c-kit+ precursors support postinfarction myogenesisin the neonatal, but not adult, heartSophy A. Jestya,1,2, Michele A. Steffeya,2,3, Frank K. Leea,2, Martin Breitbachb, Michael Hesseb, Shaun Reininga,Jane C. Leea, Robert M. Dorana, Alexander Yu. Nikitina, Bernd K. Fleischmannb,4, and Michael I. Kotlikoffa,4

aDepartment of Biomedical Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and bInstitute of Physiology 1, Life and Brain Center,University of Bonn, 53105 Bonn, Germany

Edited* by Ralph L. Brinster, University of Pennsylvania, Philadelphia, PA, and approved July 2, 2012 (received for review May 14, 2012)

We examined the myogenic response to infarction in neonatal andadult mice to determine the role of c-kit+ cardiovascular precursorcells (CPC) that are known to be present in early heart develop-ment. Infarction of postnatal day 1–3 c-kitBAC-EGFP mouse heartsinduced the localized expansion of (c-kit)EGFP+ cells within theinfarct, expression of the c-kit and Nkx2.5 mRNA, myogenesis, andpartial regeneration of the infarction, with (c-kit)EGFP+ cells adoptingmyogenic and vascular fates. Conversely, infarction of adult miceresulted in a modest induction of (c-kit)EGFP+ cells within the infarct,which did not express Nkx2.5 or undergo myogenic differentiation,but adopted a vascular fate within the infarction, indicating a lackof authentic CPC. Explantation of infarcted neonatal and adultheart tissue to scid mice, and adoptive transfer of labeled bonemarrow, confirmed the cardiac source of myogenic (neonate) andangiogenic (neonate and adult) cells. FACS-purified (c-kit)EGFP+/(αMHC)mCherry− (noncardiac) cells from microdissected infarctswithin 6 h of infarction underwent cardiac differentiation, formingspontaneously beating myocytes in vitro; cre/LoxP fate mappingidentified a noncardiac population of (c-kit)EGFP+ myocytes withininfarctions, indicating that the induction of undifferentiated pre-cursors contributes to localized myogenesis. Thus, adult postin-farct myogenic failure is likely not due to a context-dependentrestriction of precursor differentiation, and c-kit induction follow-ing injury of the adult heart does not define precursor status.

heart repair | stem cell | vasculogenesis | angiogenesis

Cardiac infarction results in permanent heart dysfunction dueto insufficient myogenesis. This myogenic failure has been at-

tributed to the absence of multipotent cardiovascular progenitorcells (CPC) in the heart, or alternatively to a context-related failureof extant CPC to adopt a cardiac fate within the infarct. Althoughthe presence of authentic progenitors in the adult heart is contro-versial (1–8), CPC in the developing heart are capable of formingall three cardiovascular lineages (9–15), and recent data indicatethat substantial cardiac regeneration occurs in the injured neonatalheart, although this regeneration has been ascribed to the expan-sion of differentiated myocytes (16). Here we directly address thequestion of whether CPC adopt cardiac fates and contribute toheart regeneration following infarction of the neonatal mouseheart, and examine the role of c-kit+ cells that are induced fol-lowing injury of the adult heart. Our finding of robust myogenesisfollowing infarction of the neonate, but no myogenesis in theidentically injured adult heart, connects myogenic potential tocells that exist at this early stage of cardiac developmental. More-over, we show that CPC partially underlie myogenic infarct repairin neonates, but that c-kit+ cells that are locally induced fol-lowing infarction of the adult mouse heart, do not adopt a cardiacfate. These results highlight the heterogeneity of c-kit–expressingcells, and the distinction between cardiovascular and vascularprogenitors.

Results and DiscussionTo determine whether neonatal hearts are capable of regener-ative cardiac myogenesis, cryoinfarctions were performed in

postnatal day (PN) 1–3 mice harboring a transcription markerfor c-kit (c-kitBAC-EGFP; ref. 14), a gene product linked to CPCstatus in the heart (10, 14, 15, 17–19). Infarction resulted in thelocalization of (c-kit)EGFP+ cells and a marked induction ofc-kit and EGFP mRNA within the area of injury, far greater thanobserved in identically infarcted adult hearts (14, 19, 20)(Fig. 1A–C and Fig. S1A). Individual (c-kit)EGFP+ cells of varyingmorphology were observed throughout the neonatal infarct, butnot in areas remote from the injury. The early cardiac tran-scription factor Nkx2.5 was identified in numerous Nkx2.5+ and(c-kit)EGFP+/Nkx2.5+ immature cells, and Nkx2.5 mRNA wasinduced within the neonatal infarct, but neither c-kit+/Nkx2.5+

cells nor Nkx2.5 message induction were observed in timematched adult infarcts (Fig. 1D and Fig. S1 B and C), excludingits expression as part of the revascularization process (10).Consistent with the (c-kit)EGFP marker identifying cells thatinclude multipotent cardiovascular precursors (9–11), (c-kit)EGFP+ cells were observed at various stages of endothelialcell (EC, Flk-1+; PECAM-1+) or cardiomyocyte (α-actinin+;Nkx2.5+) differentiation (Fig. 1 D–G). Mast cells (tryptase+ ortoluidine blue+) expressing (c-kit)EGFP were only observed atthe epicardial border and were not consistent with the markedexpansion of c-kit(EGFP)+ cells at the neonatal infarct border(Fig. S1D). Quantitative morphometry indicated that, at day 3after infarction (D3), 67.2 ± 2.2% of (c-kit)EGFP+ cells withinthe infarct and border zone were of cardiac phenotype, increasingto 87.7 ± 4.9% at D7; the great majority of the remaining (c-kit)EGFP+ cells were identified as endothelial cells (EC) (Fig. 1G).Most importantly, neonatal cryoinfarction lesions underwent

marked neomyogenesis and regeneration following injury (Fig. 2A and B); by D5, many striated cardiomyocytes were intermixedwithin areas of fibrosis. By the first week following injury, cardiacmyocytes were observed within clusters of (c-kit)EGFP+ cells,and by D21, the infarcted region was infiltrated with numerousα-actinin positive myocytes, some of which were (c-kit)EGFP+,and fibrotic areas were progressively remodeled by 3 mo (Fig. 2B–D). Quantitative analysis indicated an increase in cellularcontent within the scar area to 20.4 ± 4.1% at 3 wk and 43.2 ±3.0% at 3 mo (P < 0.001; Fig. S2A). Thus, following an ablative

Author contributions: M.A.S., F.K.L., M.B., M.H., A.Y.N., B.K.F., and M.I.K. designed re-search; S.A.J., M.A.S., F.K.L., M.B., M.H., S.R., J.C.L., and R.M.D. performed research; A.Y.N.contributed new reagents/analytic tools; S.A.J., M.A.S., F.K.L., M.B., M.H., J.C.L., R.M.D.,B.K.F., and M.I.K. analyzed data; and B.K.F. and M.I.K. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1Present address: Department of Small Animal Clinical Science, University of Tennessee,Knoxville, TN 37996-4544.

2S.A.J., M.A.S., and F.K.L. contributed equally to this work.3Present address: Department of Surgical and Radiological Sciences, School of VeterinaryMedicine, University of California, Davis, CA 95616.

4To whom correspondence may be addressed. E-mail: mik7@cornell.edu or bfleisch@uni-bonn.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1208114109/-/DCSupplemental.

13380–13385 | PNAS | August 14, 2012 | vol. 109 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1208114109

Dow

nloa

ded

by g

uest

on

Aug

ust 1

9, 2

020

infarction, c-kit transcriptionally active CPC migrate to the siteof injury and progressively differentiate into heart cells, resultingin a regenerative response to the injury.In vivo pulse-chase labeling with BrdU indicated that cardiac

myogenesis was accompanied by a marked expansion of the(c-kit)EGFP+ pool that was localized to the infarct (Fig. 2 E andF). At day 3 postinfarction (PI), the number of (c-kit)EGFP+

cardiomyocytes within the infarct was 6.7-fold greater than inremote areas; overall, 20.5 ± 4.7 and 32.1 ± 2.9% of (c-kit)EGFP+ myocytes incorporated BrdU at D3 and D5, respectively.Moreover, (c-kit)EGFP+ cells were a major component of allmyocytes showing evidence of DNA replication within the in-farct, accounting for 25.4 ± 8.5 and 42.0 ± 6.9% of all BrdU+

cardiomyocytes at D3 and D5, respectively, compared with 2.2 ±1.3 and 7.0 ± 4.1% in remote regions (Fig. 2F). By contrast,determination of histone H3 phosphorylation (pHH3) in myo-cytes, which reflects the pool of currently expanding myocytes,indicated a similar stimulation of mitosis in existing myocytes inremote and infarcted regions. pHH3+ cardiomyocytes increasedfrom a control of 0.07 ± 0.01 to 0.22 ± 0.03% and 0.23 ± 0.08%,

in remote and infarcted regions, respectively (Fig. S2 B and C).Taken together, these data are consistent with a generalized ex-pansion of existing myocytes throughout the heart, but a localizedexpansion of (c-kit)EGFP+ cells and cardiac differentiation withinthe infarct. Quantitatively, by D5, ∼32% of (c-kit)EGFP+ car-diomyocytes localized to the infarct have passed through S phase,and more than 75% of (c-kit)EGFP+ have adopted a cardiac fate(Fig. 1G), indicating that postinfarct cardiac myogenesis arisesat least partially from a local expansion of the (c-kit)EGFP+ CPCpool. The pancardiac expansion of myocytes following neonatalinfarction is not associated with c-kit expression as evidenced byminimal c-kit+/BrdU+ cells outside of the infarct zone (Fig. 2F).The (c-kit)EGFP marker reports current c-kit transcriptional

activity and thus cannot definitively identify the developmentalorigin of myocytes observed within the infarction. To unequivocallyestablish the ability of undifferentiated (c-kit)EGFP+ cells toadopt cardiac fates, we infarcted neonatal c-kitBAC-EGFP/αMHC-mCherry mice and FACS purified cells for in vitro differentiationexperiments within 6 h of infarction, to prevent dedifferentiationof myocytes. Pure populations of EGFP+/mCherry− (noncardiac)

Hoechst Nkx2.5EGFPAdultNeonate

D3

E

400

600

8002C

ells

in In

farc

t (/m

m)

Hoechst Flk-1EGFPNeonate

F D3

G

A

Neonate

Adult

D3

C

Hoechst EGFPNeonate

D3 D

D3

Hoechst Nkx2.5EGFP

+Total EGFP

+ +EGFP /PECAM-1

+ +EGFP / -Actinin

+ +EGFP /CD45

B

Fold

Incr

ease

(ove

r sha

m a

dult

LV)

20

40

c-kit

0

50

30

10

***

***

***

*** ***

0

200

D3 D5 D7 D14

***

sham D3 D5D1sham

Neo InfarctAdult InfarctD5D3D16H6H

Fig. 1. Cardiac myogenesis following infarction of the neonatal heart. (A) Induction of (c-kit)EGFP+ cells in neonatal and adult heart at 3 d after cry-oinfarction (D3). Merged fluorescent/bright-field images show localized fluorescence within the neonatal infarct. Adult fluorescent cells not visible at thiscamera gain. (B) Quantitative PCR (qPCR) of c-kit mRNA in microdissected infarcts. Note higher message in neonatal vs. adult sham, and higher inductionin neonatal infarct. Statistical comparisons to group sham. (C) Expression of (c-kit)EGFP in infarcted neonatal heart. (D) Nuclear Nkx2.5 expression in un-differentiated (c-kit)EGFP+ cells in neonatal, but not adult, heart. (E) Striated (c-kit)EGFP+ myocytes within neonatal infarct (arrows) by D3; some cells are alsoNkx2.5+. (F) Flk-1+ cells in infarct include (c-kit)EGFP+ cells (arrows). (G) Time course and phenotype of (c-kit)EGFP+ cells following infarction of the neonatalheart. Note predominant cardiac phenotype. Error bars show SEM throughout. (Scale bars: 2,000 μm in A; 100 μm in C; 20 μm in D and E; and 50 μm in F.)

Jesty et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13381

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Aug

ust 1

9, 2

020

cells began to express mCherry after incubation in cardiac dif-ferentiation medium (14) (Fig. 2G). By 12–13 d, 12.0 ± 1.4% ofcells expressed mCherry (Fig. S2 F and G), whereas no EGFP−/mCherry+ cells were observed to express EGFP in parallelexperiments. Moreover, purified EGFP+/mCherry− cells did notsimply express mCherry, but formed a contractile cardiac phe-notype with spontaneous contractions (Movies S1 and S2). FACS-purified neonate (c-kit)EGFP+ cells also adopted cardiac fateswhen injected into adult infarcts (SI Methods and Fig. S2H),whereas FACS-purified adult (c-kit)EGFP+ cells do not undergocardiac differentiation in vitro or when transplanted into adultinfarcts (14, 15). Thus, although our data do not exclude expan-sion of existing myocytes (16), they provide evidence of a pool ofprecursor cells that are capable of supporting myogenesis post-infarction in neonatal mice.We next sought to further discriminate between progenitor

and cardiomyocyte contributions to the expansion of (c-kit)EGFP+ cells, and attendant postinfarction myogenesis. We in-farcted triallelic (c-kitBAC-EGFP/ αMHC-CreER/R26-floxedSTOP-βGal) neonatal mice, in which myocytes were marked withβ-gal by tamoxifen-induced recombination before birth, resultingin a minimum of 3 d between tamoxifen exposure and infarction.Infarcted regions contained a markedly lower percentage ofrecombined cells than remote areas at D5, the time point ofmaximum (c-kit)EGFP+ cells within the infarct, and differingdistinctly from the localized expansion of (c-kit)EGFP+ cells(Fig. 2 H and I and Fig. S2D). To exclude (c-kit)EGFP+ cellsthat adopt an EC fate, we compared the percentage of recom-bined (c-kit)EGFP+ myocytes to the overall cardiomyocyte re-combination rate. In three experiments, the recombination rateof (c-kit)EGFP+ myocytes was 68.8 ± 4.2% that of EGFP−

cardiomyocytes (P < 0.01; Fig. S2E), consistent with a non-myocyte source for at least one-third of these cells, particularlywhen viewed in light of the induction of c-kit in differentiatedmyocytes following injury (14).Our data indicate a major expansion of the (c-kit)EGFP+ cells

in response to cardiac injury; however, myogenesis associatedwith these cells could reflect an influx of bone-marrow-derivedc-kit+ cells following injury, as ∼50% of c-kit+ cells have beenreported to be copositive for the hematopoietic lineage markerCD45 in adult mice (20). We observed that less than 3% of(c-kit)EGFP+ cells were CD45+ during the first week followinginfarction of the neonatal mouse (Fig. 1G). CD45+/(c-kit)EGFP− cells migrated to the infarct in large numbers, but did notparticipate in the formation of new myocytes or vascular struc-tures. Rather, these cells surrounded vessels and myocytes,persisting for weeks following the injury (Fig. 3 A–D). Unlike(c-kit)EGFP+ cells, CD45+ cells also did not coexpress cardiacα-actinin (Fig. 3B) or spatially overlap with PECAM-1+ cells(Fig. 3 C and D), further establishing (c-kit)EGFP+/CD45− cellsas the precursor pool.Although controversial in the adult heart (21, 22), neonatal

c-kit+/CD45− bone-marrow-derived stem cells could contributeto the formation of new heart cells, as neonatal cells mightpossess enhanced differentiation potential compared with adulthematopoietic stem cells. To test this possibility, we transplantedpooled marrow from 5-d-old c-kitBAC-EGFP/pCAGGS-dsRed(23) mice into irradiated adult B6 recipients, and infarcted thesemice following reconstitution. Virtually no (c-kit)EGFP+ cellswere observed in infarcts in these experiments, although dsRedcells did home to the site of injury as expected (Fig. 3E), andmicroscopic evaluation failed to reveal (c-kit)EGFP+ or dsRed+

A B

EC

GF

D21D5

D21 DEGFP -Actinin PECAM-1 Hoechst

D5H

D3 D5

-

+

c-kit/BrdU

+

+

c-kit/BrdU

-

+

c-kit/BrdU

-

+

c-kit/BrdU

+

+

c-kit/BrdU

-

+

c-kit/BrdU

***

**n.s.

*n.s.

0

5

10

15

20 InfarctRemote

%of

tota

lCar

diom

yoc y

tes

*

D94

X-galX-gal EGFP

D3

EGFP BrdU

D21D5D3

EGFP Hoechst -Actinin

I

Fig. 2. Regeneration of infarcted myocardium through expansion of cardiovascular precursor pool. (A) α-actinin staining at D3, D5, and D21 shows initialfibrosis and clusters of (c-kit)EGFP+ cells, followed by progressive muscle regeneration. (B) Myocyte clusters with many α-actinin+/(c-kit)EGFP+ cells (arrows) atD5 (Left) and fewer (c-kit)EGFP+ myocytes at D21 (Right). (C) Regression of fibrosis and myocyte clusters interspersed within fibrotic regions (trichrome). (D)Marked regeneration at D94, with further regression of fibrotic areas. (E) BrdU incorporation in infarcted neonatal heart. (Inset) BrdU incorporation ina (c-kit)EGFP+ striated myocyte. (F) Quantitative morphometry from BrdU incorporation in all myocytes. (G) Conversion of green to red fluorescence in vitrodemonstrating myogenic capacity of (c-kit)EGFP+ cells. Cells shown are at day 14 from an initial plating devoid of red cells. Same field shown as overlay of lightand green fluorescence (Upper), and light and red fluoresence (Lower) images. Separation scheme and purity are shown in Fig. S2. (H and I) Fate mapping ofmyocytes (c-kitBAC-EGFP/αMHC-CreER/R26-floxed STOP-βGal) at D5 shows limited recombination in infarct region at low (H) and high (I) magnification (red isautofluorescence). (Scale bars: 400 μm in A Left and Center; 250 μm in A Right; 20 μm in B; 200 μm in C Left; 30 μm in C Right; 1,000 μm in D Left; 25 μm in DRight; 20 μm in E; 10 μm in E Inset; 25 μm in G, 200 μm in H; and 100 μm in I.)

13382 | www.pnas.org/cgi/doi/10.1073/pnas.1208114109 Jesty et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

9, 2

020

myocytes or vascular endothelial cells, excluding hematopoieticstem cells as the myogenic/vascular precursors. This experimentis limited by the potential loss of transdifferentiation capacityof neonatal bone marrow during reconstitution (14–21 d), butreconstitution with PN2-3 neonatal marrow for 2–4 d also failedto result in the migration of any (c-kit)EGFP+ cells to the heart(SI Methods). To further confirm the cardiac origin of (c-kit)EGFP+ cells, we transplanted infarcted c-kitBAC-EGFP neonatalventricular tissue into SCID mice. Three to five days aftertransplantation, the infarcted tissue showed induction of (c-kit)EGFP transcriptional activity in cells within the transplant,many of which adopted cardiac fates (Fig. 3F). By contrast,striated myocytes were not observed in adult transplanted tissue(see below), excluding the induction of EGFP in differentiatedmyocytes. Thus, cardiac CPC migrate to the region of cardiac

injury, undergo cell division, and initiate a wave of cardiovas-cular differentiation.As c-kit+ cells participate in the formation of new vessels

within adult infarcts, which are devoid of CPCs (14, 15), ourresults indicate that at least two distinct pools of (c-kit)EGFP+

cells participate in cardiac repair. Furthermore, because marrow-derived cells appeared to play at most a very minor role in re-vascularization of the neonatal infarct (Fig. 3 A–D), our resultsmight reflect a previously unappreciated endogenous source forsuch cells in the adult heart. To further define the pool of c-kit–expressing cells participating in adult cardiac repair, we per-formed adoptive transfer experiments using adult dsRed marrowtransplanted to c-kitBAC-EGFP recipients, as well as c-kitBAC-EGFP donor marrow transplanted to dsRed recipients, and in-farcted the mice following reconstitution. As shown in Fig. 4A,

EGFP CD45 EGFP PECAM

EGFP --ActininHoechst CD45EGFP

Neonate

D3

b

a

A B

CD21

D21

D

CD45EGFP PECAMEGFP

ED3 D3

CD45

F

D21 irradiated/infarcted B6

pCAGGS dsRedBACc-kit EGFP

heart

EGFP

Fig. 3. Heart–derived (c-kit)EGFP+ cells adopt myogenic and EC fates following neonatal infarction. (A) CD45+ cells surround (c-kit)EGFP+ cells withininfarct and do not participate in angiogenesis/myogenesis. (Left) New vessel within infarct (a) formed by (c-kit)EGFP+ endothelial cells; vessel outside ofinfarct (b) has no incorporation. (Right) Myocyte clusters formed by (c-kit)EGFP+ cells, surrounded by EGFP−/CD45+ mononuclear cells. (B) At D21, someCD45+ cells persist, but are α-actinin negative. (C and D) CD45 and PECAM stained sections from D3 and D21 hearts show distinct, nonvascular location ofCD45+ cells within infarcts. (E ) Infarction of reconstituted (PN2 c-kitBAC-EGFP/(pCAGGS)dsRed marrow) B6 adults results in homing of only dsRed cells to theinfarct (green/red image). (F) (c-kit)EGFP induction and myogenic/endothelial differentiation in neonatal c-kitBAC-EGFP infarcted tissue in SCID mouse.(Left) Low magnification of infarcted area. (Right) Higher magnification shows clusters of striated (c-kit)EGFP+ myocytes (arrows) as well as endothelialcells. (Scale bars: 200 μm in A Left; 50 μm in A Center and Right; 20 μm in B; 200 μm in C; 200 μm in D Left; 50 μm in D Right; 1,000 μm in E; 100 μm in F Left;and 25 μm in F Right.)

Jesty et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13383

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Aug

ust 1

9, 2

020

dsRed-labeled marrow cells prominently occupy the infarct, butthese cells play little or no role in the formation of vascularstructures, whereas host (c-kit)EGFP+ cells markedly participatein new vessel formation. Conversely, despite complete reconsti-tution, (c-kit)EGFP+ marrow cells were rarely detected in theinfarcted hearts of cardiac mCherry (αMHC-mCherry) mice andnever adopted cardiac or vascular lineages, although numerousCD45+ cells were observed (Fig. 4 B and C). Thus, as in theneonate, there is little evidence of traffic of c-kit+ hematopoieticstem cells to the infarcted adult heart. As (c-kit)EGFP+ cells arenot found in adult mice (14), these results indicate activation ofthe reporter in resident cells that migrate to the infarct andadopt strictly vascular fates, a process similar to recent reports ofperivascular precursors that adopt EC and smooth muscle fatesin vitro, and underlie atherosclerotic injury responses (24–26).To definitively confirm that adult c-kit+ cells derive from thesurrounding, nonablated cardiac tissue, we transplanted the leftventricular freewall of adult c-kitBAC-EGFP/(pCAGGS)dsRedmice into SCID mice s.c. immediately following cryoinfarction.As in neonatal mice, (c-kit)EGFP+ cells were observed 5 d

following infarction/transplantation of adult heart tissue; how-ever, (c-kit)EGFP+ cells contributed only to the formation ofvascular structures (Fig. 4D). Highlighting the distinct molecularfeatures of the neonatal and adult processes, the expression ofsca-1, which has been implicated in angiogenic responses medi-ated by adventitial vascular precursors (24, 26), was significantlyup-regulated in adult infarcts, but is not a feature of the neonatalresponse (Fig. 4E). Thus, in adult infarcts, resident vascular pre-cursors, rather than authentic CPC, migrate from the surroundingheart tissue and differentiate to form vascular structures.Taken together, our results suggest that the failure of myo-

genesis following adult infarction is not a result of a context-dependent limit to differentiation associated with the infarctper se, but depends critically on the developmental state ofthe heart, consistent with data indicating that the early humanheart possesses significant regenerative potential (8). Recentdata indicate that early cardiac regeneration involves activationof myocyte cell cycle reentry in the entire neonatal heart, re-sulting in widespread dedifferentiation and proliferation, withoutformation of a fibrotic scar (16). Our data do not exclude the

A

B C

D E

0

2

4

6

8

10

sham D3 D5D1sham

Neo InfarctAdult InfarctD5D3D1

Fold

Incr

ease

(ove

r adu

lt LV

)

MHC-mCherryCD45

dsRed EGFP

smActin

PECAM-1

CD45

irradiated/infarcted BACc-kit -EGFPpCAGGS dsRed

BACc-kit EGFP

infarcted/irradiatedMHC-mCherry

D5

mCherry EGFPEGFP

EGFP

Fig. 4. Heart–derived (c-kit)EGFP+ cells adopt vas-cular fates following adult infarction. (A) Infarctionof reconstituted ((pCAGGS)dsRed marrow) c-kitBAC-EGFP mice indicates (c-kit)EGFP+ cells adopt endo-thelial (PECAM-1) and smooth muscle (α-smActin)fates; bone marrow-derived cells are mainly CD45+.(B) Infarction of reconstituted (c-kitBAC-EGFP mar-row) αMHC-mCherry mice reveals minimal (c-kit)EGFP+ cells homing to the heart (arrow indicatesrare EGFP+ cell). (C) CD45+ cells from c-kitBAC-EGFPmarrow home to the infarct, but do not formmyocytes; mCherry+ myocytes at border zone. (D)Adult c-kitBAC-EGFP infarcted tissue in SCID mouseundergoes (c-kit)EGFP induction, but adopt strictlyvascular fates (Right). (E) Induction of sca-1 mRNAin microdissected infarcts. (Scale bars: 500 μm in ALeft; 100 μm in A Center and Right; 100 μm in B; 25μm in C; 200 μm in D Left; and 50 μm in D Right.)

13384 | www.pnas.org/cgi/doi/10.1073/pnas.1208114109 Jesty et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

9, 2

020

participation of non-terminally differentiated cardiomyocytes inneonatal infarct repair, but the local expansion of undifferentiated,(c-kit)EGFP+ cells at the area of infarction, the induction of theearly cardiac transcription factor Nkx2.5 in these cells, and theiradoption of multiple developmental fates, coupled with a lim-ited expansion of previously differentiated heart cells at the siteof injury, clearly establishes the role of CPC in this process.Strikingly, our explant studies demonstrate the intrinsic capacityof the neonatal heart for myogenesis, a capacity that temporallycorrelates with the existence of authentic CPC (14, 15, 19). Wefurther demonstrate that the neonatal and adult heart wallscontain a reservoir of angiogenic precursors, and that activationof these cells results in c-kit expression. Finally, our data high-light the critical distinction in developmental potential of adultheart cells that express the stem cell factor receptor, c-kit.Mobilization of c-kit+ cells in the infarcted adult heart fromsurrounding heart tissue results in extensive angiogenesis/vas-culogenesis. Although incapable of cardiac myogenesis, thesec-kit+ vascular progenitors may be attractive targets for directedcardiac differentiation in vivo.

MethodsNeonatal and Adult Infarction. Tg(RP24-330G11-EGFP)1Mik Mice or combinedlines were anesthetized to a surgical plane using hypothermia (27), a ven-trolateral thoracotomy performed, and a 1-mm-diameter copper probeequilibrated in liquid nitrogen applied to the left ventricle near the apex for5–10 s and removed. Adult mice were infarcted as described (28). For neo-natal mice, the incision was at left intercostal space 7–8. Following appli-cation of the cryoprobe, the pleural cavity was filled with sterile saline, themusculature and s.c. tissue was reconstructed over the intercostal incisionwith 8-0 vicryl, and the saline was removed from the pleural space. Skinclosure was with subcuticular sutures of 8-0 vicryl to prevent maternal re-jection. Mice were recovered by rewarming, administered 10 μg of morphines.c., and placed with foster dams when strong purposeful movementwas present. Infarct regions described in this manuscript comprise the infarctand border zone areas. All procedures were approved by the Cornell In-stitutional Animal Care and Use Committee and adhered to the Guide forthe Care and Use of Laboratory Animals.

Explantation of Heart Tissue into SCID Hosts. Mice were euthanized immedi-ately following infarction reperfusion and cryoablated tissue transplantedto the dorsal s.c. space of anesthetized adult B6 SCID recipients.

BrdU Labeling.Neonates were given i.p. injections of BrdU (2.5mg/mL, 50 μg/gbody weight; BD 550891) at days 0, 1, and 2 after the cryoablation. Thehearts were harvested either 3 or 5 d postcryoablation and Stefanini fixedovernight before sectioning and analysis.

Adoptive Bone Marrow Transfer. Bone marrow transplantation was as de-scribed (29). See SI Methods.

Induced Recombination and Fate Mapping. Pregnant females carrying triallelic(c-kitBAC-EGFP/αMHC-CreER/R26-floxed STOP-βGal) pups were injected in-traperitoneally with 75 mg/kg tamoxifen at ED 17 or 18. Injected micecommonly gave birth at ED 18–20, the pups were then infarcted as describedabove. Quantitative analysis of recombination frequency was performed at5 d postinfarction (PN7) by staining for galactosidase activity with X-Galusing standard methods.

Immunohistochemistry and Quantitative Morphometry. See SI Methods.

Quantitative PCR. See SI Methods.

Statistical Analysis. Data were analyzed by one-way ANOVA, using Dunnett’scomparison with control, Tukey–Kramer multiple comparisons, or unpaired ttests. Summary values are mean ± SEM. Significant differences are reportedat the P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) levels.

ACKNOWLEDGMENTS. We thank Dr. Andrew Yen and Lavanya Sayam forFACS separation procedures; Ms. Pat Fisher and Dr. Longying Dong forimmunohistochemistry support; Dr. Jinhyang Choi for pilot bone marrowtransplantation experiments and teaching S.A.J. and S.R. this procedure;Babu Singh for help construction of the α-MHC-mCherry transgene; andRuizhe Ren for technical support. The cytometry core was supported in partby the Empire State Stem Cell Board, New York State Department of Health,Contract 123456. This work is supported by National Institutes of HealthGrants DK065992 and DK072277 (to M.I.K.), and NYSTEM Grant C023050(to A.Y.N), and European Union’s Seventh Framework Programme consor-tium CardioCell Grant 223372 (to B.F.K).

1. Sussman MA, Anversa P (2004) Myocardial aging and senescence: Where have thestem cells gone? Annu Rev Physiol 66:29–48.

2. Anversa P, Kajstura J, Leri A, Bolli R (2006) Life and death of cardiac stem cells:a paradigm shift in cardiac biology. Circulation 113:1451–1463.

3. Rubart M, Field LJ (2006) Cardiac regeneration: Repopulating the heart. Annu RevPhysiol 68:29–49.

4. Murry CE, Reinecke H, Pabon LM (2006) Regeneration gaps: Observations on stemcells and cardiac repair. J Am Coll Cardiol 47:1777–1785.

5. Hsieh PC, et al. (2007) Evidence from a genetic fate-mapping study that stem cellsrefresh adult mammalian cardiomyocytes after injury. Nat Med 13:970–974.

6. Pouly J, et al. (2008) Cardiac stem cells in the real world. J Thorac Cardiovasc Surg 135:673–678.

7. Wu SM, Chien KR, Mummery C (2008) Origins and fates of cardiovascular progenitorcells. Cell 132:537–543.

8. Bergmann O, et al. (2009) Evidence for cardiomyocyte renewal in humans. Science324:98–102.

9. Moretti A, et al. (2006) Multipotent embryonic isl1+ progenitor cells lead to cardiac,smooth muscle, and endothelial cell diversification. Cell 127:1151–1165.

10. Wu SM, et al. (2006) Developmental origin of a bipotential myocardial and smoothmuscle cell precursor in the mammalian heart. Cell 127:1137–1150.

11. Kattman SJ, Huber TL, Keller GM (2006) Multipotent flk-1+ cardiovascular progenitorcells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages.Dev Cell 11:723–732.

12. Yang L, et al. (2008) Human cardiovascular progenitor cells develop from a KDR+embryonic-stem-cell-derived population. Nature 453:524–528.

13. Christoforou N, et al. (2008) Mouse ES cell-derived cardiac precursor cells are multi-potent and facilitate identification of novel cardiac genes. J Clin Invest 118:894–903.

14. Tallini YN, et al. (2009) c-kit expression identifies cardiovascular precursors in theneonatal heart. Proc Natl Acad Sci USA 106:1808–1813.

15. Zaruba MM, Soonpaa M, Reuter S, Field LJ (2010) Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts. Circulation 121:1992–2000.

16. Porrello ER, et al. (2011) Transient regenerative potential of the neonatal mouseheart. Science 331:1078–1080.

17. Beltrami AP, et al. (2003) Adult cardiac stem cells are multipotent and supportmyocardial regeneration. Cell 114:763–776.

18. Kattman SJ, Adler ED, Keller GM (2007) Specification of multipotential cardiovascularprogenitor cells during embryonic stem cell differentiation and embryonic de-velopment. Trends Cardiovasc Med 17:240–246.

19. Fransioli J, et al. (2008) Evolution of the c-kit-positive cell response to pathologicalchallenge in the myocardium. Stem Cells 26:1315–1324.

20. Fazel S, et al. (2006) Cardioprotective c-kit+ cells are from the bone marrow andregulate the myocardial balance of angiogenic cytokines. J Clin Invest 116:1865–1877.

21. Murry CE, et al. (2004) Haematopoietic stem cells do not transdifferentiate into car-diac myocytes in myocardial infarcts. Nature 428:664–668.

22. Nygren JM, et al. (2008) Myeloid and lymphoid contribution to non-haematopoieticlineages through irradiation-induced heterotypic cell fusion. Nat Cell Biol 10:584–592.

23. Vintersten K, et al. (2004) Mouse in red: Red fluorescent protein expression in mouseES cells, embryos, and adult animals. Genesis 40:241–246.

24. Hu Y, et al. (2004) Abundant progenitor cells in the adventitia contribute to ath-erosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 113:1258–1265.

25. Pasquinelli G, et al. (2007) Thoracic aortas from multiorgan donors are suitable forobtaining resident angiogenic mesenchymal stromal cells. Stem Cells 25:1627–1634.

26. Passman JN, et al. (2008) A sonic hedgehog signaling domain in the arterial adventitiasupports resident Sca1+ smooth muscle progenitor cells. Proc Natl Acad Sci USA 105:9349–9354.

27. Phifer CB, Terry LM (1986) Use of hypothermia for general anesthesia in preweanlingrodents. Physiol Behav 38:887–890.

28. Roell W, et al. (2007) Engraftment of connexin 43-expressing cells prevents post-in-farct arrhythmia. Nature 450:819–824.

29. Choi J, Curtis SJ, Roy DM, Flesken-Nikitin A, Nikitin AY (2010) Local mesenchymalstem/progenitor cells are a preferential target for initiation of adult soft tissue sar-comas associated with p53 and Rb deficiency. Am J Pathol 177:2645–2658.

Jesty et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13385

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Aug

ust 1

9, 2

020