Ckit Cardiac Progenitors of Neural Crest Origin

+ cKit cardiac progenitors of neural crest origin

Konstantinos E. Hatzistergosa, Lauro M. Takeuchia, Dieter Saurb, Barbara Seidlerb, Susan M. Dymeckic, Jia Jia Maic, Ian A. Whitea, Wayne Balkana, Rosemeire M. Kanashiro-Takeuchia,d, Andrew V. Schallye,1, and Joshua M. Harea,1

aInterdisciplinary Stem Cell Institute, Leonard M. Miller School of Medicine, Miami, FL 33136; bDepartment of Internal Medicine, Mediziniche Klinik und Policlinik Der Technischen Universitat Munchen, Munich 81675, Germany; cDepartment of Genetics, Harvard Medical School, Boston, MA 02115; dDepartment of Molecular and Cellular Pharmacology, Leonard M. Miller School of Medicine, Miami, FL 33136; and eDepartment of Pathology and Medicine, University of Miami School of Medicine and Veterans Affairs Medical Center, Miami, FL 33125

Contributed by Andrew V. Schally, August 29, 2015 (sent for review April 27, 2015; reviewed by Roger Joseph Hajjar) The degree to which cKit-expressing progenitors generate cardio- we demonstrate that cKit marks CNCs. Furthermore, we show that myocytes in the heart is controversial. Genetic fate-mapping studies their relatively small contribution to myocardium during embryogen- suggest minimal contribution; however, whether or not minimal esis is not related to poor cardiomyogenic capacity, but rather to contribution reflects minimal cardiomyogenic capacity is unclear be- changes in the cardiac activity of the bone morphogenetic protein cause the embryonic origin and role in cardiogenesis of these pro- (BMP) pathway that prevent their differentiation into cardiomyocytes. genitors remain elusive. Using high-resolution genetic fate-mapping approaches with cKitCreERT2/+ and Wnt1::Flpe mouse lines, we show Results kit kit that cKit delineates cardiac neural crest progenitors (CNC ). CNC Genetic Lineage-Tracing of cKit+ CPs. We used a well-characterized + + possess full cardiomyogenic capacity and contribute to all CNC de- cKitCreERT2/ mouse line to lineage-trace cKit CPs (23–25). + rivatives, including cardiac conduction system cells. Furthermore, by cKitCreERT2/ are healthy, fertile, and express the white spotting modeling cardiogenesis in cKitCreERT2-induced pluripotent stem cells, kit phenotype (12, 23, 24, 26) (Fig. 1A). we show that, paradoxically, the cardiogenic fate of CNC is regu- We first investigated whether cKit marks mesodermal CPs (e.g., lated by bone morphogenetic protein antagonism, a signaling path- first- or second-heart field CPs; or primitive hemogenic lineage) way activated transiently during establishment of the cardiac (1), by administering pregnant mice carrying cKitCreERT2;IRG em- crescent, and extinguished from the heart before CNC invasion. To- + bryos with tamoxifen (TAM) from embryonic days (E)7.5 to E8.5 gether, these findings elucidate the origin of cKit cardiac progenitors (Fig. 1B and Table S1). At E18.5, EGFP expression was detected in

and suggest that a nonpermissive cardiac milieu, rather than minimal MEDICAL SCIENCES kit mesodermal cells (13, 14, 21, 26), including gonads, blood, and cardiomyogenic capacity, controls the degree of CNC contribution lungs (Fig. 1 C and D). At this stage of labeling (21), EGFP was to myocardium. + rarely detected in the heart, and EGFP heart cells were non- cardiomyocytes with rare colocalization with the cardiac tran- cardiac stem cells | cardiac neural crest | cardiomyogenesis | BMP signalling scription factor Gata4 (Fig. 1 E and F). Next, to test whether cKit marks other cardiomyogenic line- eart development is a highly regulated process during which ages (e.g., proliferating cardiomyocytes; or CPs of the epicardial, Hcell lineage diversification and growth programs are dynam- CNC, and definitive hemogenic lineages) (1), we administered ically coordinated in temporal and spatial manners (1). These TAM to pregnant mice at selected time points during E9.5–E12.5 programs are activated sequentially, in parallel, or intersect to give rise to distinct heart domains. For example, the myocardial lineage Significance originally develops from cardiac progenitors (CPs) of mesodermal origin (2–5), which form the first and second heart fields. How- ever, later during morphogenesis, the cardiomyogenic program A high-resolution genetic lineage-tracing study in mice reveals diverges and activates cardiomyocyte proliferation signals, along that cKit identifies multipotent progenitors of cardiac neural with CPs from the hemogenic endothelium, epicardial, cardio- crest (CNC) origin. Normally, the proportion of cardiomyocytes pulmonary, and cardiac neural crest (CNC) lineages, to produce produced from this lineage is limited, not because of poor new cardiomyocytes (1, 6–11). Gauging the relative contribution differentiation capacity as previously thought, but because of of each lineage for scaling their cardiomyogenic—and consequently stage-specific changes in the activity of the bone morphoge- therapeutic—capacity is a challenge. For example, many of the CP netic protein pathway. Transient bone morphogenetic protein antagonism efficiently directs mouse iPSCs toward the CNC lineages are heterogeneous and incompletely characterized, and + therefore cannot always be traced under a straightforward genetic lineage and, consequently, the generation of cKit CNCs with fate-mapping experiment. Furthermore, it is unknown whether and full capacity to form cardiomyocytes and other CNC derivatives how changes in the cardiac milieu (i.e., morphogens, tissue com- in vitro. These findings resolve a long-standing controversy position, and size) regulate the final proportions of heart muscle regarding the role of cKit in the heart, and are expected to lead derived from each lineage. to the development of novel stem cell-based therapies for the cKit is a receptor tyrosine kinase that marks several cell lineages, prevention and treatment of cardiovascular disease. including neural crest (NC), hematopoietic, and germ-line stem cells – Author contributions: K.E.H., A.V.S., and J.M.H. designed research; K.E.H., L.M.T., and (12 15). Following the seminal description by Beltrami et al. (16) of R.M.K.-T. performed research; K.E.H., D.S., B.S., S.M.D., J.J.M., I.A.W., and J.M.H. contrib- clusters of cKit cells in the postnatal mammalian heart, several uted new reagents/analytic tools; K.E.H., L.M.T., R.M.K.-T., and J.M.H. analyzed data; and laboratories, including ours, suggested that cKit marks CPs (16–19), K.E.H., D.S., W.B., A.V.S., and J.M.H. wrote the paper. a finding that led to the clinical testing of these cells for heart repair Reviewers included: R.J.H., Mount Sinai School of Medicine. (20). Recently, a straightforward genetic fate-mapping study showed Conflict of interest statement: K.E.H. and J.M.H. report having a patent for cardiac cell- that a relatively small proportion of murine myocardium is derived based therapy. K.E.H. and J.M.H. own equity in Vestion Inc. and are members of the + from cKit CPs, leading to the conclusion that the cardiomyogenic scientific advisory board and consultants of Vestion, Inc. J.M.H. is a board member of + Vestion Inc. Vestion Inc. did not participate in funding this work. The other authors report capacity of cKit CPs is functionally insignificant (21). However, the + no conflicts. identity of cKit CPs and the mechanisms controlling their differ- 1To whom correspondence may be addressed. Email: [email protected] or andrew. entiation into cardiomyocytes remain controversial (22). Here, by [email protected]. using a high-resolution genetic lineage-tracing strategy, as well as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. induced pluripotent stem cell (iPSC)-based models of cardiogenesis, 1073/pnas.1517201112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1517201112 PNAS | October 20, 2015 | vol. 112 | no. 42 | 13051–13056 Downloaded by guest on October 1, 2021 CreERT2/+ Fig. 1. cKit lineage-tracing. (A) Phenotype CreERT2/+ of cKit mice. (B) Summary of the experimental design. (C–F) administration of TAM during E7.5–E8.5 (n = 10) marks testicular (C, arrowheads), pulmonary (D) and, rarely, immature cells in the myocardium (E and F, arrowheads). (G–J) Live tissue CreERT2/+ CreERT2 imaging of cKit (G), IRG (H), and cKit ; IRG (I) E18.5 littermates subjected to TAM during E9.5–E11.5 (n = 7). Widespread EGFP epifluorescence in ventricles and atria (I and J), lungs (J), OFT (J, arrow), epicardium (J, arrowheads). (K–N) Lineage- tracing in cKitCreERT2;R26RlacZ mice (n = 8). (O)Sum- mary of cKit genetic fate-mapping. Panels F and J are confocal tile-scans. Panels K–N are photomerged image tiles. (Scale bars, 10 μminD and F;200μminJ; 500px in K–N.) (Magnification, 100× in C, E,andG–I.)

(Table S1). Cre-mediated recombination resulted in EGFP tdTomato in various NC-derived tissues of E12.5 Wnt1-Cre;RC:: expression in embryonic melanoblasts, craniofacial cells (27), tdTomato embryos, including the NT and the heart, (Fig. S3). How- − + + + neural tube (NT), dorsal root ganglia (DRGs), blood, gastroin- ever, compared with the cKit /tdTomato cells, cKit /tdTomato testinal cells, gonads, and pulmonary cells (Fig. 1 G–J). Unlike population exhibited a weak expression of tdTomato (Fig. S3). the fate-map of E7.5–E8.5 cKit-expressing cells, EGFP epifluor- We therefore performed NC lineage-restricted genetic fate- escence is detected within the cardiac outflow tract (OFT), epi- mapping of cKit. We generated a novel mouse carrying two cardium, and myocardium (Fig. 1 G–J). recombinase systems (Cre-loxP and Flp-FRT), which enables + To rule out a limited transgene expression, we also performed intersectional genetic fate-mapping of cKitCreERT2/ in the Wnt1- fate-mapping using the R26 promoter-driven R26RlacZ allele. The expressing CNC lineage and its derivatives (34). Two previously results were similar using this reporter compared with EGFP (Fig. established dual-recombinase responsive indicator alleles, the 1 K–N). RC::Fela (35) and RC::Frepe (36), and a novel Wnt1-Flpe recom- To identify the original population of CPs, we administered binase driver line, the Wnt1::Flpe4351,wereused(Fig.2A). TAM at selected time points between E9.5 and E13.5, and col- Timed-pregnant cKitCreERT2;Wnt1::Flpe;RC::Fela or cKitCreERT2; lected embryos 24 h after the last injection (Fig. S1A). Using live Wnt1::Flpe;RC::Frepe mice were administered TAM during E8.5– epifluorescence and immunofluorescence (IF) imaging, we de- E11.5 and embryo analysis was performed at selected time points tected Cre-recombined cells in the NT, skin, lungs, gut, cono- between E10.5 and E18.5 (Table S1). Wnt1::Flpe-mediated re- truncus, OFT, and epicardium, but not within the myocardium combination resulted in extensive labeling with the flp indicators in + (Fig. S1). These findings suggest that the cKitCreERT2/ -labeled heart the craniofacial region, melanoblasts, gut, DRG, OFT, as well as in cells during the period of TAM-induced recombination do not arise cells within the epicardium and myocardium (Fig. 2 and Fig. S4). + within the myocardium [i.e., differentiated cardiomyocytes (28) or When both Wnt1::Flpe and cKitCreERT2/ were expressed, Flpe in- transdifferentiating cardiac fibroblasts (29) and hemogenic pro- dicators persisted in all NC-derived tissues, including the DRG, genitors (6, 7)], and derived from an extracardiac CP lineage. skin,heart,andOFT(Fig.2andFig. S4 A–E). However, expres- + + + Finally, similar to cKitCreERT2/ reporters, cKit IF in cKit / sion of intersectional indicators was also detected in the craniofa- embryos marked cells within the NT, skin, lung, OFT, and epi- cial region, melanocytes, DRG, OFT, as well as in cells within the cardium, but not differentiated cardiomyocytes (Fig. S2). epicardium and myocardium (Fig. 2 and Fig. S4). Importantly, Collectively, our findings suggest that cKit marks a CP lineage expression of intersectional indicators in the lung was not docu- that emerges at ∼E9.5 and contributes to the development of the mented (Fig. 2N), consistent with the hypothesis that the heart and + mouse heart. lung cKit progenitors are of different origins. These studies il- + + lustrate a lineal relationship between the cKit CPs and Wnt1 Intersectional Genetic Fate-Mapping of cKit and Wnt1 Protooncogenes. kit + CNCs (CNC ). Because our findings are consistent with a CNC origin of cKit CPs, we used a well-established CNC-specific mouse, the Wnt1-Cre; CNCkit Derivatives in the Heart. Expression of Cre-reporters was RC::tdTomato (30–32), to examine the expression of cKit in CNC CPs detected in all expected cardiac NC derivatives (37), including (8, 10, 11, 31–33). IF against cKit illustrated its colocalization with the OFT (Figs. 1J and 3 A and B, and Figs. S2B and S5), the

13052 | www.pnas.org/cgi/doi/10.1073/pnas.1517201112 Hatzistergos et al. Downloaded by guest on October 1, 2021 Fig. 2. Intersectional genetic fate-mapping of cKit and Wnt1.(A) schematic of the two different approaches of the study. (B–D) Live epifluorescence imaging (B), followed by salmon-gal histochemical CreERT2 detection of nLacZ (C and D), in an E10.5 cKit ; RC::Fela embryo exposed to TAM during E8.5–E9.5. The flp- and intersectional indicators are not expressed in the absence of Flpe and Flpe/Cre-mediated recom- CreERT2 bination, respectively. (E–H)AWnt1::Flpe;cKit ; RC::Fela littermate exhibits widespread GFP epifluorescence (E) and a few salmon-gal+ cells in the NT and heart. (I–L) Live embryo imaging of mCherry and GFP CreERT2 epifluorescence in a E17.5 Wnt1::Flpe;cKit ;RC:: + kit Frepe embryo. EGFP CNC in the craniofacial region (I), skin (J), OFT (K), and the epicardial wall of the heart (L). (M and T)X-gal+ CNCkit derivatives in the OFT (M and G), heart (N, O, R, and S), and epicardium CreERT2 (P and T) of E17.5 Wnt1::Flpe;cKit ;RC::Fela MEDICAL SCIENCES embryos. Arrows in M–P are depicted in higher magnification in panels Q–T, respectively. Panels B, E are photomerged image tiles. OFT, outflow tract; Ht, heart; Lu, lung. (Scale bars, 50 μminM–P and 300px in I–L.) (Magnification, 100× in B–H and Q–T.)

tunica media of the aortic arch (Fig. 3C), cardiac and aortic mice were crossed with mice carrying an Isl1 nuclear lacZ valves (Fig. 3 D and E), atria (Fig. 1K), inflow tract, satellite glial (Isl1nLacZ) allele (40). When pregnant mice were administered progenitors, and sensory cells (Fig. S5 A and B and Movie S1). TAM from E9.5–E11.5, (Table S1), colocalization of EGFP and Consistent with their CNC origin (38), CNCkit contributed to nLacZ was documented in cells of the NT, DRGs, and the OFT endothelium and smooth muscle layers of the OFT (Fig. 3 A and (Fig. S7 D–J) in E12.5 embryos. B), although coronary vascular cell differentiation was not ob- Transient BMP Antagonism Induces Cardiomyogenesis in CNCkit. To served (Fig. 3F). + In agreement with previous reports in zebrafish (8, 11) and determine the full cardiomyogenic capacity of cKit CPCs mice (10, 21, 31–33), our analysis with the Wnt1-Cre (Fig. S6 and (Fig. 1), we established iPSCs from cKitCreERT2;IRG mice + Movie S2) and cKitCreERT2/ alleles suggests that CNCs contrib- (iPSCkit). We induced cardiomyogenesis in iPSCs with either ute to the myocardial lineage. Particularly, we documented con- ascorbic acid (AA), which drives cardiogenesis partly via an in- + + tribution of CNCkit to atrial and ventricular cardiomyocytes termediate differentiation stage into cKit /Nkx2.5 CPs (15, 41), + (29.9% ± 3.1% of total EGFP derivatives) (Fig. 3 K and L), and or BMP antagonism, a signaling pathway that regulates the de- pericardial, endocardial, and epicardial cells (Figs. 1 I, J, and N, velopment of both mesodermal and NC lineages (Fig. 4A) (2, 5, and 3 G–K, and Movie S3). The majority of CNCkit-derived 31, 42–46). cardiomyocytes was localized in the interventricular septum Both AA or BMP antagonists enhanced cardiac differentiation (Figs. 1J and 3 K and L), which, unlike the left and right ven- into spontaneously contracting embryoid bodies (EBs) compared tricular myocardium, is partly derived from mesoderm posterior with controls (Fig. 4B)(P = 0.0073). When EBs were treated − 1 homolog nonexpressing (Mesp1 ) CPs of undefined origin (9). with 4-OH TAM during differentiation (Fig. 4A), we detected that 79.05% ± 4.9% of the beating EBs generated via transient kit + CNC Identity. To better characterize the identity of CNCKit,we BMP antagonism were EGFP , compared with 39.03% ± 7.7% + studied the expression of micropthalmia-associated transcription and 43.14% ± 6.9% EGFP beating EBs following treatment factor (Mitf), a direct target and transactivator of cKit signaling, with AA or vehicle, respectively (Fig. 4 B–E and Movie S4)(P < expressed not only in cranial NC derivatives and mast cells but 0.0001), suggesting that endogenous BMP signaling may limit + also in cardiomyocytes (39). IF analysis demonstrated that Mitf is cardiomyogenesis from cKit CPs. also expressed in CNCkit and their cardiomyocytic derivatives Compared with control, AA, and Noggin (NOG)-treated iPSCkit, + (Fig. S7 A–C). However, EGFP cells in the heart did not ex- Dorsomorphin (Dorso) enhanced cardiomyogenesis by signifi- press the melanocyte-specific markers tyrosinase or trp1, sug- cantly repressing Brachyury transcription (Fig. 4F and Fig. S8), + gesting that Mitf CNCkit derivatives in the heart are not while up-regulating ISL1, NKX2-5 (Fig. 4F) (43). In addition, we melanocytes (Fig. S7C). detected a significant up-regulation in CNC-related genes, in- Next, we investigated the expression of Isl1, a homeobox cluding PAX3 and WNT1 (Fig. 4G and Fig. S8 E–H), whereas the transcription factor that specifies the majority of the mammalian expression of the proepicardial genes WT1, TCF21,andTBX18 (2), CP lineages, including CNCs (36). Accordingly, cKitCreERT2;IRG as well as the coronary endothelium marker KDR (Fig. 4G and

Hatzistergos et al. PNAS | October 20, 2015 | vol. 112 | no. 42 | 13053 Downloaded by guest on October 1, 2021 kit Fig. 3. Heart derivatives of CNC .(A and B) Colocalization of X-gal with SM1 (arrowhead) and + Pecam1 (arrows) in the OFT. (C) X-gal cells (arrow- + head) within the aortic tunica media. (D) EGFP derivatives within the mitral valve (arrowhead) and aortic valve (arrow). (E) EGFP+ derivatives in the kit aortic valve (arrowheads). (F) CNC are associated with, but do not contribute to, coronary vasculature. + + (G)EGFP/cmlc2v ventricular cardiac myocytes. + + (H)EGFP cardiac derivatives coexpress Gata4 .(I and J) EGFP+ pericardium (arrows), epicardium (arrowheads), and endocardium (J,yellowarrow).(K)Cardiomyocytic + versus noncardiomyocytic EGFP derivatives in the + heart. (L) Distribution of EGFP cells in the heart [n = 3 embryos; 11 sections (K and L)].AoV,aorticvalve;CM, cardiomyocytes; cmlc2v, cardiac muscle light chain 2v; LA, left atrium; LV, left ventricle; LCA, left coronary artery; MV, mitral valve; SM1, smooth muscle myosin heavy chain. Values represent means ± SEM.

Fig. S8), were significantly down-regulated compared with controls. cardiomyogenic capacity, which our findings now suggest is Finally, expression of cKit increased significantly over time (Fig. undermined by developmental changes in the activity of BMP and S8D), although the level of expression was similar between the Wnt pathways preceding their invasion in the heart (5, 31, 45). It is different treatment groups (Fig. 4G). As previously shown (15, 41), also noteworthy that a pool of multipotent postmigratory NC + IF analysis confirmed that myocardial specification of cKit CPs progenitors (47, 48), some of which express cKit (49), has been commenced via coexpression of NKX2.5 (Fig. 5 and Fig. S9 A–D). recorded in other tissues; hence, it would be interesting to ex- Remarkably, BMP antagonism enhanced the development of amine their relationship to CNCkit. + + EGFP /NKX2.5 progenitors by ∼sevenfold (Fig. 5, Fig. S9 A–D, Our study differs from a recent cardiac genetic fate-map of and Movie S5)(P = 0.0057). Moreover, iPSCkit-derived CNCkit cKit, using different cKit alleles (21). First, in contrast to findings + gave rise to all CNC derivatives, including EGFP smooth muscle presented here and elsewhere (6, 15, 29), van Berlo et al. (21) + + cells (Fig. S9 E and F), Isl1 (Fig. S9 G and H), and Pax3 CPs reported that cKit CPs contribute extensively to coronary en- (Fig. S9 I and J), while innervating the beating EBs with neuro- dothelium. However, it is noteworthy that mutations in the + + filament-M and Tuj1 neurons (Fig. S9 K–N). mouse W/cKit locus have not been associated with tangible car- + diovascular defects (12), as would be likely if cKit CPs com- Discussion prised a major source of coronary vascular cells. Second, van The major findings are that cKit marks CPs of CNC origin, which Berlo et al. (21) concluded that the minimal cardiomyocyte enter the embryonic mouse heart at ∼E9.5 and contribute a contribution of cKit CPs reflects minimal differentiation capac- relatively small proportion of myocardium and other derivatives ity. However, although our study agrees that the in vivo car- + of the CNC, but not coronary vascular cells. In addition, we show diomyocyte contribution of cKit CPs is lower than expected that CNCkit CPs with cardiomyocyte differentiation capacity can be from previous reports (50, 51), we show that this is not a result of derived in vitro from mouse iPSCs following transient antagonism minimal differentiation capacity, but rather, because of their of the BMP pathway, which drives the stage-specific differentiation developmental origin in the CNC, which comprises a minor of iPSCs toward the cardiac mesodermal and CNC lineages. contributor of cardiomyocytes to the mammalian heart. Impor- Our findings confirm previous developmental studies in mice tantly, using iPSC modeling we demonstrate that differentiation showing that, during gastrulation, cKit is expressed in extraem- of CNCkit to cardiomyocytes requires the BMP signaling path- bryonic mesoderm and embryonic ectoderm, but not mesodermal way, which also directs differentiation of mesodermal CPs to the CPs (4, 13, 14, 42). Furthermore, the findings are in agreement myocardium. This finding suggests that, although CNCkit hold + with previous reports supporting the existence of cKit CPs, which full cardiomyocyte differentiation capacity, their in vivo contri- do not contribute to coronary endothelium (15), as well as with bution is repressed by spatiotemporal changes in BMP activity, recent endothelial lineage fate-mapping analyses suggesting that which render the cardiac milieu nonconducive for cardiomyocyte + the coronary endothelium is unlikely to originate from cKit cells differentiation during CNC invasion to the heart (45). (6, 29). Our findings have several important implications. First, they Notably, although much controversy exists over the contribu- resolve the current controversy over the existence and car- + + tion of CNCs to the myocardium (8, 10, 11, 30–33), our studies diomyogenic capacity of cKit CPs (22). We show that cKit CPs + with the cKitCreERT2/ , Wnt1-Cre, and Wnt1::Flpe alleles strongly invested within the developing heart are fully capable of pro- support the hypothesis that the mammalian CNC holds full ducing new cardiomyocytes, both in vivo and in vitro. Therefore,

13054 | www.pnas.org/cgi/doi/10.1073/pnas.1517201112 Hatzistergos et al. Downloaded by guest on October 1, 2021 kit Fig. 4. Transient BMP antagonism in iPSC induces kit CNC and suppresses the epicardium. (A) Schematic of the experimental approach. (B) Quantification of the percentage of beating EBs, and the percentage of beating EBs that are EGFP+ following Cre-recombination. (C) Live fluorescent imaging of a vehicle-treated spontaneously beating EB, coexpressing EGFP and DsRed. (D and E) Confocal microscopy of EBs following treatment with AA (D) or Dorso (E), illustrates that the + CreERT2/+ EGFP cKit derivatives within the EBs are cTnnT+ cardiomyocytes. (F) Gene-expression analysis of Brachyury during the time-course of iPSCkit differentiation into cardiomyocytes following treatment with vehicle,Dorso,orNOG.(G) Comparison of the expression profiles of cardiac mesoderm- and CNC-related genes in day 11 EBs, in response to treatment with vehicle, AA, or Dorso. Compared with controls, AA and Dorso enhance cardiomyogenesis via a significant in- duction in CNC-related genes while suppressing proepicardial and endothelial progenitor genes. In addition, Dorso significantly enhances the expression of ISL1 and NKX2.5. cTnnt, cardiac troponin T; values repre- ±

sent means SEM. MEDICAL SCIENCES

+ coupled with the findings from many laboratories that cKit CPs opportunity to study and understand the biology and function of are present in the postnatal heart, they represent an important CNCs, as well as to test their regenerative capacity in novel cell- therapeutic target for heart regeneration (17–19, 52, 53). For based therapeutic strategies. Finally, considering the technical limi- example, the activity of BMP in the damaged myocardium could tations often associated with conditional gene-targeting approaches, be modulated pharmacologically, or via transplantation of cells our findings do not exclude the possibility that, in addition to CNCkit, capable of regulating BMP activity, to support production of + + the adult heart contains other cKit cells with full cardiovascular myocardium from endogenous or exogenously supplied cKit CPs (18, 52). differentiation capacity, as those reported by others (21, 28, 50, 51), Second, the findings advance our understanding of the cellular which may have remained undetectable with our reagents. In conclusion, our findings support the hypothesis that the and molecular mechanisms underlying mammalian cardiomyo- + genesis, by illustrating a previously unknown relationship be- mammalian heart is invested with a cKit CP lineage, with full tween the spatiotemporal modulation of the BMP pathway and capacity to generate cardiomyocytes in vivo and in vitro, and the generation of myocardium from mesodermal and CNC CPs. therefore provide an important therapeutic target for the pre- Third, the generation of CNCs from iPSCs provides a unique vention and treatment of heart disease. Modulation of the activity

kit + + Fig. 5. Derivation of CNC from mouse iPSCs. (A–C) Representative confocal immunofluorescence images illustrating EGFP /NKX2.5 derivatives within cTnnT+ EBs of vehicle-treated (A), AA-treated (B), or Dorso-treated (C)iPSCkit. Note that several of the EGFP+ cells in the vehicle- and AA-treated groups are NKX2.5− (asterisks). (Insets) Higher magnification. (D) Quantitation of EGFP+/NKX2.5+ cells between groups (n = 9 per group). Values represent means ± SEM.

Hatzistergos et al. PNAS | October 20, 2015 | vol. 112 | no. 42 | 13055 Downloaded by guest on October 1, 2021 of the BMP pathway in the heart may enhance the therapeutic RC::Fela and RC::Frepe mice were developed as previously described (35, 36). kit CreERT2 regeneration of damaged myocardium from CNCkit. iPSC were generated from adult cKit /IRG tail-tip fibroblasts. Geno- typing, TAM injections, gene-expression analysis, lineage-tracing, and Materials and Methods histological analysis of mouse embryos was performed as previously described (24). See SI Materials and Methods for more detailed discussion. All animals were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facility at the University of Miami, nLacZ ACKNOWLEDGMENTS. We thank Dr. Sylvia Evans for providing the Isl1 Miller School of Medicine, and procedures were performed using Institutional mice. This study was funded by National Institutes of Health Grants Animal Care and Use Committee-approved protocols according to NIH stan- R01 HL107110, R01 HL094849, R01 HL110737, R01 HL084275, and 5UM cKitCreERT2/+ Wnt1- dards. mice were developed as previously described (24). The HL113460 (to J.M.H.); grants from the Starr foundation and the Soffer Family LacZ Cre, RC::tdTomato, IRG,andR26R mouse lines were purchased from Jackson Foundation (to J.M.H.); and Deutsche Forschungsgemienschaft Grant SA Laboratories. The Isl1nLacZ mice have been described elsewhere (40). Wnt1::Flpe, 1374/1-3 (to D.S.).

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