Resident c-kit þ cells in the heart are not cardiac stem cells

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1 Reeived 3 Jul 05 Aepted Sep 05 Pulished 30 Ot 05 DOI: 0.038/nomms970 OPEN Resident -kit þ ells in the hert re not rdi stem ells Nisht Sultn, *, Lu Zhng, *, Jinyun Yn, *, Jiqiu Chen, Weiin Ci, Sheguft Rzzque, Dongtk Jeong, Wei Sheng 3, Lei Bu 4, Mingjing Xu 5, Guo-Ying Hung 3, Roger J. Hjjr, Bin Zhou 6, Anne Moon 7 & Chen-Leng Ci Identifying on fide popultion of rdi stem ells (CSCs) is ritil step for developing ell-sed therpies for hert filure ptients. Previously, rdi -kit þ ells were reported to e CSCs with potentil to eome myordil, endothelil nd smooth musle ells in vitro nd fter rdi injury. Here we provide further insights into the nture of rdi -kit þ ells. By trgeting the -kit lous with multiple reporter genes in mie, we find tht -kit expression rrely o-lolizes with the expression of the rdi progenitor nd myogeni mrker Nkx.5, or tht of the myordil mrker, rdi troponin T (TnT). Insted, -kit predominntly lels rdi endothelil ell popultion in developing nd dult herts. After ute rdi injury, -kit þ ells retin their endothelil identity nd do not eome myogeni progenitors or rdiomyoytes. Thus, our work strongly suggests tht -kit þ ells in the murine hert re endothelil ells nd not CSCs. Deprtment of Developmentl nd Regenertive Biology, The Blk Fmily Stem Cell Institute, nd The Mindih Child Helth nd Development Institute, Ihn Shool of Mediine t Mount Sini, New York, New York 009, USA. Deprtment of Mediine, Crdiovsulr Reserh Center, Ihn Shool of Mediine t Mount Sini, New York, New York 009, USA. 3 Crdiovsulr Center, Children s Hospitl of Fudn University, Shnghi 00, Chin. 4 Leon H. Chrney Division of Crdiology, Deprtment of Mediine, New York University Shool of Mediine, New York, New York 006, USA. 5 Deprtment of Biohemistry nd Moleulr Biology, Sylvester Comprehensive Cner Center, University of Mimi Miller Shool of Mediine, Mimi, Florid 3336, USA. 6 Deprtment of Genetis, Alert Einstein College of Mediine of Yeshiv University, Bronx, New York 046, USA. 7 Weis Center for Reserh, Geisinger Clini, Dnville, Pennsylvni 78, USA. * These uthors ontriuted eqully to this work. Correspondene nd requests for mterils should e ddressed to C.-L.C. (emil: henleng.i@mssm.edu). NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved.

2 NATURE COMMUNICATIONS DOI: 0.038/nomms970 Myordil loss or dysfuntion from ishemi hert disese is the leding use of mortlity worldwide. Crdi stem ell (CSC) trnsplnttion is potentil therpeuti intervention for ptients with hert filure 6. For these resons, onsiderle efforts hve een mde to identify speifi mrkers of stem ells with rdiomyogeni potentil 4,7 0. Among the vrious mrkers employed to identify resident CSCs, -kit hs plyed prominent role,. Crdi -kit-positive (-kit þ ) ells were the first popultion of puttive CSCs desried s eing negtive for the lood linege mrker (Lin ) in the dult rt hert, long with possessing selfrenewing, lonogeni nd multipotent hrteristis 7,3,4. -kit þ rdi ells were lso shown to e neessry nd suffiient for myordil regenertion fter rdi injury in rts nd mie 5. While these findings present enourging therpeuti possiilities, ontroversy regrding their use remins due to divergent oservtions out the myogeni potentil of resident -kit þ ells in the mmmlin hert 6 8. A reent linege tring study of -kit progeny reveled tht -kit þ ells hve extremely limited potentil to differentite into rdiomyoytes during ging nd fter injury in mie 9. However, douts hve een st on this study, questioning not only the effiieny of Cre/LoxP reomintion for linege tring ut lso potentilly unrelile or insensitive expression of oth the Cre drivers nd reporter mouse models employed 0,. Thus, importnt questions remin unnswered. For exmple, wht is the ext nture of rdi -kit þ ells? Do these ells give rise to multiple rdi lineges during development nd fter hert filure? Do the enefits (if ny) of -kit þ ell-sed therpies rise from n ility to differentite into rdiomyoytes, or do -kit þ ells generte prrine ftors or prellulr environment tht promotes reovery? Given tht -kit þ ells re eing linilly tested on humn ptients with ishemi rdiomyopthy 3, fully ddressing these questions is ritil. Determining the identity of -kit þ ells in the mmmlin hert is the foundtion for nswering these questions. To overome the limittions of ntiody-sed immunostining or trnsgeni mouse lines with prtil regultory elements tht my not e suffiiently sensitive nd/or fithfully reprodue the endogenous expression of gene, we generted series of mouse models tht llow preise hrteriztion of the identity of -kit þ ells nd their progeny in mouse herts. Using these models, we unexpetedly found tht rdi -kit-expressing ells re tully supopultion of endothelil ells in the developing nd dult hert in mie. -kit þ ells rrely express the rdi progenitor mrker Nkx.5 or the differentited rdiomyoyte mrker rdi troponin T (TnT), nor do they eome rdiomyoytes during development or fter injury. Our results strongly suggest tht -kit þ ells in the mmmlin hert re tully endothelil ells nd not CSCs. Results -kit is expressed in the developing nd dult mouse hert. We first generted knok-in mouse model, -kit HB-tdTomto/þ, y gene trgeting (Fig. nd Supplementry Fig. ). In this niml, the HB-tdTomto ssette ws inserted into the -kit strt odon without deleting ny genomi sequenes, therey expressing tdtomto under the ontrol of the full omplement of endogenous -kit regultory elements. Sine tdtomto is fused to histone HB gene 4, its expression is lolized to the nuleus. To onfirm the fidelity of the -kit HB-tdTomto signl to the endogenous -kit expression pttern, we performed whole-mount RNA in situ hyridiztion on the wild-type mie from emryoni dy (E) 9.5 to E3.5. By ompring -kit HB-tdTomto signls to -kit mrna expression, we found tht the signls overlpped in ll known s of -kit expression 5,6, suh s the phryngel rhes, liver, umilil ord nd melnoytes (Supplementry Fig. ). Furthermore, HB-tdTomto expression ws deteted in other orgns, inluding the lung, stomh, intestine nd spleen (Supplementry Fig. e), s well s the neurl tue nd yolk s during emryogenesis. This finding is onsistent with previous reports of -kit expression in these orgns 5,6. Immunostining of setioned -kit HB-tdTomto/þ mouse tissues reveled tht the -kit HB-tdTomto -positive ells o-lolized with -kit ntiody in the liver, lung nd melnoytes (Supplementry Fig. 3). Further support for the sensitivity nd fidelity of this reporter is the oservtion tht ells with low -kit expression deteted y ntiody exhiited right HB-tdTomto fluoresene (Supplementry Fig. 3,). Next, we exmined the lotion of -kit þ ells in the herts of -kit HB-tdTomto/þ mie (Fig. ). Endordil ells with nuler tdtomto expression were oserved s erly s E8.5 nd 9.5 (Fig.,). Strting from E.5, ells with strong -kit HB-tdTomto expression were dispersed throughout the hert, with the highest density in the inner lyers of the tril nd ventriulr hmers t ll emryoni stges tested (Fig. d,e). At postntl dy (P), P30, P60 nd P0, -kit HB-tdTomto expressing ells were onsistently deteted in ll hmers of the hert (Fig. f i). The rod distriution of -kit HB-tdTomto -positive ells in the hert from emryoni stges to dulthood is inonsistent with previous studies reporting tht -kit þ ells represent smll popultion of CSCs in the mmmlin hert 7, 5,7. -kit þ ells do not express Nkx.5 or TnT fter E3.5. In the initil hrteriztion of rdi resident -kit þ ells in the dult rt, -kit þ ells were shown to ontin mixed popultion of ells exhiiting erly stges of myogeni differentition s demonstrted y the tive expression of the erly rdi trnsription ftors Nkx.5, Gt4 nd Mef in the nuleus nd of sromeri proteins in the ytoplsm of these ells 7,5. To determine whether -kit HB-tdTomto -positive ells express the rdi progenitor mrker Nkx.5, we rossed Nkx.5 HB-GFP/þ knok-in mie 8 with -kit HB-tdTomto/þ mie to otin ompound heterozygotes (-kit HB-tdTomto/þ ;Nkx.5 HB-GFP/þ ). HB GFP expression in Nkx.5 HB-GFP/þ mie fithfully repitultes the endogenous Nkx.5 pttern 8. We exmined rdi tissues throughout the emryoni (E ) nd postntl (P 0) stges (Supplementry Fig. 4). All histologil setions from E9.5 to 3.5 herts nd more thn 30 setions from E4.5 to P0 herts were inspeted (n ¼ 3 for eh stge). However, no -kit HB-tdTomto nd Nkx.5 HB-GFP doule-positive ells were found (Supplementry Fig. 4,d g), exept t E.5, wherein only doule-positive ells were deteted in the ventriulr septum (Supplementry Fig. 4, B0.007% of totl Nkx.5 HB-GFP -positive ells). To determine whether ny -kit þ ells produe sromeri or myordil proteins 7,5, we pplied TnT HB-GFP/þ knok-in mouse model with insertion of n HB GFP ssette into the strt odon of TnT (Tnnt; Supplementry Fig. 5). On exmining hert setions from -kit HB-tdTomto/þ ;TnT HB-GFP/þ ompound heterozygous nimls t emryoni nd postntl stges (E8.5 P0), we did not detet ny ells in whih oth mrkers were ololized (Supplementry Fig. 5), with the exeption of E3.5, where n verge of 5 doule-positive ells were found within the ventriulr septum (Supplementry Fig. 5d, B0.009% of totl TnT HB-GFP -positive ells). These oservtions revel tht -kit þ ells in -kit HB-tdTomto/þ mie very rrely o-express either Nkx.5 or TnT in the emryoni hert nd do not o-express these mrkers in foetl or dult herts. NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved.

3 NATURE COMMUNICATIONS DOI: 0.038/nomms970 ARTICLE E -kit ATG E HB-tdTomto-pA E -kit HB-tdTomto -kit HB-tdTomto/+ OFT E8.5 d OFT E9.5 e E.5 f RA VS E4.5 g VS -kit HB-tdTomto /DAPI P h RA P30 i P60 P0 Figure Crdi -kit expression in -kit HB-tdTomto/þ mie. () Digrm of the -kit HB-tdTomto/þ knok-in llele. ( i) Setions of -kit HB-tdTomto/þ herts t emryoni dys (E) 8.5, 9.5,.5 nd 4.5 ( e) nd t postntl (P) dys, 30, 60 nd 0 (f i)., e, g nd i re high-mgnifition imges (without DAPI) of the res outlined in, e, g nd i, respetively. -kit HB-tdTomto ells re denoted y rrows., left tri;, left ventrile; OFT, outflow trt; RA, right tri;, right ventrile; VS, ventriulr septum. n ¼ 3 for eh stge. Sle r, 00 mm. Crdi -kit þ ells re supopultion of endothelil ells. To further determine the identity of -kit þ ells, we performed immunostining with ntiodies ginst the endothelil mrker PECAM (CD3) nd the smooth musle mrker, -SMA. Surprisingly, t ll the stges exmined (E8.5 P0), -kit HB-tdTomto -positive ells were PECAM þ (Fig. -f) ut -SMA (Fig. g,h). This finding suggests tht rdi -kit HB-tdTomto -positive ells re endothelil ells. Quntittive flow ytometri nlysis of 4-month-old herts demonstrted tht B43% PECAM þ ells in the ventriles were lso -kit þ (Supplementry Fig. 6). Thus, our results indite tht -kit HB-tdTomto -positive ells represent suset of rdi endothelil ells. tdtomto is right fluoresent protein 9,30. We were onerned tht the long stility of tdtomto ould omplite the detetion of trnsient -kit expression. To onfirm the identity of -kit þ ells identified y -kit HB-tdTomto/þ, we generted nother reporter line, -kit nlz-hb-gfp/þ, y inserting LoxP-nlZ-4XPolyA-LoxP-HB GFP ssette into the -kit strt odon (Fig. 3 nd Supplementry Fig. 7). HB GFP is not deteted in this line unless the nlz-4xpolya stop ssette is removed y Cre-medited reomintion. We performed whole-mount X-gl stining on -kit nlz-hb-gfp/þ emryos nd found tht the -kit nlz signl ws not only relily repitulted y -kit mrna expression, ut lso onsistent with the HB tdtomto expression ptterns in -kit HB-tdTomto/þ mie (Supplementry Fig. ). Furthermore, X-gl stining of whole-mount nd setioned herts t E5.5 P90 redily deteted rod distriution of -kit nlz -positive ells throughout the hert (Fig. 3,d,f,h, nd j), inluding the endordium (Fig. 3,h), similr to the pttern oserved in -kit HB-tdTomto/þ mie. X-gl stining of ompound heterozygous littermte herts ering n endothelil-speifi Tie-Cre llele (-kit nlz-hb-gfp/þ ;Tie Cre ) ould not detet -kit nlz -positive ells (Fig. 3,e,g,i nd k; less thn 0 rndomly distriuted -kit nlz -positive ells were found in the dult hert, representing B0.000% of totl -kit þ ells). Consistent with the endothelil nture of -kit þ ells in the hert, -kit HB-GFP -positive ells generted y Tie Cre exision were ll o-stined with nti-pecam ntiody (Supplementry Fig. 8). Thus, the -kit nlz-hb-gfp/þ reporter line onfirms the endothelil identity of rdi -kit þ ells. NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms & 05 Mmilln Pulishers Limited. All rights reserved.

4 NATURE COMMUNICATIONS DOI: 0.038/nomms970 -kit HB-tdTomto/+ OFT E8.5 E9.5 E6.5 d e f P P60 P0 g h -kit HB-tdTomto /PECAM/DAPI α-sma/dapi P0 P0 Figure Crdi -kit HB-tdTomto ells re PECAM þ endothelil ells. (,) At E8.5 nd E9.5, -kit HB-tdTomto ells re endordil (PECAM þ ). ( f) -kit HB-tdTomto ells express PECAM t E6.5 () nd t P 0 (d f). Arrows indite PECAM þ nd tdtomto þ doule-positive ells. Arrowheds indite PECAM þ nd tdtomto ells. (g,h) Crdi smooth musle ells (-SMA þ ) re tdtomto t P0 (rrowheds). h re highmgnifition imges of the res outlined in h (without DAPI), respetively. n ¼ 3 for eh stge. Sle r, 00 mm. To further ddress the issue of stility of oth HB tdtomto nd nlz proteins, we nlysed rdi -kit ells with the third reporter llele -kit MerCreMer/þ, in whih n induile MerCreMer ssette ws inserted into the -kit strt odon (Fig. 4 nd Supplementry Fig. 9). -kit MerCreMer/þ ; ROSA6R tdtomto/þ mie were susequently generted y rossing with ROSA6R tdtomto/þ mie. In the sene of tmoxifen tretment, no tdtomto-expressing ells were deteted in the dult herts. To onfirm whether -kit is tively expressed in the postntl hert, we injeted tmoxifen t P30, P60 or P90 for 3 onseutive dys (dys, nd 3), nd immeditely olleted rdi tissues for nlysis t dy 4 (P30-34, P60-64) or 4 (P90-04). This tretment onsistently resulted in tdtomto lelling of lrge numer of ells in the hert (Fig. 4,d,e) tht lso expressed PECAM (Fig. 4). This result further onfirms tht rdi -kit þ ells re endothelil (Figs nd 3), nd supports the previous oservtion tht rdi -kit þ ell progeny re endothelil 9. -kit þ endothelil ells re identified y immunostining. -kit HB-tdTomto/þ, -kit nlz-hb-gfp/þ nd -kit MerCreMer/þ nimls re heterozygous null for -kit (-kit þ/ ). Hploinsuffiieny of -kit ould ffet -kit regultion in vivo 0,3 33, possily leding to etopi rdi expression. To determine whether etopi -kit expression ours in the reporter mouse herts, we performed immunostining t emryoni (E.5 5.5) nd postntl stges (P 60) using -kit ntiody on mie of four different genotypes: wild type, -kit HB-tdTomto/þ (-kit þ/ ), -kit HB-tdTomto/MerCreMer (-kit / ) nd -kit MerCreMer/MerCreMer (-kit / ). Using -kit ntiody, we frequently deteted ells in wild-type herts tht were dully lelled with -kit nd PECAM (Supplementry Fig. 04,d4,g nd Supplementry Fig.,f,h,i). In -kit HB-tdTomto/þ nimls, -kit ntiody immunoretivity o-lolized with -kit HB-tdTomto (Supplementry Fig. 0, e,h nd Supplementry Fig.,), lthough the immunofluoresene ws deresed ompred with tht in wild-type 4 NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved.

5 NATURE COMMUNICATIONS DOI: 0.038/nomms970 ARTICLE E -kit ATG LoxP E LoxP E X Cre nlz-4xpa HB-GFP-pA -kit nlz-hb-gfp E HB-GFP-pA E -kit HB-GFP -kit nlz-hb-gfp/+ -kit nlz-hb-gfp/+ ;Tie Cre -kit nlz-hb-gfp/+ -kit nlz-hb-gfp/+ ;Tie Cre h i E5.5 P60 d e P f g j k P30 P90 Figure 3 -kit nlz ells re of Tie endothelil linege. () Digrm of the -kit nlz-hb-gfp/þ reporter llele (). The -kit HB-GFP/þ llele is generted when the nlz ssette is removed y Cre exision (). ( k) X-gl stining of -kit nlz-hb-gfp/þ nd -kit nlz-hb-gfp/þ ;Tie Cre littermte herts t E5.5 (,, setions) nd t P 90 (d k). Arrows indite omprle s to X-gl þ or X-gl stining. Arrowheds indite rre X-gl þ ells on -kit nlz-hb-gfp/þ ;Tie Cre herts, suggesting tht most -kit þ ells lose the nlz gene euse they re in the Tie Cre linege. f k re high-mgnifition imges of the res outlined in f k, respetively. n ¼ 3 5 for eh stge. Sle r, 400 mm (lk) nd 00 mm (white). nimls. Redued -kit immunoretivity in -kit HB-tdTomto/þ tissues is onsistent with the -kit þ/ geneti kground (theoretilly 50% -kit protein redution in -kit þ/ ). Importntly, -kit ntiody stining ws ompletely undetetle in -kit / mutnt herts or lungs, even with Tyrmide Signl Amplifition (TSA) mplifition (Supplementry Figs 0,f nd d,e), demonstrting the speifiity of the ntiody stining. Therefore, immunostining with -kit ntiody lso revels tht rdi -kit þ ells re endothelil nd indites tht no etopi rdi -kit expression ours in the new knok-in mouse models employed. Resident -kit þ ells rrely differentite into rdiomyoytes.to further determine the myogeni potentil of -kit þ ells during hert formtion, we pplied TnT nlz-hb-gfp/þ rdiomyoyte-speifi reporter mie with the LoxP-nlZ-4X PolyA-LoxP-HB-GFP ssette trgeted into TnT strt odon. TnT HB-GFP expression is deteted in rdiomyoytes when Cre is expressed in the myordium or myogeni preursor ells (Fig. 4f). We rossed -kit MerCreMer/þ mie with TnT nlz-hb-gfp/þ mie nd injeted tmoxifen in -kit MerCreMer/þ ;TnT nlz-hb-gfp/þ nimls. After two doses of tmoxifen dministrtion (dys nd ) to pregnnt mie (E.5 emryos) or four doses (dys,, 3 nd 5) to P30, P60 nd P90 mie, we olleted herts for nlysis t E3.5 or t P37, P67 nd P97, respetively. All rdi setions were ssessed for TnT HB-GFP -positive ells. On verge, pproximtely 50, 34, 56 nd 66 ells were found in herts (n ¼ 3 for eh group) t E3.5, P37, P67 nd P97, respetively (Fig. 4g), representing o0.04% of rdiomyoytes t orresponding stges (o0.007% fter P90). This finding demonstrtes tht the myogeni potentil of -kit þ ells, if ny, is extremely low in oth emryoni nd postntl herts. Previous studies hve reported tht within 4 weeks of myordil infrtion in dult mouse herts, the numer of -kit/nkx.5 doule-positive myogeni preursors signifintly inresed in the injured, nd some of these myogeni preursors trnsformed into prolifertive rdiomyoytes 7,5.To diretly investigte the differentition potentil of rdi -kit þ ells post myordil infrtion, we ligted the left nterior desending (D) oronry rtery of -kit HB-tdTomto/þ ; Nkx.5 HB-GFP/þ mie ( 5 months old, n ¼, Fig. 5,). Exmintion of rdi setions t, 3, 7,, 30 nd 60 dys post-surgery (dps) reveled mny -kit HB-tdTomto -positive ells NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms & 05 Mmilln Pulishers Limited. All rights reserved.

6 NATURE COMMUNICATIONS DOI: 0.038/nomms970 E -kit ATG E MerCreMer E -kit MerCreMer X R6R tdtomto Injet tmoxifen t dy,, 3, nd ollet smples t dy 4 4 -kit MerCreMer /+ tdtomto /+ ;R6R d P30 34 P60 64 e R6R tdtomto /PECAM f P90 04 E -TnT ATG LoxP LoxP E E nlz-4xpa HB-GFP-pA -TnT nlz-hb-gfp X -kit MerCreMer Injet tmoxifen for -4 dys, nd ollet smples t dy 3 7 g -kit MerCreMer /+ ;-TnT nlz-hb-gfp/+ x50 x34 x56 x66 E P30 37 P P Figure 4 Ative -kit endothelil expression nd myogeni potentil ssyed y trnsient indution of Cre tivity in -kit MerCreMer/þ mie. () Digrm of the -kit MerCreMer/þ llele. -kit MerCreMer/þ nimls were rossed to the ROSA6R tdtomto reporter line to otin -kit MerCreMer/þ ; ROSA6R tdtomto/þ.( e) Cre tivity ws trnsiently indued in -kit MerCreMer/þ ;ROSA6R tdtomto/þ nimls t P30, P60 nd P90 y tmoxifen injetion on dys 3. Herts were hrvested on dys 4 nd 4. Mny tdtomto þ ells (rrows in, d nd e) were deteted in herts t P34 (), P64 (d) nd P04 (e). These tdtomto þ ells were PECAM þ (, rrows, P30-34)., d nd e re high-mgnifition floresent imges of the res outlined in, d nd e (right field), respetively. (f) Digrm of the TnT nlz-hb-gfp/þ llele nd linege tring using -kit MerCreMer/þ ;TnT nlz-hb-gfp/þ mie. Cre tivity ws trnsiently indued y tmoxifen injetion for 4 dys on dys,, 3 nd 5 (dys nd for E.5). Smples were olleted on dy 7 (dy 3 for E.5). (g) TnT HB-GFP ells were deteted t E3.5, P37, P67 nd P97 (rrows), with the totl numer in the whole hert noted t the upper right orner. Sle r, mm (lk) nd 00 mm (white). TnT HB-GFP /DAPI in the infrted (Fig. 5 f). However, no -kit HB-tdTomto nd Nkx.5 HB-GFP doule-positive ells were found in the injured re t ny stge tested (Fig. 5 f, f). To further determine the ell identity of these -kit þ ells, we performed D ligtion on Tie Cre ;-kit nlz-hb-gfp/þ mie ( 4 months old, n ¼ 3). -kit HB-GFP -positive ells were redily deteted in the infrted, demonstrting tht they retined their endothelil nture fter injury (Fig. 6). A reent study reported tht supopultion of endothelil ells yields progeny with CSC hrteristis in the dult mouse hert 34. This supopultion purportedly rises from endothelil mesenhyml trnsition nd gives rise to rdiomyoytes tht ontriute to hert renewl 34. To determine whether -kit þ endothelil ells produe CSCs tht further differentite into rdiomyoytes following rdi injury, we performed D ligtion on TnT MerCreMer/þ ; -kit nlz-hb-gfp/þ ;ROSA6R tdtomto/þ mie ( 4 months old, n ¼ 4, Fig. 6). TnT MerCreMer/þ medites speifi nd effetive myordil reomintion fter tmoxifen indution 35. If -kit nlz-hb-gfp/þ ells eome rdiomyoytes nd if -kit expression is mintined in these ells, then -kit HB-GFP -positive ells would e deteted. However, fter tmoxifen ws injeted t 3 7 dps nd 3 35 dps (three tmoxifen tretments for eh period), we deteted no -kit HB-GFP -positive ells in the infrted (Fig. 6), lthough myordil reomintion ws widely deteted in nd djent to the infrted (s reveled y ROSA6R tdtomto stining, Fig. 6). Furthermore, exmintion of dult -kit MerCreMer/þ ;TnT nlz-hb-gfp/þ mie fter D ligtion (3 5 months old, n ¼ 3, Fig. 6d) reveled o0 TnT HB-GFP -positive ells per hert (B0.00% of totl myordil ells) throughout the injured (Fig. 6e). TnT HB-GFP -positive ells ould lso e deteted in remote uninjured s (B30 ells, B0.003% of totl myordil ells, Fig. 6e), suggesting tht the TnT HB-GFP -positive ells found in the injured re likely not response to rdi injury. These rdi injury mouse models reveled tht the myordil potentil of -kit þ endothelil ells, if ny, is extremely low. However, these dt do not prelude the possiility tht -kit rdi endothelil ells my hve the potentil for endothelil mesenhyml trnsition nd myordil differentition. A rre popultion of rdi -kit þ ells re rdiomyoytes. In the linege tring experiments used to determine the 6 NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved.

7 ARTICLE NATURE COMMUNICATIONS DOI: 0.038/nomms970 -kit tdtomto/+;nkx.5 HB-GFP/+ Infrted 60 dps Infrted Trihrome D 3 dps d dps e 30 dps -kit tdtomto/nkx.5 HB-GFP/DAPI f 60 dps Figure 5 -kit þ ells do not o-express Nkx.5 in the injured. () Digrm of D ligtion. () Msson trihrome stining shows the infrted of -kithb-tdtomto/þ ;Nkx.5HB-GFP/þ hert t 60 dys post-surgery (dps). nd re high-mgnifition imges of the numered outlined res in. ( f) No -kithb-tdtomto/nkx.5hb-gfp doule-positive ells were found in the infrted s t 3 (), (d), 30 (e) nd 60 dps (f). /, d/d, e/e, nd f/f re high-mgnifition imges of the numered outlined res in, d, e nd f, respetively. Sle r, 500 mm (lk) nd 50 mm (white). myordil potentil of -kitþ ells during development nd fter rdi injury in -kitmercremer/þ ;TnTnlZ-HB-GFP/þ niml models, very smll numer of TnTHB-GFP-positive ells ws deteted (Fig. 4g, B66 56 ells; nd Fig. 6e, B0 ells). In ll ses, the numer ws extremely low when ompred with the totl numer of -kithb-tdtomto-positive ells (o0.005%) or myordil ells (o0.05%) in whole herts. The origin of these rre ells is unknown. These ells my e derived from unommitted ells originlly expressing -kit, or they ould e rdiomyoytes tht express oth -kit nd TnT due to rre stohsti event. To explore these possiilities, we exmined TnTMerCreMer/þ ;-kitnlz-hb-gfp/þ dult mouse herts ( 4 months old, uninjured) fter tmoxifen injetion for onseutive dys (dys nd ). At dys 3, 7 nd 30, we deteted B0 30 -kithb-gfp-positive ells per hert fter exmining ll the hert setions (n ¼ 3, Supplementry Fig. ). This result suggests tht very smll numer of resident -kit ells re rdiomyoytes (B0.005% of totl -kitþ ells nd B0.00% of totl myordil ells in the hert). Notly, the numer of -kithb-gfp-positive ells deteted in TnTMerCreMer/þ ; -kitnlz-hb-gfp/þ herts (B0 30, Supplementry Fig. ) is less thn the numer of TnTHB-GFP-positive ells in herts (B66 56, -kitmercremer/þ ;TnTnlZ-HB-GFP/þ Fig. 4g3,g4). This is proly due to muh higher levels of TnT expression thn -kit expression nd/or to differentil sensitivity of the reporters to Cre-medited reomintion. Disussion Currently, the identity nd differentition potentil of resident -kitþ ells in the mmmlin hert re the entrl questions in rdi regenertive mediine. For more thn dede, rdi NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved. 7

8 NATURE COMMUNICATIONS DOI: 0.038/nomms970 Tie Cre ;-kit nlz-hb-gfp/+ -kit HB-GFP DAPI -kit HB-GFP -kit HB-GFP 30 dps 3 TnnT MerCreMer/+ ;-kit nlz-hb-gfp/+ ;R6R td-tomto/+ Trihrome -kit HB-GFP DAPI -kit HB-GFP 60 dps d Trihrome -kit MerCreMer/+ ;TnnT nlz-hb-gfp/+ e Tnnt HB-GFP DAPI < 0x Remote Tnnt HB-GFP DAPI 60 dps Figure 6 Cell type nd linege of -kit þ ells in the injured hert. () -kit HB-GFP -positive ells were present in the infrted of Tie Cre ; -kit nlz-hb-gfp/þ herts t 30 dps. is green hnnel of, nd 3 is high-mgnifition imge of the re outlined in. () Msson trihrome stining of TnT MerCreMer/þ ;-kit nlz-hb-gfp/þ ;ROSA6R tdtomto/þ herts t 60 dps shows the infrted. () Adjentsetionof. ROSA6R tdtomto signl indites myordil ells fter tmoxifen indution (). No -kit HB-GFP ells were oserved in the infrted zone (rrows). is green hnnel of. (d) Msson trihrome stining of -kit MerCreMer/þ ;TnT nlz-hb-gfp/þ herts t 60 dps. (e) Adjent setion of d shows few TnT HB-GFP ells (o0) tht were found in the infrted zone (e, rrowhed). TnT HB-GFP ells were lso present in remote, uninjured (e, rrowhed). Sle r, 00 mm. -kit þ ells hve een desried s multipotent ell popultion with regenertive pity 7, 5,7. If -kit expression defines stem or undifferentited stte, then -kit should not mintin to e expressed in ny differentited rdi ell type suh s the endothelium or myordium. However, our studies of three informtive reporter lleles in mie (-kit HB-tdTomto/þ, -kit nlz-hb-gfp/þ nd -kit MerCreMer/þ ;ROSA6R tdtomto/þ ) omined with -kit immunostining onsistently reveled tht -kit tively lels n endothelil popultion in mouse herts during development nd into dulthood. These results rgue ginst the urrent prdigm tht -kit is mrker of CSCs or tht rdi -kit þ ells re unommitted 7,3,4. A reent -kit linege tring study y vn Berlo et l. 9 reveled tht -kit þ ells rrely eme rdiomyoytes, insted entering into n endothelil ell fte. Our study supports this oservtion. However, vn Berlo s study leves the possiility tht -kit my lel rdi stem or progenitor ells tht possess endothelil potentil. Conerns were lso rised regrding the fidelity nd sensitivity of the mouse models employed in vn Berlo s study 0. Here with new set of mouse models, we demonstrted the endothelil nture of rdi resident -kit þ ells. Our oservtions explin the -kit endothelil linege findings y vn Berlo et l., nd lso explin the low myordil potentil of these ells (euse they re in ft endothelil ells). Ative -kit expression in the ommitted endothelium during hert formtion indites tht -kit is not n pproprite mrker of resident CSCs, inluding CSCs destined for n endothelil fte. Our studies of myordil infrtion injury mouse models suggest tht -kit þ endothelil ells rrely (if ever) de-differentite into CSCs to ontriute to myordil repir. Future studies re wrrnted to determine the mehnisms y whih -kit þ ells ontriute to hert repir (if ny) sed on their endothelil identity. Methods Mouse models. -kit HB-tdTomto/þ, -kit nlz-hb-gfp/þ nd -kit MerCreMer/þ knok-in mouse models were generted y inserting LoxP-4XPloyA-LoxP-HBtdTomto-FRT-Neo-FRT, LoxP-nlZ-4XPloyA-LoxP-HB-GFP-FRT-Neo-FRT nd MerCreMer-FRT-Neo-FRT ssettes, respetively, into the strt odon of the -kit lous (with disruption of endogenous ATG) through homologous reomintion in 9/SvJ ES ells. In the trgeting onstruts, the insertion ssettes re flnked y 3.7 k 5 0 nd 3.8 k 3 0 homologous rms (Supplementry Figs,7,9). The trgeting vetors were linerized nd eletroported individully in mouse ES ells. ES ells were sreened y long-rnge PCR (Rohe, Ct ) with two pirs of primers (P þ P nd P3 þ P4, Supplementry Figs,7,9). The sequenes of the PCR frgments from the positive ES ells were further verified y DNA sequening. The mle himeri mie rrying the trgeted ssette in their germ line were rossed with Blk Swiss femles to generte F heterozygous mie. The Neo ssette flnked y two FRT sites ws removed y rossing F mie with Flippse deleter mie 36. -kit LoxP-4XPloyA-LoxP-HB-tdTomto/þ (-kit STOP-HB-tdTomto/þ ) mie were rossed with Protmine-Cre 37 to remove the 4XPloyA stop ssette nd to otin -kit HB-tdTomto/þ. The P 4 sequenes re: P, 5 0 -GGGTCTTCCTATA TCTCCCTAGCT-3 0 ;P(-kit STOP-HB-tdTomto/þ ), 5 0 -CCAAATAAGCTTGGA TCCGGAACC-3 0 ;P(-kit nlz-hb-gfp/þ ), 5 0 -ATTCGCGTCTGGCCTT CCTGTAGC-3 0 ;P(-kit MerCreMer/þ ), 5 0 -CTCTTCTTCTTGGGCATGGTCTG C-3 0 ; P3, 5 0 -TACCTGCCCATTCGACCACCAAGC-3 0 ; nd P4, 5 0 -ACCTCAC ACAGAACCTCCAGCAAT-3 0. Nkx.5 HB-GFP/þ, TnT MerCreMer/þ nd ROSA6R tdtomto/þ (R6R tdtomto/þ ) mouse lines were previously desried 8,35,38.ForTnT nlz-hb-gfp/þ mouse line, LoxP-nlZ-4XPloyA-LoxP-HB-GFP ssette ws trgeted to the TnT strt odon 8 NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved.

9 NATURE COMMUNICATIONS DOI: 0.038/nomms970 ARTICLE (mnusript ws sumitted). The TnT HB-GFP/þ mouse ws otined y rossing TnT LoxP-nlZ-4XPloyA-LoxP-HB-GFP/þ (TnT nlz-hb-gfp/þ )miewithprotmine- Cre mie 37. Nkx.5 HB-GFP/þ nd TnT HB-GFP/þ mie were rossed with -kit HBtdTomto/þ to otin -kit HB-tdTomto/þ ;Nkx.5 HB-GFP/þ nd -kit HB-tdTomto/þ ; TnT HB-GFP/þ ompound heterozygous mie. The ompound heterozygous mie hd norml hert development. Tmoxifen (Sigm, Ct. T5648) ws intrperitonelly injeted into mie (0. mg g ody weight). Genomi DNA ws prepred from yolk ss or tils for genotyping. Mouse husndry ws onduted in ordne with n pproved protool y Ihn Shool of Mediine t Mount Sini Institutionl Animl Cre nd Use Committee (IACUC) nd ws in ompline with institutionl nd governmentl regultions (PHS Animl Welfre Assurne A3-0). X-gl stining. For whole-mount stining, the tissues were fixed in 4% prformldehyde for 30 min on ie. After the tissues were quikly wshed twie in PBS, they were stined in X-gl solution (5 mm potssium ferriynide, 5 mm potssium ferroynide, mm MgCl, nd mg ml X-gl) overnight t room temperture. For setion stining, the hert tissues were fixed in 4% prformldehyde for 30 min, wshed with PBS, soked in 30% surose overnight nd then emedded in optiml utting temperture ompound (Tissue-Tek). Coronl setions of herts were prepred using ryostt. The setions were re-fixed in 4% prformldehyde for 5 7 min followed y stining with X-gl solution t 37 C overnight. At lest three mie from eh stge were exmined. RNA in situ hyridiztion. Whole-mount RNA in situ hyridiztion of mouse emryos ws performed using Wilkinson s protool 39. Immunofluoresene. Mouse tissues were fixed in 4% prformldehyde for 30 min, wshed with PBS, soked in 30% surose overnight nd then emedded in optiml utting temperture. Cryosetions of hert (oronl) were ut to 8 mm thikness. The primry ntiodies used in this study were rt nti-pecam (CD3; :00, BD Biosienes, Ct ), got nti--kit (CD7; :0 to :40 for postntl herts nd :40 to :00 for emryoni herts, R&D systems, AF356) nd mouse nti--sma (:00, Sigm, Ct. A58). Alex Fluor 488- or 594- onjugted seondry ntiodies (:500; Invitrogen) were pplied to detet the orresponding primry ntiodies. A TSA kit (Perkin Elmer, Ct. NEL7400KT) ws pplied to mplify fluoresent signls resulting from -kit ntiody stining. Horserdish peroxidse onjugted nti-got IgG (:500; Am, Ct. 970) ws used s seondry ntiody when TSA ws pplied to enhne immunostining. Flow ytometry. Mouse ventriulr endothelil ells were otined y enzymti dissoition of the hert following stndrd perfusion proedures 40 with modifitions. Briefly, dult mie (4 months old) were injeted with heprin 0 min efore hert exision nd nesthetized y isoflurne inhltion. Herts were quikly removed from the hest nd perfused with C þ -free solution ontining ollgense type II (Worthington, Lkewood, NJ, USA). Ventriles were ut into smll piees nd gently mined with Psteur pipette. Dissoited ells were trnsferred to 50 ml Flon tue nd kept in Tyrode s solution t room temperture for 5 0 min. Ventriulr rdiomyoytes settled on the ottom of the tue. Most non-rdiomyoyte ells were then olleted without disturing the rdiomyoyte lyer for flow ytometri nlysis. The ells were wshed in PBS with 0.5% ovine serum lumin (BSA). The ell suspension ws djusted to onentrtion of 0 6 ells ml, nd single ells were inuted in loking uffer (PBS with F loking IgG nd % BSA) for 30 min t room temperture. PECAM/CD3-APC onjugted ntiody (BD, Ct. 5684) ws dded to the loking uffer (5 ml per 0 6 ells). The ells were inuted with gentle shking for 30 min t room temperture in the drk. Red lood ell lysis uffer ws dded, nd then the smples were inuted t room temperture for 0 min to eliminte red lood ells. The ells were susequently wshed twie nd then resuspended in PBS with 0.5% BSA for flow ytometry (Bekmn Coulter MoFlo Cytomtion). Myordil infrtion. Myordil infrtion ws indued y D oronry rtery ligtion in mie of oth genders with ody weights rnging from 5 to 34 g ( 6 months old) 4. Briefly, mie were nesthetised intrperitonelly with ketmine (0.065 mg g ody weight), epromzine (0.00 mg g ody weight) nd xylzine (0.03 mg g ody weight). After thorotomy, D ligtion ws performed with 7-0 silk suture 3 4 mm from the tip of the left urile. The suessful performne of D ligtion ws verified y visul inspetion of the olour of the pex. The hest ws losed with 6-0 silk suture, nd the skin ws losed with 4-0 silk sutures. All mie were housed under identil onditions nd were given wter nd food d liitum. Cell ounting. Speifi genotype mie were pplied to ount the numer of -kit þ (-kit HB-tdTomto/þ ), Nkx.5 þ (Nkx.5 HB-GFP/þ ) nd TnT þ (TnT HB-GFP/þ ) ells in the herts. Cryosetions (0 mm, oronl) were ut through the hert. For emryoni stges, every fifth setion ws olleted. For herts older thn P30, in every 0 setions ws olleted. Cells from five representtive setions were ounted oth mnully nd utomtilly using ImgeJ softwre with imges quired on fluoresene mirosope. By ompring the numers quired y mnul ounting nd ImgeJ utomti ounting, the thresholds of prtile size nd intensity were set in ImgeJ. Cells from the remining setions were ounted y ImgeJ with the sme threshold. The totl ells were lulted y dding the ell numers for ll setions. The numer of rdiomyoytes in the dult hert ws divided y onsidering tht 85 90% of these ells re inuleted in mie 4. As result, B Nkx.5 þ or TnT þ ells were lulted in E mouse herts, B myordil ells were lulted in the dult mouse herts, nd B kit þ were lulted in the dult mouse herts (P30 P90). Referenes. Bui, A. L., Horwih, T. B. & Fonrow, G. C. Epidemiology nd risk profile of hert filure. Nt. Rev. Crdiol. 8, 30 4 (0).. Segers, V. F. & Lee, R. T. Stem-ell therpy for rdi disese. Nture 45, (008). 3. Pssier, R., vn Lke, L. W. & Mummery, C. L. Stem-ell-sed therpy nd lessons from the hert. Nture 453, 3 39 (008). 4. Lflmme, M. A. & Murry, C. E. Hert regenertion. Nture 473, (0). 5. Ptszek, L. M., Mnsour, M., Ruskin, J. N. & Chien, K. R. Towrds regenertive therpy for rdi disese. Lnet 379, (0). 6. Xin, M., Olson, E. N. & Bssel-Duy, R. Mending roken herts: rdi development s sis for dult hert regenertion nd repir. Nt. Rev. Mol. Cell Biol. 4, (03). 7. Beltrmi, A. P. et l. Adult rdi stem ells re multipotent nd support myordil regenertion. Cell 4, (003). 8. Ci, C. L. et l. Isl identifies rdi progenitor popultion tht prolifertes prior to differentition nd ontriutes mjority of ells to the hert. Dev. Cell 5, (003). 9. Yng, L. et l. Humn rdiovsulr progenitor ells develop from KDR þ emryoni-stem-ell-derived popultion. Nture 453, (008). 0. Bu, L. et l. Humn ISL hert progenitors generte diverse multipotent rdiovsulr ell lineges. Nture 460, 3 7 (009).. Anvers, P., Kjstur, J., Rot, M. & Leri, A. Regenerting new hert with stem ells. J. Clin. Invest. 3, 6 70 (03).. Torell, D., Ellison, G. M., Mendez-Ferrer, S., Inez, B. & Ndl-Ginrd, B. Resident humn rdi stem ells: role in rdi ellulr homeostsis nd potentil for myordil regenertion. Nt. Clin. Prt. Crdiovs. Med. 3(suppl ): S8 3 (006). 3. Linke, A. et l. Stem ells in the dog hert re self-renewing, lonogeni, nd multipotent nd regenerte infrted myordium, improving rdi funtion. Pro. Ntl Ad. Si. USA 0, (005). 4. Berzi, C. et l. Humn rdi stem ells. Pro. Ntl Ad. Si. USA 04, (007). 5. Ellison, G. M. et l. Adult -kit(pos) rdi stem ells re neessry nd suffiient for funtionl rdi regenertion nd repir. Cell 54, (03). 6. Jesty, S. A. et l. -kit þ preursors support postinfrtion myogenesis in the neontl, ut not dult, hert. Pro. Ntl Ad. Si. USA 09, (0). 7. Hsieh, P. C. et l. Evidene from geneti fte-mpping study tht stem ells refresh dult mmmlin rdiomyoytes fter injury. Nt. 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10 NATURE COMMUNICATIONS DOI: 0.038/nomms Bernex, F. et l. Sptil nd temporl ptterns of -kit-expressing ells in WlZ/þ nd WlZ/WlZ mouse emryos. Development, (996). 7. Smith, A. J. et l. Isoltion nd hrteriztion of resident endogenous -Kit þ rdi stem ells from the dult mouse nd rt hert. Nt. Proto. 9, (04). 8. Zhng, L. et l. Mesoderml Nkx.5 is neessry nd suffiient for erly seond hert field development. Dev. Biol. 390, (04). 9. Shner, N. C., Steinh, P. A. & Tsien, R. Y. A guide to hoosing fluoresent proteins. Nt. Methods, (005). 30. Dy, R. N. & Dvidson, M. W. The fluoresent protein revolution, xvi (340 Bo Rton, 04). 3. Molkentin, J. D. Letter y Molkentin regrding rtile, The sene of evidene is not evidene of sene: the pitflls of Cre Knok-Ins in the -Kit Lous. Cir. Res. 5, e e3 (04). 3. Ndl-Ginrd, B., Ellison, G. M. & Torell, D. Response to Molkentin s letter to the editor regrding rtile, the sene of evidene is not evidene of sene: the pitflls of Cre knok-ins in the -kit lous. Cir. Res. 5, e38 e39 (04). 33. Duttlinger, R. et l. W-ssh ffets positive nd negtive elements ontrolling -kit expression: etopi -kit expression t sites of kit-lignd expression ffets melnogenesis. Development 8, (993). 34. Fioret, B. A., Heimfeld, J. D., Pik, D. T. & Htzopoulos, A. K. Endothelil ells ontriute to genertion of dult ventriulr myoytes during rdi homeostsis. Cell Rep. 8, 9 4 (04). 35. Yn, J. et l. Genertion of tmoxifen induile Tnnt(MerCreMer) knok-in mouse model for rdi studies. Genesis 53, (05). 36. Rodriguez, C. I. et l. High-effiieny deleter mie show tht FLPe is n lterntive to Cre-loxP. Nt. Genet. 5, (000). 37. O Gormn, S., Dgenis, N. A., Qin, M. & Mrhuk, Y. Protmine-Cre reominse trnsgenes effiiently reomine trget sequenes in the mle germ line of mie, ut not in emryoni stem ells. Pro. Ntl Ad. Si. USA 94, (997). 38. Mdisen, L. et l. A roust nd high-throughput Cre reporting nd hrteriztion system for the whole mouse rin. Nt. Neurosi. 3, (00). 39. Wilkinson, D. G. In Situ Hyridiztion: Prtil Approh, xviii 4 p. (Oxford University Press, 998). 40. Lin, X. et l. Suellulr heterogeneity of sodium urrent properties in dult rdi ventriulr myoytes. Hert Rhythm 8, (0). 4. Chen, J. et l. Ner-infrred fluoresent imging of mtrix metlloproteinse tivity fter myordil infrtion. Cirultion, (005). 4. Ahuj, P., Sdek, P. & MLelln, W. R. Crdi myoyte ell yle ontrol in development, disese, nd regenertion. Physiol. Rev. 87, (007). Aknowledgements We thnk Drs Brue Gel nd Json Kovi for their ritil reding of the mnusript nd Dr Kevin Kelly in the Trnsgeni Core t Mount Sini for the help in generting the mouse models. Author ontriutions C.-L.C. nd J.Y. designed the study; C.-L.C., N.S. nd A.M. disussed the results nd wrote the pper; N.S., L.Z. nd0 J.Y. performed the primry experiments nd nlysed the dt; J.C., W.C., S.R., D.J., W.S., L.B., M.X., G.-Y.H., R.J.H., B.Z. nd A.M. helped to perform the experiments nd provided tehnil ssistne. Additionl informtion Supplementry Informtion ompnies this pper t ntureommunitions Competing finnil interests: The uthors delre no ompeting finnil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to ite this rtile: Sultn, N. et l. Resident -kit þ ells in the hert re not rdi stem ells. Nt. Commun. 6:870 doi: 0.038/nomms970 (05). This work is liensed under Cretive Commons Attriution 4.0 Interntionl Liense. The imges or other third prty mteril in this rtile re inluded in the rtile s Cretive Commons liense, unless indited otherwise in the redit line; if the mteril is not inluded under the Cretive Commons liense, users will need to otin permission from the liense holder to reprodue the mteril. To view opy of this liense, visit 0 NATURE COMMUNICATIONS 6:870 DOI: 0.038/nomms970 & 05 Mmilln Pulishers Limited. All rights reserved.