Expansion and maintenance of human embryonic stem cell derived endothelial cells by TGFb inhibition is Id1 dependent

Transcription

1 Expnsion n mintnn o humn mryoni stm ll riv nothlil lls y TGF inhiition is I1 pnnt Dylon Jms 1, Hyung-song Nm 2,7,8, Mro Snl 1,3,8, Dnil Noln 1, Tylr Jnovitz 1, Mrk Tomishim 4, Lornz Stur 4, Gsng L 4, Dvi Lyn 1, Rort Bnzr 2, Niki Zninovi 5, Zv Rosnwks 5, Sin Y Rny 1,6 & Shhin Rii 1 Prvious orts to irntit humn mryoni stm lls (hescs) into nothlil lls hv not hiv sustin xpnsion n stility o vsulr lls. To in vsulogni vlopmntl pthwys n nhn irntition, w us n nothlil ll spii VE-hrin promotr riving grn luorsnt protin (GFP) (hvpr-gfp) to srn or tors tht promot vsulr ommitmnt. In phs 1 o our mtho, inhiition o trnsorming growth tor (TGF)β t y 7 o irntition inrss hvpr-gfp + lls y tnol. In phs 2, TGFβ inhiition mintins th prolirtion n vsulr intity o purii nothlil lls, rsulting in nt 36-ol xpnsion o nothlil lls in homognous monolyrs, whih xhiit trnsriptionl proil o I1 high VEGFR2 high VE-hrin + phrinb2 +. Using n I1-YFP hesc rportr lin, w show tht TGFβ inhiition sustins I1 xprssion in hesc-riv nothlil lls n tht I1 is rquir or inrs prolirtion n prsrvtion o nothlil ll ommitmnt. Our pproh provis srum-r mtho or irntition n long-trm mintnn o hesc-riv nothlil lls t sl rlvnt to linil pplition. Humn mryoni stm lls (hescs), whih sl-rnw ininitly 1, or plntiul sour o nothlil lls or thrputi rvsulriztion. Howvr, w stuis hv intii spii vlopmntl stimuli suiint to support th spiition n mintnn o lrg numrs o untionl n vsulr-ommitt nothlil lls rom hescs 2 7. Although smll numrs o hesc-riv nothlil lls hv n gnrt in short-trm ulturs, ths lls hv not n sujt to sustin xpnsion, ngiogni proiling or intrrogt s to th stility o vsulr t. As rsult, molulr pthwys tht mintin vsulr intity n long-trm xpnsion o hesc-riv nothlil lls rmin unknown. To tt th mrgn o nothlil lls rom irntiting hescs in rl tim, w gnrt ll lin or nothlil ll spii ling tring. W lon 1.5-kilos rgmnt rom tril rtiiil hromosom (BAC) ontining th gnomi lous o th humn nothlil ll spii gn VE-hrin (CDH5). Th promotr squn o this gn, nompssing rgion upstrm o xon 1, ws insrt into lntivirl vtor upstrm o GFP (hvpr-gfp; Fig. 1). Humn nothlil lls trnsu with this vtor show roust xprssion o GFP, in ontrst to trnsu humn msnhyml n irolsti lls, whih i not xprss GFP (Supplmntry Fig. 1 ). Enothlil-spii xprssion o th rportr ws lso vint in trnsu, spontnously irntiting hescs (RUES1 lin; Fig. 1 n Supplmntry Fig. 1 j): hvpr-gfp + lls wr orgniz into pillry-lik struturs xprssing nothlil ll mrkrs, inluing VE-hrin, CD31 n CD34 (Supplmntry Figs. 1 g n 2,), n wr ngtiv or lph smooth musl tin (α-sma) n CD45, mrkr o hmtopoiti lls (Supplmntry Figs. 1h j n 2). Using th hvpr-gfp hesc rportr lin, w trk th hronology n gomtry o vsulogni irntition in irntiting myroi ois y tim-lps onol mirosopy. Bginning t y 5, w osrv th spiition n mrgn o hvpr-gfp + lls (Supplmntry Vio 1 n Supplmntry Fig. 3), n y y 8, hvpr-gfp + lls o-xprssing vsulr nothlil growth tor rptor (VEGFR)2 n CD31 (Fig. 1) orm motil vssl-lik struturs (Supplmntry Vio 2). Ths t vlit th ility o th hvpr-gfp rportr onstrut to spiilly intiy n trk hesc-riv nsnt nothlil lls. W us th rportr lin to vlop hmilly in, srum-r mtho or nhning vsulr irntition. In phs 1, htrognous mryoi oy ulturs o hvpr-gfp hescs wr squntilly stimult with on morphognti protin (BMP)4, tivina, irolst growth tor (FGF)-2 n VEGF-A 8 10 (Fig. 1). Although ths growth onitions promot ormtion o hvpr-gfp + struturs (Supplmntry Fig. 4 n Supplmntry Vios 3 n 4), th yil o issoit hvpr-gfp + nothlil lls otin y luorsn-tivt ll sorting (FACS) ws low, n ths w isolt nothlil lls oul not xpn without th mjority o lls ssuming non-nothlil ll phnotyp 1 Howr Hughs Mil Institut, Ansry Stm Cll Institut, Dprtmnt o Gnti Miin, Will Cornll Mil Collg, Nw York, Nw York, USA. 2 Progrm in Cnr Biology n Gntis, Mmoril Slon-Kttring Cnr Cntr, Nw York, Nw York, USA. 3 Division o Mil Onology, Dprtmnt o Miin, Mmoril Slon-Kttring Cnr Cntr, Nw York, Nw York, USA. 4 Dvlopmntl Biology Progrm, Mmoril Slon-Kttring Cnr Cntr, Nw York, Nw York, USA. 5 Ronl O. Prlmn n Clui Cohn Cntr or Rproutiv Miin, Nw York, Nw York, USA. 6 Bionginring Progrm, Hostr Univrsity, Hmpst, Nw York, USA. 7 Prsnt rss: Will Cornll Mil Collg, Nw York, Nw York, USA. 8 Ths uthors ontriut qully to this work. Corrsponn shoul rss to S.R. (srii@m.ornll.u). Riv 1 Dmr 2009; pt 8 Jnury 2010; pulish onlin 17 Jnury 2010; oi: /nt1605 ntur iothnology volum 28 numr 2 rury

2 1.5 k hvpr Intron k ATG hvpr-gfp 10 4 LTR Exon 1 64 p Exon p α-cd31 U3 U5 GA RRE GFP WPRE U3 U5 Suspnsion Ahrnt Dy ( 1) α-vegfr2 Isoltion BMP-4 Ativin A FGF-2 Phs 1 irntition Msorml n vsulr spiition VEGF-A SB EC mpliition Phs 2 isoltion hvpr-gfp Nu VEC hvpr-gfp Propiium ioi 10 1 g h % 1.8% hvpr-gfp i % hvpr-gfp + lls Dy 14 Group Isolt P <.001 P < N 7 + N 7 + Figur 1 Squntil TGFβ tivtion ollow y inhiition uring phs 1 irntition promots tnol xpnsion o hvpr-gfp + hesc-riv lls. () A 1.5-k rgmnt o th puttiv humn VE-hrin promotr (hvpr) rgion ws isolt rom BAC lon n pl upstrm o GFP in lntivirl xprssion vtor (hvpr-gfp). () Spontnously irntiting mryoi ois xhiit xprssion o hvpr-gfp in tuulr struturs. Inst, mrg o GFP n rightil viws. () Flow ytomtri nlysis show hvpr-gfp + lls wr positiv or th vsulr mrkrs CD31 n VEGFR2. () Shmti igrm showing th squn in whih BMP4, tivina, FGF-2, VEGF-A n SB wr n rmov rom irntition ulturs. EC, nothlil ll. () Ahrnt hvpr-gfp ulturs stimult with SB (10 µm) rsult in irntition o hescs into monolyrs o hvpr-gfp + hrnt lls. Inst, hvpr-gfp + lls lon. () Humn VPr-GFP + lls wr immuno-positiv or VE-hrin. Blu, nulr ountrstin. Inst, hvpr-gfp + lls lon. (g,h) Th proportion o hvpr-gfp + lls ws msur y low ytomtry t y 14 tr ultur in th sn ( SB; g) n prsn (; h) o SB (i) Msurmnt o hvpr-gfp + lls t y 14 whn mryoi ois wr ultur ithr in groups or s isolt mryoi ois n SB ws t y 0, y 7 or not t ll (N). Error rs rprsnt s.. o xprimntl vlus prorm in triplit. Sl rs, µm. (t not shown). W thror srn or iotiv smll moluls tht woul improv vsulr irntition. ning o >20 moluls ssoit with rly vlopmntl signling pthwys (Supplmntry Tl 1) show tht th TGFβ-inhiitory molul SB (r. 11) rprouily inrs th yil o hvpr-gfp + lls. Aing SB to irntition ulturs t y 7 rsult in th ormtion o hvpr- GFP + VE-hrin + monolyrs (Fig. 1,), whih, upon issoition, yil tnol mor hvpr-gfp + nothlil lls thn ulturs stimult y ytokins lon (Fig. 1g i). No hvpr-gfp + lls wr gnrt i SB ws t th onst o irntition (y 0), suggsting tht vsulr ommitmnt pns on tiv TGFβ/tivin/nol signling or y 7. Kinti nlysis o irntition suggst shit rom pluripotnt phnotyp (Ot3/4 + ; Fig. 2) to vsulr phnotyp (CD31 + ; Fig. 2,) through msorml intrmit (rhyury + ; Fig. 2). Aition o SB to irntiting hesc ulturs t y 7 lrt th rution o Ot3/4 n rhyury n inrs th numr o hvpr-gfp + CD31 + lls ginning t out y 9, whil ruing xprssion o α-sma (Fig. 2,). Atr isoltion rom htrognous ulturs y FACS, nothlil lls grown in th sn o TGFβ inhiition rtin high xprssion o CD31 ut lso xprss α-sma (Supplmntry Vio 5), initing tht ths nothlil ll lik lls h not ssum trminlly ommitt vsulr t. Exprssion o α-sma in hesc-riv nothlil lls suggst gr o plstiity tht is not prsnt in trminlly irntit nothlil lls (humn umilil vin nothlil lls; HUVEC, Fig. 2). In, xtn ultur (>10 tr FACS isoltion) o hescriv nothlil lls in th sn o TGFβ inhiition yil sustntil numr o lls o-xprssing VE-hrin n α-sma (Fig. 2). On xplntion or th inrs prntg o nothlil lls in SB stimult ulturs is mintnn o th vsulrommitt stt tr spiition. To tst th pity o TGFβ inhiition to promot xpnsion o pur popultions o hesc-riv nothlil lls, w issoit y 14 irntition ulturs, isolt 162 volum 28 numr 2 rury 2010 ntur iothnology

3 hvpr-gfp + lls y FACS n xpn thm or n itionl 5 with or without SB (Phs 2, Fig. 2 i). SB trt ulturs yil mor lls in th 5- prio, n highr prntg o th popultion rtin n α-sma CD31 + VE-hrin + phnotyp (Fig. 2 h). In ition to prsrving th vsulr phnotyp, SB lso inrs ll prolirtion, s init y highr prntg o phospho-histonh3 + (PHH3) mitoti nothlil lls (Fig. 2i n Supplmntry Vios 6 n 7). In ggrgt, TGFβ inhiition in phs 1 n 2 rsult in 36-ol xpnsion in th totl numr o vsulr-ommitt hesc-riv nothlil lls, with 7.4 CD31 + VE-hrin + nothlil lls gnrt rom vry on hesc input ovr 20, ompr with 0.2 nothlil lls pr input hesc riv rom ontrol ultur onitions (Fig. 2j). Similr lvls o xpnsion o hesc-riv nothlil lls wr hiv with our itionl hesc lins n on inu pluripotnt stm ll lin using th sm protool xpt tht ithr SB or solul TGFβRII Rltiv xprssion (.u.) 10,000 1,000 j 10 1 Brhyury SB Brhyury + SB Nu SMA VEC Ot3/4 SB Ot3/4 + SB Nu Unirntit hescs Non-vsulr lls Smooth musl lls Enothlil lls Rltiv xprssion (.u.) VEC αsma SB αsma + SB CD31 SB CD31 + SB Nu VEC Cll numr (k) <10k ECs g k 10k 5k Prnt hvpr-gfp + lls Totl lls CD31 + lls h hvpr-gfp + SB hvpr-gfp + + SB () Totl numr o αsma + lls ~10k ECs CD31 + SB CD SB Dy Dy Dy Prnt o PHH3 + lls i : Prnt CD31 + lls 50k hescs ~60k ECs () ~360k ECs 1:7.4 Dy 0 Dy 7 () Dy 14 (puriition) Phs 1 Phs 2 Htrognous irntition ultur Homognous isolt ultur Dy 20 Rtio o ESCs/ECs Figur 2 TGFβ inhiition tr nothlil ll isoltion uring phs 2 inrss yil n prsrvs vsulr intity o purii nothlil lls. ( ) Humn VPr-GFP hescs wr squntilly stimult with ytokins ( SB) n SB () (Fig. 1 n Onlin Mthos) n ulturs wr ssss or th prvln o pluripotny (Ot3/4) n msorml trnsripts (rhyury) (), CD31 n α-sma trnsripts () n nothlil ll mrkrs hvpr-gfp n CD31 () t multipl tim points uring irntition. Th sonry xis in shows vlus or lls shown in soli rs. () Isolt nothlil lls tht wr ultur in th sn o SB wr stin or oth VE-hrin n α-sma n show rr lls tht wr positiv or oth mrkrs (rrowh in th inst). Inst, α-sma lon. ( i) Humn VPr-GFP + lls wr isolt rom irntition ulturs t y 14 y FACS n urthr ultur in th sn () or prsn () o SB (g) Flow ytomtri ssssmnt o CD31 ws prorm tr 5 o isolt ultur (totl lls r shown in whit n CD31 + lls r shown in lk in th r grph). (h) Atr isoltion n 5 o ultur in th prsn or sn o SB431542, th inin o α-sma + lls ws msur. (i) Atr 5 o ultur ollowing isoltion, unstimult ulturs show ru inin o lls positiv or phospho-histonh3 (PHH3 + ), rltiv to SB stimult ulturs. Th mn inins o α-sma n phospho-histonh3 positiv lls wr otin y ounting positivly stin lls in multipl prlll wlls. (j) Th yil o nothlil lls (ECs) rom hescs is shmtiz rltiv to 50,000 hesc input t y 0. Th rltiv irn in nothlil ll (ECs) numr is init t y 14 (upon isoltion rom irntition ulturs), n y 20 (tr xpnsion in isolt onitions). Th rtio o input hescs to ommitt hesc-riv nothlil lls tr 20 is lso shown. Rltiv trnsript unn ws msur y QPCR n normliz to th houskping gn β-tin (ACTB). Error rs in ( n g i) rprsnt s.. o xprimntl vlus prorm in triplit. Sl rs, µm. ntur iothnology volum 28 numr 2 rury

4 hesc riv Umilil or riv Exprssion 14 i1:yfp FACS α-cd31 ntioy hvpr-gfp EBs 14 () 10 4 Sort hvpr-gfp+ 14 () Sort hvpr-gfp+ 24 () CD31 + i1 low CD31 + i1 high 10 4 l1 promotr: YFP Enothlium (HUVEC) Smooth musl lls l1:yfp Cor loo CD34 + lls : Blow ttion thrshol Gns CXCR4 EphrinB2 EphB4 Lyv-1 Pooplnin Prox-1 VEGFR1 (Flt1) VEGFR2 VEGFR3 (Flt4) CD34 CD133 (Prominin1) E-Sltin P-Sltin PECAM (CD31) Thromomoulin VE-hrin vonwillrn tor HOXA9 l1 l2 l3 α-smooth musl tin l1:yfp l1:yfp MFI (k) l1 low () III I CD31 + l1 low I 3 Dys () l1 low () IV l1:yfp MFI II 4 Dys l1 high () CD31 + l1 high V CD31 MFI II 5k 5k 11.2k 21.7k 18.3k 53.8k III IV V VI Totl lls l1 high () VI CD31 MFI (k) Figur 3 Molulr proiling o hesc-riv nothlil lls rvls signtur in y high I1 xprssion. Humn VPr-GFP mryoi ois n highly purii hvpr-gfp + lls wr ompr to mtur vsulr lls y mirorry nlysis. () RNA ws xtrt or mirorry nlysis rom humn VPr-GFP mryoi ois ultur in th prsn o rominnt ytokins lon until y 14; isolt nothlil lls (99.8% pur) rom hvpr-gfp mryoi ois ultur in th prsn o rominnt ytokins n th TGFβ inhiitor SB until y 14; isolt nothlil lls (>95% pur) rom hvpr-gfp mryoi ois ultur in th prsn o rominnt ytokins n th TGFβ inhiitor SB until y 14, ollow y 10 itionl ultur in th prsn o ytokins n SB431542; HUVECs; humn umilil vin smooth musl lls; n CD34 + umilil or loo lls. Exprss tors r isply in n orr rry, s ll, with highly xprss tors shown in r, minimlly xprss tors shown in lu n tors or whih trnsripts r low signiint xprssion lvl shown in gry. ( ) Following th nothlil ll irntition protool (Fig. 1), I1- YFP hesc-riv lls wr sort y FACS, sprting th CD31 + popultion into I1-YFP high -xprssing lls ( (grn) n ) n I1-YFP low -xprssing lls ( (r) n ). Insts, rightil viws on th y tr isoltion o I1-YFP high () n I1-YFP low () lls. () Atr 3 ultur in th prsn o SB431542, oth popultions wr trnsrr to onitions with n without SB or n itionl 4 ( n SB, rsptivly). () Totl lls n mn luorsn intnsity (MFI) msurmnts o I1:YFP (lk) n CD31 + (whit) wr msur or: CD31 + I1 low (I) n CD31 + I1 high (II) popultions upon isoltion; n or our popultions ollowing ultur onitions (s shown in, III VI). Sl rs, µm. rptor oys ws us intrhngly to inhiit tivtion o th tivin/nol rnh o TGFβ suprmily signling (Supplmntry Fig. 5 ). Ths rsults monstrt tht th t o TGFβ inhiition shown or th RUES1 lin is pplil to othr pluripotnt ll lins. To in th vsulogni trnsriptionl signtur o hesc-riv nothlil lls t irnt tim points uring phss 1 n 2, w rri out Aymtrix mirorry nlyss o svrl hesc-riv popultions n mtur ll typs (Fig. 3). Th yil o rshly isolt phs 1 nothlil lls in th sn o TGFβ inhiition ws insuiint or mirorry nlyss, unrsoring th vlu o our pproh or gnrting suiint xpning (phs 1) n vsulr-ommitt (phs 2) nothlil lls or molulr proiling. Phs 1 hesc-riv nothlil lls show inrs lvls o tors typil o rtril-lik nothlil lls (VEGFR2, VEGFR1, I1, CD31, CD34, VE-hrin, vwf, thromomoulin, phrinb2 n E-sltin) ut not o lymphti nothlil lls (Prox1 n pooplnin). Mrkrs o vsulr prognitor lls, inluing CD133 n I1 (rs ), wr lso highly xprss in phs 1 nothlil lls n ownrgult upon in vitro ultur. Trnsription tors xprss primrily in ommitt nothlil lls, inluing HoxA9 (r. 18), wr not xprss in phs 1 nothlil lls. Aoringly, w in omprhnsiv vsulogni xprssion proil o th hesc-riv nothlil ll popultion s VE-hrin + VEGFR2 high I1 high thromomoulin high phrinb2 + CD133 + HoxA9, whrs mtur nothlil lls wr intii y VE-hrin + VEG FR2 low I1 low phrinb2 + CD133 HoxA9 + phnotyp. I1 ws on o numrous trnsription tors uprgult in phs 1 nothlil lls. Bus it hs n shown to moult irntition n mintnn o vsulr ll t 19, w ous on I1 s potntil mitor o th pro-ngiogni t o TGFβ-inhiition osrv in our stuy. To trk I1 xprssion in liv hesc irntition ulturs, w us 164 volum 28 numr 2 rury 2010 ntur iothnology

5 α-cd %(+/ 0.3) VEGFR2 14.6%(+/ 2.6) α-vegfr2 0.56%(+/ 0.2) VEGFR2 6.2%(+/ 1.4) Rltiv I1 xprssion g I1 I1 75k 50k 25k h Totl lls CD31 + lls HUVEC hvprgfp 14+8 () VE-hrin Nu hvpr-gfp hvpr-gfp GIB4 Cll numr I1 I1 i Figur 4 TGFβ inhiition uprgults I1 xprssion n is nssry or th inrs yil o untionl nothlil lls pl o in vivo nongiognsis. (,) Humn VPr-GFP hescs tht wr stly trnsu with ontrol () or I1-spii () shrnas wr irntit oring to th protool shown in Figur 1 n ssss t y 14 or th prvln o VEGFR2 + (lu) n hvpr-gfp + (grn) lls. Th insts show plots o si sttr on th y xis n hvpr-gfp on th x xis. () Control n I1-spii shrnas wr to HUVEC or rshly isolt (t y 14) hvpr-gfp + lls, n th rltiv I1 trnsript lvls wr msur tr 3. *, P < Error rs, s.. o xprimntl vlus prorm in triplit. () Control n I1-spii shrnas wr to rshly isolt hvpr-gfp + lls, whih wr ultur in th sn or prsn o SB Atr 5, th totl ll numr n proportion o CD31 + lls ws msur y low ytomtry. Error rs, s.. o xprimntl vlus prorm in triplit., srml ontrol shrna. ( g) Humn VPr-GFP + lls wr isolt y FACS t y 14 n xpn in monolyr ultur () or 8 whil rtining xprssion o oth th nognous VE-hrin () n th hvpr-gfp trnsgn (g). Pnl shows mosi viw o on wll o 24-wll ish. A mgnii viw o th oxs in n r shown in n g, rsptivly. (h,i) Expn lls wr injt in Mtrigl plugs into immunoiint mi n xis tr 10 ollowing intrvitl lling o untionl vsultur with ltin (GIB4, lu). h, Viw o hvpr-gfp + lls lon; i, viw o hvpr-gfp + lls mrg with GIB4 + lls. Sl rs, µm. stl BAC trnsgni hesc lin 20 ontining yllow luorsnt protin rivn y th I1 promotr (I1-YFP) (Fig. 3 ) (Nm, H.S. n Bnzr, R., unpulish t). Dirntit nothlil lls wr isolt t y 14 rom I1-YFP ulturs (Fig. 1), su-rtionting th CD31 + popultion into I1-YFP high-xprssing (Fig. 3) n low-xprssing (Fig. 3) lls, n ths popultions wr srilly xpn or 7 with or without th TGFβ inhiitor (Fig. 3,). Flow ytomtri nlysis o ths lls rvl irt rltionship twn uprgultion o I1 xprssion n TGFβ inhiition. Notly, lthough SB inrs th prntg o th CD31 + popultion, th mn luorsn intnsity o CD31 on ths lls ws lowr thn tht o unstimult lls. Ths t suggst tht TGFβ inhiition inrs xpnsion o hesc-riv nothlil lls y mintining high lvls o I1 xprssion n prsrving n immtur prolirtiv phnotyp. To trmin th rquirmnt or I1 in miting nothlil ll ommitmnt, w trnsu hvpr-gfp + lls with lntivirl short hirpin (sh)rna trgt ginst th I1 trnsript (Fig. 4,). In th prsn o SB431542, knokown o I1 ru th numrs o oth VEGFR2 + vsulr prognitors n hvpr-gfp + lls t y 14. Whn th I1 shrna onstrut ws introu tr isoltion o th hvpr-gfp + rtion (Fig. 4), it liit mrk rs in CD31 + nothlil lls tr 5 o SB trtmnt (Fig. 4). Ths rsults intii TGFβ inhiition mit I1 uprgultion s primry tor in promoting nothlil ll xpnsion n mintining long-trm vsulr intity. To monstrt tht our ultur nothlil lls oul orm untionl vssls, w grw purii hvpr-gfp + lls rom y 14 irntition ulturs or n itionl 8 in th prsn o SB Ths nothlil lls show high prolirtiv potntil (up to tn ll ivisions) n gnrt homognous hvpr-gfp + VE-hrin + monolyrs (Fig. 4 g) with rtntion o hvpr-gfp luorsn t th singl-ll lvl (rrowhs in Fig. 4g). Ths lls wr suutnously injt in Mtrigl plugs into nonos (NOD)/svr omin immunoiint (SCID) mi n 10 ltr xtrt tr intrvnous injtion o ltin into liv nimls. In Mtrigl plugs, hvpr-gfp + lls o-loliz with ltin + lls, orming himri vssls long with host lls (Fig. 4h i n Supplmntry Vios 8 n 9). Ths t init tht th nothlil lls gnrt y our mthos oul untion in vivo. A prrquisit to thrputi vsulriztion using hesc-riv lls is gnrtion o unnt url nothlil lls tht upon xpnsion mintin thir ngiogni proil without irntiting into nonnothlil ll typs. Hr, w show tht irntition o hescs into lrg numr o stl n prolirtiv nothlil lls n hiv y rly-stg TGFβ-mit msorm inution ollow y TGFβ inhiition ginning t y 7 (phs 1) n tr isoltion t y 14 (phs 2). Using this pproh, w hiv 36-ol nt xpnsion o ommitt nothlil lls. Th inrs yil llow trnsriptionl nlysis, whih rvl molulr signtur tht shs light on th rgultory inluns tht govrn mryoni vsulognsis. In, gns noing ntur iothnology volum 28 numr 2 rury

6 tors ssoit with vsulr prognitor intity (I1 high, VEGFR2 high, CD133) 12 17,19 s wll s vsulr mrkrs (PECAM, VE-hrin, phrinb2) wr highly xprss in hesc-riv nothlil lls n, mong ths tors, I1 ws oun to t ownstrm o TGFβ inhiition to inrs nothlil ll yil y promoting prolirtion n prsrving vsulr ommitmnt. Ths stuis stlish TGFβ moultion o I1 xprssion s trminnt o hesc-riv nothlil ll intity n st th stg or lrg-sl gnrtion o uthnti long-lsting humn nothlil lls or thrputi vsulriztion. Our us o vsulr-spii hvpr-gfp n I1-YFP hesc rportr lins in smll-molul srns llow th isovry o th TGFβ inhiitor SB s ky stimulus or humn nothlil ll irntition n prolirtion in srum-r onitions. In murin ESCs, TGFβ n srum tors promot smooth musl ll irntition, whrs inhiition o this pthwy promots ormtion o CD31 + lls 21. Our t show tht stg-spii TGFβ inhiition, ginning on y 7 t point ollowing TGFβ-mit msorm inution, inrss th mitoti inx n mintnn o hesc-riv nothlil lls y uprgultion o I1 xprssion. Dirntition o hvpr-gfp hescs with TGFβ inhiition gnrt nothlil lls t yils tnol grtr thn thos o lls irntit with ngiogni tors lon, n tr puriition, TGFβ inhiition support nothlil ll xpnsion or up to tn ll ivisions whil rtining th ngiogni sur phnotyp. Th ility o TGFβ inhiition to inrs nothlil ll yil in oth irntiting (phs 1) n purii (phs 2) ulturs rsult in 36-ol inrs in th solut numr o hesc-riv nothlil lls, with 95% o th popultion mintining nothlil ll intity. As suh, w hv stlish mns o gnrting homognous popultion o stl nothlil lls in rtios tht grtly x hesc input n r rlvnt to thrputi vsuloplsty. Exprssion o I1 hs n shown to inhiit ll irntition n growth rrst in multipl ll typs 22. Th TGFβ signling pthwy, through th tors Sm3 n ATF3, hs n shown to rprss I1 promotr tivity 23. Th link twn TGFβ signling, I1 n prsrvtion o prolirtion n phnotypi intity o hesc-riv nothlil lls provis insight into th molulr mhnisms tht rgult vsulr ontogny uring humn vlopmnt. In, ths rsults point towr iphsi rol or TGFβ signling uring vsulognsis, whry rly tivtion o this pthwy is rquir or spiition o msorml prognitors, n inhiition tr vsulr ommitmnt untions to inrs mitoti inx n prvnt th loss o nothlil intity. Our pproh or vsulr monitoring n irntition my nl th intiition o s-yt unrogniz vsulogni n ngiogni moultors or prlinil stuis im t th ll-s thrputi rvsulriztion o ishmi tissus. METHODS Mthos n ny ssoit rrns r vill in th onlin vrsion o th ppr t Assion os. GEO: GSE ACKNOWLEDGMENTS W thnk A. Brivnlou or proviing th RUES1 hesc lin. D.J., M.S. n G.L. r Fion n Stnly Druknmillr Fllows o th Nw York Stm Cll Fountion. S.R. is support y Howr Hughs Mil Institut; Ansry Stm Cll Institut; Aninr n Nwmns Own Fountion; Ntionl Hrt, Lung, n Bloo Institut R01 grnts HL n HL097797; Qtr Ntionl Prioritis Rsrh Progrm; n Empir Stt Stm Cll Bor n Nw York Stt Dprtmnt o Hlth, NYS C Author Contriutions D.J. sign n prorm th xprimnts n wrot th mnusript. H.-s.N. n R.B. sign n rt th I1-YFP BAC trnsgni vtor. M.S. prorm xprimnts n ontriut to th mnusript. D.N. prorm low ytomtri xprimnts. T.J. prorm molulr loning. M.T. n L.S. gnrt th I1- YFP BAC trnsgni hesc lin. L.S. n G.L. gnrt th FD ipsc lin. N.Z. n Z.R. gnrt th hesc lins WMC2, WMC8 n WMC9. D.L. n S.Y.R. sign xprimnts n prorm t nlysis. S.R. sign xprimnts n wrot th mnusript. Not: Supplmntry inormtion is vill on th Ntur Biothnology wsit. COMPETING INTERESTS STATEMENT Th uthors lr no ompting innil intrsts. Pulish onlin t Rprints n prmissions inormtion is vill onlin t 1. Thomson, J.A. t l. Emryoni stm ll lins riv rom humn lstoysts. Sin 282, (1998). 2. Ymhr, K. t l. Augmnttion o novsulriztion in hinlim ishmi y omin trnsplnttion o humn mryoni stm lls-riv nothlil n murl lls. PLoS ONE 3, 1666 (2008). 3. Son, M. t l. Pthwy or irntition o humn mryoni stm lls to vsulr ll omponnts n thir potntil or vsulr rgnrtion. Artrioslr. Throm. Vs. Biol. 27, (2007). 4. Lu, S.J. t l. Gnrtion o untionl hmngiolsts rom humn mryoni stm lls. Nt. Mthos 4, (2007). 5. Golmn, O. t l. A oost o BMP4 lrts th ommitmnt o humn mryoni stm lls to th nothlil ling. Stm Clls 27, (2009). 6. Nours, M.B. t l. VEGF inus irntition o untionl nothlium rom humn mryoni stm lls: implitions or tissu nginring. Artrioslr. Throm. Vs. Biol. 30, 80 89(2009). 7. Bi, H. t l. BMP4 rgults vsulr prognitor vlopmnt in humn mryoni stm lls through sm-pnnt pthwy. J. Cll Biohm. pulish onlin, oi:10.2/j (30 Novmr 2009). 8. Hur, T.L., Kousko, V., Fhling, H.J., Plis, J. & Kllr, G. Hmngiolst ommitmnt is initit in th primitiv strk o th mous mryo. Ntur 432, (2004). 9. Lvnrg, S., Zoln, J., Bsvith, Y. & Lngr, R. Enothlil potntil o humn mryoni stm lls. Bloo 110, (2007). 10. Yng, L. t l. Humn riovsulr prognitor lls vlop rom KDR+ mryonistm-ll-riv popultion. Ntur 453, (2008). 11. Inmn, G.J. t l. SB is potnt n spii inhiitor o trnsorming growth tor-t suprmily typ I tivin rptor-lik kins (ALK) rptors ALK4, ALK5, n ALK7. Mol. Phrmol. 62, (2002). 12. Ghling, U.M. t l. In vitro irntition o nothlil lls rom AC133-positiv prognitor lls. Bloo 95, (2000). 13. Klly, M.A. & Hirshi, K.K. Signling hirrhy rgulting humn nothlil ll vlopmnt. Artrioslr. Throm. Vs. Biol. 29, (2009). 14. Pihv, M. t l. Exprssion o VEGFR-2 n AC133 y irulting humn CD34(+) lls intiis popultion o untionl nothlil prursors. Bloo 95, (2000). 15. Rii, S. & Lyn, D. Cnr. A w to lip th ngiogni swith. Sin 319, (2008). 16. Go, D. t l. Enothlil prognitor lls ontrol th ngiogni swith in mous lung mtstsis. Sin 319, (2008). 17. Lyn, D. t l. Impir rruitmnt o on-mrrow-riv nothlil n hmtopoiti prursor lls loks tumor ngiognsis n growth. Nt. M. 7, (2001). 18. Rossig, L. t l. Histon tyls tivity is ssntil or th xprssion o HoxA9 n or nothlil ommitmnt o prognitor lls. J. Exp. M. 201, (2005). 19. Ruzinov, M.B. & Bnzr, R. I protins in vlopmnt, ll yl n nr. Trns Cll Biol. 13, (2003). 20. Plntonkis, D.G. t l. BAC trnsgnsis in humn mryoni stm lls s novl tool to in th humn nurl ling. Stm Clls 27, (2009). 21. Wt, T. t l. TGF-t rptor kins inhiitor nhns growth n intgrity o mryoni stm ll-riv nothlil lls. J. Cll Biol. 163, (2003). 22. Jnkovi, V. t l. I1 rstrins myloi ommitmnt, mintining th sl-rnwl pity o hmtopoiti stm lls. Pro. Ntl. A. Si. USA 104, (2007). 23. Kng, Y., Chn, C.R. & Mssgu, J. A sl-nling TGFt rspons oupl to strss signling: Sm nggs strss rspons tor ATF3 or I1 rprssion in pithlil lls. Mol. Cll 11, (2003). 166 volum 28 numr 2 rury 2010 ntur iothnology

7 ONLINE METHODS Humn ESC ultur. Th xprimnts lint in this rport wr prorm primrily with th rntly pprov RUES1 hesc (gnrous git rom A. Brivnlou 24 ), n orroort using WMC2, WMC7, WMC8, gnrt t Will Cornll Mil Collg (ourtsy o Z. R./N.Z.) n H9 (I1-YFP, ourtsy o R.B./H.-s.N. n L.S./M.T.) n IPS (ourtsy L.S./G.L.). Th prmissions or us o ths ll lins wr otin tr omprhnsiv rviw y th Cornll- Rokllr-Slon Kttring Institut ESC rsrh ovrsight ommitt. Th uning or xution o ths stuis ws sur rom pprov non-rl uning rsours. Humn ESC ultur mium onsist o Avn DMEM/F12 (Gio) supplmnt with 20% knokout srum rplmnt (Invitrogn), 1 non-ssntil mino is (Gio), 1 l-glutmin (Invitrogn), 1 pniillin/ strptomyin (Invitrogn), 1 β-mrptothnol (Gio) n 4 ng/ml FGF-2 (Invitrogn). Humn ESCs wr mintin on Mtrigl using hesc mium onition y mous mryoni irolsts (Chmion). Lntivirl vtors n trnsution. Suprntnts ontining intious prtils wr ollt 40 n 68 h tr trnstion o HEK 293T with hvpr-gfp long with ssory vtors s prviously sri 25. Virl suprntnts wr onntrt y ultrntriugtion n us to trnsu unirntit RUES1 hescs. Atr two pssgs, hescs wr isggrgt y uts to orm singl lls, whih wr isolt n xpn to orm multipl prlll ulturs, h ontining rltivly onsistnt lvl o virl inorportion. Atr xpnsion, ths ulturs wr irntit s hrnt mryoi ois n srn or th prsn o GFP + lls. I1-YFP hesc rportr lin n lntivirl I1 shrna knokown. A tril rtiiil hromosom (BAC) ws moii to pl YFP unr ontrol o th nognous I1 promotr lous. This onstrut ws ltroport into th H9 hesc lin, slt or BAC intgrtion using ntiioti rsistn n sulon. Clons wr ssss n slt s on xprssion o YFP in I1 hesc rivtivs tr spontnous irntition. Th I1 n ontrol shrna lntivirl onstruts wr otin rom Opn Biosystms n virl prtils wr ssml oring to th mnuturr s rommntions (plko Lntivirl Pkging Systm). Emryoi ois. Humn VPr-GFP hescs wr grown to onlun on Mtrigl (BD Biosins) n thn inut in 5 units/ml isps (Gio) until olonis wr ompltly th rom th sustrt. Humn VPr-GFP mryoi ois wr wsh n ultur in hesc mium on ultr-low tthmnt plts (Corning) n ultur in th onitions sri, with rplmnt o ytokin-supplmnt mium vry 48 h. Emryoi ois wr ix in 4% prormlhy n rozn or ryostioning n stining. Enothlil irntition protools. Emryoi ois wr gnrt n ultur in s hesc mium, supplmnt with th ytokins s shown. Squntil ministrtion o ytokins ws implmnt (Fig. 1). Brily, mryoi ois wr gnrt in hesc s mium without FGF-2. On th morning tr gnrtion o mryoi ois (y 0), mium ws supplmnt with 20 ng/ml BMP4 (R&D Systms) (rmov t y 7); on y 1, mium ws supplmnt with 10 ng/ml tivina (R&D Systms) (rmov t y 4); on y 2, mium ws supplmnt with 8 ng/ml FGF-2 (Pproth) (rmin or th urtion o ultur); on y 4, mryoi ois wr trnsrr to hrnt onitions on Mtrigl-ot plts n mium ws supplmnt with 25 ng/ml VEGF-A (Pproth) (rmin or th urtion o ultur); on y 7, SB (Toris) ws t 10 µm onntrtion n rmin or init urtion. Culturs wr issoit using 0.5% Trypsin/EDTA (Gio) or Auts (Biosin). Asolut yil s wll s rtio o input hescs to irntit nothlil lls ws lult rom th numr o liv lls rovr rom irntition ulturs t ys 0, 14 n 20. Purii nothlil lls oul rozn n thw in 10% DMSO with >90% rovry. Quntittiv PCR. Totl RNA ws prpr rom ultur lls using th RNsy xtrtion kit (Qign) n rvrs trnsri using Suprsript II rvrs trnsripts (Invitrogn) oring to th mnuturr s instrutions. Rltiv quntittiv PCR ws prorm on 7500 Fst Rl Tim PCR Systm (Appli Biosystms) using ithr TqMn PCR mix long with I1 n β-tin primr pirs, or SYBR Grn PCR mix (Appli Biosystms). Humn-spii SYBR grn primr pirs us wr: PECAM, 5 -tttgtgt 3, r, 5 gggtgtttggttggtttt-3 ; Ot3/4 -, 5 -tgggtttgtggggttt-3, r, 5 -tgttttt-3 ; Brhyury, 5 -gtgggttggttggg-3, r, 5 -gttgggtgtgg-3 ; n -SMA,, 5 -tttgttggtggtggt-3, r, 5 -ggtggtttggtgg-3. Cyl onitions wr: on yl t 50 C or 2 min ollow y 1 yl t 95 C or 10 min ollow y 40 yls t 95 C or 15s n 60 C or 1 min. Primrs wr hk or mpliition in th linr rng n primr issoition n vrii. Thrshol yls o primr pros wr normliz to th houskping gn β-tin (ACTB) n trnslt to rltiv vlus. Enothlil ll isoltion n low ytomtry. Enothlil lls wr isolt rom irntition ulturs using Mgnti Ativt Cll Sorting (MACS; Miltnyi Bioth) with n ntioy ginst CD31 onjugt to mgnti miros. Altrntivly, lls wr isolt y virtu o th xprssion o GFP/YFP or luorophor onjugt ntioy to humn CD31 or VEGFR2 (BD) using FACSAriII (BD). Mirorry nlysis. Th Aymtrix Humn Gnom U rry ws us to nlyz gn xprssion. In ri, using Qign RNsy kits, totl RNA ws xtrt rom: Humn VPr-GFP mryoi ois tht wr ultur in th prsn o rominnt ytokins lon until y 14; MACS-sort nothlil lls isolt rom hvpr-gfp mryoi ois ultur in th prsn o rominnt ytokins lon until y 14; MACS-sort nothlil lls isolt rom hvpr-gfp trnsu mryoi ois ultur in th prsn o rominnt ytokins n th TGFβ inhiitor SB until y 14; MACS-sort nothlil lls isolt rom hvpr-gfp mryoi ois ultur in th prsn o rominnt ytokins n th TGFβ inhiitor SB until y 14, ollow y 10 itionl ultur in th prsn o ytokins n SB431542; humn umilil vin nothlil lls; humn umilil vin smooth musl lls; n CD34 + umilil or loo lls. Th Suprsript hoi kit (Invitrogn) ws us to mk DNA with T7-(T)24 primr inorporting T7 RNA polymrs promotr. Th iotin-ll RNA ws m y in vitro trnsription (Enzo Dignostis). Frgmnt RNA ws hyriiz to th gn hips, wsh, n stin with strptviin phyorythrin. Th pro rrys wr snn with th Gnhip Systm onol snnr n Aymtrix Mirorry suit 4.0 s us to nlyz th t. Mtrigl plug. Humn VPr-GFP mryoi ois wr irntit or 14 y our irntition protool ollow y xpnsion in th prsn o SB or 10 n injt suutnously into NOD/SCID mi in suspnsion o Mtrigl. Atr 2 wks, Grioni simpliioli IB4 ltin n/or Ulx uropus gglutinin ltin wr ministr intr-vitlly to Mtrigl plug ring mi n plugs wr hrvst, ix ovrnight in 4% prormlhy n quilirt in 30% suros or rzing n ryostioning. Immunoluorsn. Cryostions wr immunoytohmilly stin s prvious sri 24. Brily, smpls wr prmiliz in PBST n lok in 5% onky srum. Smpls wr inut or 2 h in primry ntiois loking solution, wsh 3 tims in PBS n inut in CY3-onjugt sonry ntiois (Jkson Lortoris) or 1 h. Atr wshing, som stions wr ountrstin or nuli is y TO-PRO3 (Invitrogn) or mounting n imging y onol mirosopy. Primry ntiois inlu CD31 (DAKO), CD34 (DAKO), Phospho-HistonH3, Smooth Musl Atin (DAKO) n VE-hrin (R&D). All imging ws prorm using Ziss 510 META onol mirosop. Liv imging n 3D rnring. Humn VPr-GFP mryoi ois wr ultur in TOKAI-HIT liv ll-imging hmr on Ziss 510 META onol mirosop. Lsr intnsity n intrvl wr optimiz to nsur viility o lls or th urtion o th xprimnts. Thr-imnsionl ronstrution n rnring o optil z-stks ws prorm using Improvision Voloity sotwr. 24. Jms, D., Noggl, S.A., Swigut, T. & Brivnlou, A.H. Contriution o humn mryoni stm lls to mous lstoysts. Dv. Biol. 295, (2006). 25. Nlini, L. t l. In vivo gn livry n stl trnsution o noniviing lls y lntivirl vtor. Sin 272, (1996 ntur iothnology volum 28 numr 2 rury