae ycnoa irsoy ro niae h oaiaino A-.( JAM-A. of localization the indicates Arrow microscopy. confocal by imaged were JAM-A for stained 10 day at Cells ) lns In planes. ln.The plane. x-z x-y P lnstkna niae wieln)o h corresponding the on line) (white indicated as taken planes , x-z ofcliae fhHC dy0), (day hFHPCs of images confocal .5cmae ihdy0 h aaaepeetda h mean the as presented are data The 0. day with compared 0.05 x-z ofcliae,Z- appeared ZO-1 images, confocal ofcliae,ZO-1/F-actin images, confocal x-z cross-sections P , .5cmae ihdy0 ( 0. day with compared 0.05 ( A CF nhptcts(ae-lscysie l,20) i.3B Fig. 2003). al., et (Zamek-Gliszczynski 6)-carboxy-2,7-dichlorofluorescein hepatocytes an (and in probenecid, (CDF) 5 of 2- of (MRP) presence transport protein or mediated resistance-associated absence multidrug the the the of in the inhibitor 10 into evaluate day added 5(6)-carboxy- at was HLCs, To cultures in (CDFDA) HLCs. transporters diacetate drug 2,7-dichlorofluorescein hFHPC-derived of activity the 3A, functional (Fig. at among microvilli cultures with BC in arrow) displayed studied sections Ultrathin were 10. ultra-structures day their structures, like 2E). (Fig. 0 that day to that at relative 6, respectively, cells day Paracellular 69%, 5, the and reveal day 81% of onward. 101%, at were cells after 8 results differentiated day in and 6 FITC-dextran of The day rates flux from measure TJ leakage. sensitive declined more of a permeability apical-to-basolateral be measure may a therefore instantaneous and of over min measured an 30 is of is the period diffusion In FITC-dextran which the differentiation. integrity, to TER, upon functional determine FITC-dextran examined to To compared apical-to-basolateral was contrast cultures 2D). TJs, and increased (Fig. across of 5 leakage 0) day significantly function at (day dynamic cells were cells in TER undifferentiated 10 of values day The fluxes. paracellular infcn eraei D a bevdi h Clmni the in lumen A BC the probenecid. in of observed lumen was absence CDF BC the in the decrease in in significant presented HLCs was hFHPC-derived CDF among fluorescent the that shows utrdclsdul-tie o O1adFatnwr mgdi the in imaged were F-actin and ZO-1 for double-stained cells Cultured ) oeaiewehrtehHCdrvdHC omdBC- formed HLCs hFHPC-derived the whether examine To x-y lnsaeson rosidct h ooaiaino ZO-1 of colocalization the indicate Arrows shown. are planes 6 E ..( s.d. .Brirfnto esrda aaellrfue using fluxes paracellular as measured function Barrier ). n 5 3). C lrti section Ultrathin ) D are function Barrier ) x-y Journal of Cell Science oiiestaining. positive ouecrirfml 0(oimbl cdc-rnpre aiy,mme ;SC25 ouecrirfml 2(rai aincriietranspo cation/carnitine (organic 22 family carrier solute SLC22A5, 1; member family), co-transporter acid (sodium/bile 10 family carrier solute BC,APbnigcset,sbfml CT/R) ebr2 Y12 yohoeP5 A;CPA,ctcrm 4034 NT nicotinamide NNMT, 3A4; P450 cytochrome CYP3A4, 1A2; P450 cytochrome CYP1A2, 2; member N (CFTR/MRP), C sub-family cassette, ATP-binding ABCC2, C ( BC. irvlu.M,mcoils E,ruhedpamcrtclm t iohnra DD sitraie yhHCdrvdfntoa Ls cleav HLCs, functional hFHPC-derived by ( internalised without CDF is fluorescent CDFDA as mitochondria. BC Mt, into excreted reticulum; and endoplasmic esterases rough intracellular rER, microvillus; Mv, microvillus. tgswr oprdb endarm hc e othe to led which genes diagram, regulated significantly Venn 514 sets: a gene three by of two classification the compared from derived were lists (814 the stages among genes affected regulated The were in II. significantly downregulated) genes stage 865 microarray 1835 and upregulated were I; The (970 and stage affected significantly in II). genes downregulated) I) 966 (stage 1780 and (stage upregulated HLCs that S2): mature HLCs revealed to Fig. immature the HLCs material to in immature (supplementary transitions expression hFHPCs major patterns undifferentiated gene two expression A identified of differentiation. gene filtering hFHPCs comparison of Plot stage the pairwise Volcano on based a data microarray performed changes, expression gene we global on perspective initial an obtain To differentiation hFHPC annotation during functional analysis hepatocyte and expression natural gene Differential resembles that way a functional to development. in rise gives HLCs protocol polarized differentiation this (OATP) that polypeptide suggest transporting anion surface organic (Ko basal the the demonstrates of 3E higher Fig. staining 3D). significantly drug (Fig. of were hFHPCs that in as HLCs than well hFHPC-derived as reveal enzymes in expression I/II phase transporters gene of level of mRNA the comparisons that secretion Quantitative canalicular in functions 3C). transporter that (Fig. hepatocytes a of MRP2, in pore by CDF apical the of mediated excretion was the HLCs that hFHPC-derived suggesting probenecid, of presence HLCs. hFHPC-derived in polarization functional and expression Gene 3. Fig. mtytaseae PC,aoiorti -.( C-I. apolipoprotein APOC1, -methyltransferase; nge l,20)i FP-eie Ls hs results These HLCs. hFHPC-derived in 2000) al., et ¨nig D uniaiecmaio ftetasrpino hs n hs Iezmsa ela rgtasotr nhHC n hi rgne.SLC10A1, progenies. their and hFHPCs in transporters drug as well as enzymes II phase and I phase of transcription the of comparison Quantitative ) E FP-eie LssandfrOT eeiae ycnoa irsoy rosidct OATP- indicate Arrows microscopy. confocal by imaged were OATP for stained HLCs hFHPC-derived ) B n ih( with and ) Lsaei epn ihtecagsi elpeoye,and phenotypes, cell in changes the with keeping in mature are and HLCs HLCs immature hFHPCs, undifferentiated of patterns S5. Table material supplementary two in the listed by within shared are genes annotations stages regulated the overrepresented of classification significantly functional most the ten ‘cellular S4- The ‘cell-cell Table material and 2). supplementary groups: 4D; functional morphogenesis’, (Fig. process’ included structure developmental The downregulated ‘anatomical S4-1). the signalling’, Table the of material and process’ classification supplementary metabolic II, acid metabolic 4C; stage amino (Fig. ‘alcohol ‘cellular in and included: found ‘glycolysis’, groups process’, genes functional analysis gene regulated similar upregulated A for S3-2). performed process’ Table was material metabolic supplementary ‘cellular 4B; and ‘cell (Fig. included S3-1). organization’, I adhesion’, stage ‘organelle Table structure in cycle’, groups ‘cell material functional ‘anatomical gene supplementary downregulated as The and 4A; the such (Fig. to organization’, morphogenesis’ led groups, I matrix stage functional ‘extracellular in found of genes upregulated identification the significantly explore Analysis the differentiation. to hFHPCs of subsets in genes gene regulated the the of to functions according performed was genes (supplementary clustering S2). hierarchical Table S3B; the (supplementary Fig. by material II subsets stage into gene classified were to four genes overlapping unique 514 The I; S3A). stage were Fig. to material unique genes were genes regulated 1266 stages; 1321 two the by shared were ( A C ae oehr hs eut ugs httegn expression gene the that suggest results these together, Taken expressed differentially of analysis annotation gene Functional lrti eto hwn Caogtecls rosidct the indicate Arrows cells. the among BC a showing section Ultrathin ) Mpoeei.Arw niaetefursetCFtasotdinto transported CDF fluorescent the indicate Arrows probenecid. mM 4 ) eaoyeplrt omto 5803 formation polarity Hepatocyte tr,mme 5; member rter), dby ed Journal of Cell Science o peuae ee nqet tg I ( II. stage to unique genes upregulated for B)trsfrurgltdgnsuiu osaeI ( I. stage to unique genes upregulated for terms (BP) 6.Aogtecmo ee,sxgns( ( genes genes eight six genes, whereas common the (29 Table were CDC42EP3 material genes genes Among (supplementary 19 stages 69 and S6). two and II, the stage between I (40 common in genes stage downregulated) 63 in 40 found differentiation) 0010769:upregulated, downregulated) We in 23 examination. GO further involved upregulated, for morphogenesis; morphogenesis selected cell cell were GO of of the morphogenesis; regulation cell within regulation 0000902: hFHPCs genes (GO 0022604: during categories expressed mechanisms GO generation differentially molecular defined polarity the the of hepatocyte differentiation, understanding regulate better that a gain morphogenesis To cell in hepatocyte expressed involved differentially genes the on of analysis network regulators Functional of effects specific differentiation. the reflecting differentiation. hFHPC during differentially expressed genes for terms process biological GO enriched Most 4. Fig. 5804 rmitnneo elplrt’ mmrn organization’, ‘membrane polarity’, cell hepatocyte of and maintenance ‘establishment in using involved or genes were categorized genes candidate further The were candidate analysis. the GO genes examine candidate II the To the cells. polarization, neighboring between produce stage cells on that exert correlations in forces and the internally by upregulated both driven is which and morphogenesis, I S4). stage Fig. material (supplementary in contrast, In downregulated II. stage in downregulated MYH10 salsmn feihla elplrt spr fcell of part is polarity cell epithelial of Establishment IL6 and ora fCl cec 2 (23) 125 Science Cell of Journal , , NOX4 ANTXR1 EFNB1 TGFB3 and , , eeurgltdi tg and I stage in upregulated were ) FBLIM1 RAC2 DFNB31 eedwrgltd nbt stages; both in downregulated, were , and GDF7 D otsgiiatB em o oneuae ee nqet tg II. stage to unique genes downregulated for terms BP significant Most ) THY1 SPG3A , ITGB4 eeupregulated, were ) B otsgiiatB em o oneuae ee nqet tg .( I. stage to unique genes downregulated for terms BP significant Most ) and , ADM LIMA1 VEGF , , APOE MBP were , , PRKCI r novdi yokltnognzto.Oegn subset gene ( One organization organization. membrane in in involved involved are DOCK2 epnil o salsigmttcsideorientation; spindle mitotic establishing for responsible with I, stage in downregulated were elaia/aa polarity. apical/basal cell and PARD3 genes the Among organization, TGFB3 junction transport’. cell in ‘vesicle-mediated involved in involved Tbe1.Itrsigy he eesbesivle nthe identified. in were involved polarity cell subsets of organization’PTK7 gene maintenance ‘cytoskeleton three or establishment Interestingly, and 1). organization’, (Table junction ‘cell novdi J,and TJs, in involved II. stage in downregulated were DLC1 novdi deesjunctions. adherens CENPA in involved cluster, functional genes 16 and 5, Fig. in shown network As functional ( STRING. to submitted using were reconstruction genes candidate the level, processes biological polarization. key cell the for to required that assigned showed were analysis genes GO candidate the the Thus, TJs. epithelial of formation the FYN obte iulz h ucinlitrcin ttenetwork the at interactions functional the visualize better To PRKCI MYH10 , , eedwrgltdi tg ;and, I; stage in downregulated were PARD3 RHOJ , , eeurgltdi tg I; stage in upregulated were and EPHB3 and CCDC99 epnil o salsigo anann epithelial maintaining or establishing for responsible ULK1 Fg A eecutrdtgte.Wti this Within together. clustered were 5A) (Fig. ) NDE1 , and PLXNB1 , EFNB1 a peuae nsaeI l fwihare which of all I, stage in upregulated was ) PRKCI CDC42 eedwrgltdi tg I l fwhich of all II, stage in downregulated were and FYN , , KNTC2 NDE1 PVRL1 DLC1 eeurgltdi tg ,with I, stage in upregulated were , , MYH10 CDC42 CDC42 ( , , A , atcpt nmitnneof maintenance in participate CDC42 PRKCI otsgiiatbooia process biological significant Most ) CDC42 KNTC2 CLASP1 SMAD3 , , C and THY1 PARD3 PARD3 otsgiiatB terms BP significant Most ) DAB2 , SHROOM2 , , , TPM1 PRKCI CENPA , CENPA , SMAD7 NTN1 , KNTC2 , GJA1 APOE and and , RAC2 , r novdin involved are and and , , CDC42EP3 PRKCI CDC42 PVRL1 PRKCI and , ( CCDC99 CCDC99 NDC80 , MYH10 LIMA1 TGFB3 CAP2 PTK7 and and are are ), , , , , Journal of Cell Science .Rglto fclua opnn organization: component cellular of Regulation 4. .Ctseeo organization: Cytoskeleton 5. .Rglto fcl size: cell polarity: of cell Regulation of 2. maintenance or Establishment II 1. Stage in genes Downregulated 0 yokltnorganization: Cytoskeleton 10. .Etbiheto aneac fcl oaiy(nldn pclbslcl polarity): cell apical/basal (including polarity cell of maintenance or Establishment I 1. Stage in genes Upregulated ssoni i.7,w ucsflyrdcdthe reduced successfully we their 7A, Fig. reducing of in assessed. formation By shown were the differentiation As on function. hFHPC effects during their cellular polarity hepatocyte siRNA, unique using polarity level hepatocyte their expression in on involved based genes expressed differentially principle, of proof a As the in involved polarity genes hepatocyte candidate of analysis Functional genes levels. expression of of the expression range from wide the calculations a index represented across gene accurately our data that reveal microarray data The in presented 6. real-time are Fig. coefficients and correlation their microarray with the results RT-PCR of Comparison RT-PCR. real-time ( ( function morphogenesis on cell hepatocyte based in the selected roles from genes calculated their expressed indexes differentially gene six the of microarray, accuracy the validate To by genes of RT-PCR expression real-time differential the of Confirmation attachments kinetochore- 5B). (Fig. and integrity kinetochore organization: Cytoskeleton 8. .Atnctseeo organization: cytoskeleton Actin 6. organization: Cytoskeleton 5. .Cl-eljnto organization: junction Cell-cell 3. .Rglto fclua opnn organization: component cellular of Regulation 5. .Atnctseeo organization: cytoskeleton Actin 9. size: cell of Regulation shape: 3. cell of Regulation 2. .Rglto fcl shape: cell polarity: of cell Regulation of 2. maintenance or Establishment II 1. Stage formation: in bundle genes filament Upregulated actin of Regulation 7. .Clua opnn assembly: component Cellular 6. organization: component Cellular 4. .Clua opnn organization: component Cellular 3. .Rglto fclua opnn size: component cellular of Regulation 7. 1 ci yokltnorganization: cytoskeleton Actin 11. 1 euaino ci yokltnorganization: cytoskeleton actin of Regulation formation: 11. bundle filament Actin 10. size: component cellular of Regulation 4. .Etbiheto aneac fcl polarity: cell of maintenance or Establishment I 1. Stage in genes Downregulated .Clua opnn organization: component Cellular 4. organization: junction Cell shape: 3. cell of Regulation 2. .Cl ucinorganization: junction Cell 5. .Clua opnn organization: component Cellular 7. organization: Membrane 6. .Rglto ellrcmoetorganization: component cellular Regulation 8. .Clua opnn assembly: component Cellular 9. IL7R CHRNB2 NOX4 , , KIF3A KIAA1893 and , NFATC1 DFNB31 , IL6 APOE , , al .Slce ifrnilepesgnsivle nteHC oaiyformation polarity HLCs the in involved genes express differential Selected 1. Table HPRT1 NGFB APBB1 NUMBL DAB2 PTK7 CDC42EP3 PALM2-AKAP2 DLC1 BCL6 CAP2 KNTC2 SMAD3 THY1 , BBS1 CDC42 , FGD6 BBS1 , , , SHROOM2 NTN1 APOE and HES1 , DAB2 and , , CDC42 FGD6 , ARL6 GJA1 STK4 , SMO EPHB3 LIMA1 THY1 FGD6 TTL , LIMA1 , LIMA1 CDC42 , SMAD7 VEGF , , PARD3 , LIMA1 , LIMA1 , , , , ABCA1 FYN PDGFB , and , , , LIMA1 LIMA1 GLI2 TGFB3 BCL6 MYH10 , CDC42 SLITRK5 , PRKCI LIMA1 SLC1A3 , , , CLASP1 , DLC1 LIMA1 , ANTXR1 and CDC42EP3 GDF7 , , THY1 and PTK7 , , FBLIM1 , RUFY3 CENPA , CDC42 , THY1 EGR2 , , GAS7 DKFZP586P0123 CAP2 CLASP1 KNTC2 , GAS7 eeslce rmthe from selected were , , SMAD3 , , NGFB APBB1 , PALM2-AKAP2 CHRNB2 LIMA1 TGFB3 SEMA3A and and TGFBR3 , ANTXR1 PRKCI , CPS1 , , CCDC99 , STXBP1 eecnimdby confirmed were ) ANTXR1 CABP4 and ANTXR1 SEMA5A , , , , MYH10 , , TGFB3 and RAC2 , GJA1 MBP and ITGB4 , DOCK2 CCDC99 , MYH10 , , , APOE NTN1 CDC42 FBLIM1 and SHROOM2 DAB2 and and , CDH4 , , and DLC1 and , MBP , , , DOCK2 , , WNT3A , UNC5B KNTC2 APOE PLXNB2 , DLC1 , DOCK2 DMD ULK1 PTK7 S100A4 , PRKCI MYH10 PCK2 , TGFB3 ULK1 PDGFB and and , , , , TGFB3 , CENPA DLC1 PVRL1 CDC42EP3 and JAK2 , , , RHOJ , NDE1 , ITGB4 APOE and GAS7 and , COL18A1 , , DLC1 )or and VEGF , and GAS7 RHOJ RXRA , , NRXN3 , , , RAC2 SEMA3A KLF7 TGFBR3 TPM1 CSPG2 and PTPRF PLXNB1 , , , , MYH10 MYH10 , MYH10 , SMAD7 , and MYH10 CCDC99 ROBO3 THY1 , , , and L1CAM omdln h ope rhtcuedrn hepatocyte during architecture leads which complex interior, cell in the and key proteins surface modeling of cell is organization the to requires specific on hepatocytes lipids that the and process in and multistage interactions polarity a cell-cell and membrane program developmental of Generation Discussion that demonstrate data differentiation. hFHPC These during formation cells. polarity either control of in reduction pole nucleus apical the between localized and was apparatus Golgi the while cells, of reduction The control. PARD3 itiue ntectpamin randomly cytoplasm was the apparatus actin in apical Golgi distributed and the assembly F-actin Furthermore, on concentration. effects the inactivated exhibited with which cells 7D, Fig. PARD3 in shown As differentiationprotocol. two-stage our using hepatocytes into differentiation 7B,C). of (Fig. expression post-transfection a days over 6 fluxes at paracellular min the 30–120 of increased period and TER the decreased ONECUT2 NOX4 , TTLL3 and and SHROOM2 oietf opooycags FPswt reduced with hFHPCs changes, morphology identify To TTL , , , , PTK7 , OFD1 PDGFB , OFD1 CLASP1 , TTC3 SMAD3 , NDE1 ULK1 LAMB1 , SPON2 THY1 xrsinlvlb 09%cmae otetransfection the to compared 80–90% by level expression eeual oaqietetpcleihla morphology, epithelial typical the acquire to unable were , , , PARD3 , PDGFB PRKCI , PARD3 , , PDGFB , RAC2 , , and , FBLIM1 ADM RAC2 ITGA1 NRP2 PTK7 TGFB3 and , , , eaoyeplrt omto 5805 formation polarity Hepatocyte RHOJ and and , RAC2 , SPG3A , , CENPA RAC2 , PRKCI PVRL1 GDF7 PTK7 APOE TPM1 TTLL3 and , and TPM1 , and , COL4A3BP , , , PARD3 EFNB1 TTLL3 VEGF PTPRF LRRC4C or TPM1 PTK7 and PARD3 , PTK7 , , UCHL1 VANGL2 DFNB31 GAP43 , eecalne oundergo to challenged were , NUMBL CLASP1 and and , a nii hepatocyte inhibit may , TRAPPC4 ETV4 and PARD3 , , STXBP1 PARD3 EFNB1 ADM , BDNF , , SPG3A APOE and significantly knockdown , PTK7 IL6 VEGF , , , HIF1A EGFR SH2B and , , , Journal of Cell Science ifrnito ohmrhlgclyadfntoal and hFHPCs that observed al., We functionally 2008). et Zorn, 2001; (Kinoshita al., et development and Kamiya 1999; hepatocyte natural morphologically the hepatocyte resembles functional both generate hepatocyte to differentiation mimics which efficient hepatocytes, hFHPC-derived is in polarity hepatocyte DEX and in OSM, involved generate mechanisms to molecular was polarization. study the this of hFHPCs explore polarity the goal from hepatocyte The hepatocytes polarized 2008). functional human al., complex et (Decaens developing for al., few a et available only because Jozefczuk polarity partly hepatocyte 2010; unclear for mechanism is al., exact generation the 2011), et 2009; al., al., Synnergren et Fu et Chiao 2010; 2011; Kidambi 2007; al., 2009; al., et al., al., et et Saulnier Bonora-Centelles et Imamura 2008; (Matsui 2003; al., process et al., this et for Michalopoulos 2007). mechanisms 2002; al., suggesting and accumulated et routes been specific Takashi recently no 2004; has of information color Boyer, Every Although and (arrow). panel (Wang associa the clusters. functional in identified differentiation predicted depicted the the algorithms, indicate represent clustering B edges KMEANS and The using A proteins. clustered cluster. are were a nodes proteins to network These corresponds The lines. differentiation. colored hFHPC differently during seven STRING. morphogenesis with cell visualization in network involved protein Predicted 5. 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Tepass, and P. K. Harris, M. I. Arias, and J. P., Lippincott-Schwartz, Y., J. Wakabayashi, Lahad, D., Fu, W., K. Cheng, K., L. Nolden, G., D. Rosen, X., Sui, M., A. Eder, A. S. Duncan, oao .T,Go T., D. M. Cassio, Donato, and B. Grosse, M., Durand, C., Decaens, ha,E,Eaa,M,Xn,Y,Xog . mt . iln .T,Gen .S., J. Glenn, T., M. Millan, M., Kmet, A., Xiong, Y., Xing, M., Elazar, E., Chiao, riemn .T,Hfenn . yse . on,D,Se .P,Yty R., Yutzy, P., A. See, D., Johns, L., Nyasae, S., Heffernan, T., L. Braiterman, aia . ioht,T n iaia A. Miyajima, and T. Kinoshita, A., Kamiya, J. Adjaye, and L. Chavez, A., Prigione, J., Jozefczuk, M. Takeichi, and T. Ishiuchi, h xeiet FPstasetdwt o-agtn iNswr sdas used were siRNAs non-targeting with transfected control. experimental hFHPCs experiment. the ooaCnels . oe,R,Mrbt . ao,A,Croel . atl,J V. J. Castell, F., Carbonell, A., Lahoz, V., Mirabet, R., Jover, A., J. Bonora-Centelles, Nance, and M. A. Wehman, A., Achilleos, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.110551/-/DC1 online available material Science Supplementary Natural Natural Z.]. Beijing H. the National to and 5102013 Li]; the number Liying [grant to from Foundation 81030009 to grants and 31171310 30971472, Z. by numbers H. [grant China supported of Foundation was Science work This Funding Bio-tech, KangChen by performed China. Shanghai, were experiments Microarray Acknowledgements 11.5 samples. if SPSS significant using replicate considered tests were with ANOVA Differences one-way times software. using statistics three performed was repeated analysis Statistical independently were Experiments Statistics in .adGpa S. Gupta, and H. Qian, nrdn eaoye oniigwt hne nmrhlg n ucino tight of function and morphology in changes with junctions. coinciding hepatocytes rodent N. in Sawada, and K. Hirata, resources. bioinformatics DAVID using lists gene Protoc. large of analysis integrative oprsnadvsaiaino ilgcllssuigae-rprinlVenn area-proportional using lists biological of visualization diagrams. and comparison Traffic al. et cancer. N. ovarian E. polarity in apical-basal Atkinson, of overexpression L., loss 12519-12524. E through C. prognosis cyclin Warneke, poor and to H., contributes K. PKCiota Atypical Lu, M., Kango-Singh, tmltshptct oaiaintruhacAMP-Epac-MEK-LKB1-AMPK a through polarization pathway. hepatocyte stimulates rat and human Dev. intact in plates. activities 96-well P450IIB1 on cultured and hepatocytes P450IA1 cytochrome polarity? measuring hepatocyte study to used best be could eai el eie rmhmnebyncse cells. stem embryonic human from derived cells J. hepatic Baker, and H. W. Wong, seta n niioyt eeomn fhptcplrt nWFBcells. WIF-B in polarity hepatic Physiol. al. of Liver. et development Gastrointest. P. to Physiol. 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