Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs.

Dear Editor,Programmed cell death has an essential role in development and homeostasis of mammalians. Fas-associated death domain (FADD) interacts with the death domain of receptors, leading to the activation of caspase-8, which subsequently activates several downstream caspases and finally executes apoptosis.1 Ablation of Caspase-8 or Fadd resulted in embryonic lethality at around E10.5, which implicates a non-apoptosis function of these proteins in embryonic development.2 Recently, extensively genetic studies have shown that embryonic lethality caused by Fadd (or Caspase-8) deletion can be rescued by Ripk3 or Ripk1 ablation.2 However, it remains unclear which targeted cell type is responsible for the lethality of Fadd−/− mice.Mice with conditional deletion of Fadd in immune cells, skin or intestine produced no lethality.2 Given the fact that mice with Caspase-8 deficiency in endothelium, employing Tie1-cre promotor, resemble the embryonic lethality of Caspase-8 germline knockout associated with cardiac defects,3 we hypothesized that embryonic lethality of Fadd knockout might also attribute to the loss of Fadd in endothelial cells. To directly test this hypothesis, we took advantage of the mice that expressed a functional FADD:GFP fusion gene to reconstitute Fadd−/− mice, and generated tissue-specific Fadd deletion mice using cre-recombinase under the control of tissue-specific promoter, which were reported previously.4First, we specifically deleted FADD:GFP in cardiomyocytes and cardiac progenitor cells by crossing the mice (Fadd−/−Fadd:gfp+) individually with transgenic mice expressing the cTnt-cre and Nkx2.5-cre. cTnt-Cre efficiently delete target genes in myocardium and Nkx2.5-cre targets cardiomyocyte progenitors.5, 6 We found that both Fadd−/−Fadd:gfp+cTnt-cre+ and Fadd−/−Fadd:gfp+Nkx2.5-cre+ mice develop normally at E11.5 (Figure 1a). These data indicate that it is loss of Fadd in other types of cells causing embryonic death of Fadd−/− mice, not cardiomyocytes or cardiac progenitor cells. We then generated Fadd deficiency in Tie-2 expressing cells by crossing Fadd−/−Fadd:gfp+ mice with a transgenic line that expresses cre-recombinase under the control of Tie-2 promoter. In contrast, Fadd−/−Fadd:gfp+Tie2-cre+ mice died at E11.5 with the same cardiovascular defects as Fadd−/− mice, such as vessel defect and pericardial bleeding, suggesting that hemodynamic failure resulting in embryonic death could be owing to abnormal cardiovascular development (Figure 1a). Whole-mount staining for endothelial cell marker PECAM and FADD surrogates GFP showed that FADD:GFP was expressed in endothelial cells in Fadd−/−Fadd:gfp+ mice (yellow). However, FADD:GFP was not detected in endothelial cells of Fadd+/−Fadd:gfp+Tie2-cre+ embryo (Figure 1b), whereas FADD:GFP was still expressed in non-endothelial cells such as cardiomyocytes (Figure 1b). These data indicate that Tie2-cre efficiently ablates FADD:GFP in endothelial cells. Compared with normal embryos, Fadd−/−Fadd:gfp+Tie2-cre+ displayed a low degree of trabeculation in the walls of the common ventricular chamber and endocardial cushion defect by reduced endothelial-to-mesenchymal formation (Figure 1c), suggesting that loss of Fadd in endothelial cells causes endocardium-related cardiac development defect. Although the lethality of Fadd−/−Fadd:gfp+Tie2-cre+ is caused by the same pathology as Fadd−/− embryo at E11.5,7, 8 we asked whether this lethality of Fadd−/−Fadd:gfp+Tie2-cre+ is mediated by RIPK3 as Fadd−/− mice. Therefore, we crossed Ripk3 knockout allele to the Fadd−/−Fadd:gfp+Tie2-cre+ mice and found that embryonic lethality of Fadd−/−Fadd:gfp+Tie2-cre+ mice at E11.5 was rescued by Ripk3 deletion. Furthermore, Fadd−/−Fadd:gfp+Tie2-cre+Ripk3−/− embryos displayed normal degree of trabeculation in the walls of the common ventricular chamber and normal cushion development as Fadd−/−Fadd:gfp+ embryos (Figure 1c). Given that Tie2 is predominantly expressed in endothelial cells and hematopoietic cells, embryonic lethality of Fadd−/−Fadd:gfp+Tie2-cre+ mice at E11.5 might be owing to Fadd deletion in both endothelial and hematopoietic populations. In addition, hematopoietic stem cells are mainly derived from aortic endothelial cells during early embryonic development, and the role of FADD in hematopoietic development could also be secondary to the defect in endothelial cells. More specific genetic tools that could distinguish hematopoietic cells and endothelial cells are needed to dissect the roles of FADD in these two populations. Taken together, these results demonstrated that RIPK3-mediated signaling in Tie-2 expressing cells was responsible for the embryonic lethality of Fadd−/− with cardiac failure. Further, mechanistic study of cell death in these cell populations will be important for understanding the function of cell death during embryonic development.The authors declare no conflict of interest.We thank Dr Jianke Zhang (Thomas Jefferson University, Philadelphia, PA, USA) for providing Fadd−/−Fadd:gfp+ mice. This work was supported by grants from the Ministry of Science and Technology of China (2016YFSF110034) and the National Natural Science Foundation of China (31571426, 91339104). HZ was supported by Thousand Young Talents Program of the Chinese government.

The origin of fat cells or adipocytes is a fundamental biological question with important ramifications for human health and disease1. Epicardial fat is associated with increased risk of cardiovascular diseases such as coronary atherosclerosis2. However, the origin of these epicardial fat cells remains largely unknown. Epicardial progenitors play a pivotal role in the developing heart, by secreting paracrine signals and by differentiating into fibroblasts, smooth muscle cells, and potentially endothelial cells and cardiomyocytes3,4,5,6. Some of these developmental programs are reactivated during postnatal heart injury7,8, and reactivated adult epicardial progenitors provide pro-angiogenic protection and undergo limited epithelial-to-mesenchymal transition in cardiac injury7,9. Since epicardial fat is physically adjacent to epicardial cells, we hypothesized that some of these cells originate from the epicardium during development and in postnatal heart injury.We first generated Wt1-CreER; Rosa26RFP/+ double transgenic mouse line. In the absence of tamoxifen, we found little detectable labeling of epicardial progenitors and their derivatives7. After tamoxifen induction, translocation of CreER into nucleus allowed removal of a loxP-flanked transcriptional stop cassette, thus leading to indelible RFP labeling of epicardial cells and their descendants, epicardium-derived cells (EPDCs, Supplementary information, Figure S1A). We injected tamoxifen at embryonic day 10.5 (E10.5) to label epicardial progenitors and collected hearts during postnatal weeks 8 to 12 (P8w to P12w) to analyze their contribution to epicardial fat (Figure 1A). We found that fetal EPDCs contribute significantly to intramyocardial vessels in adult hearts (Supplementary information, Figure S1A). Interestingly, some EPDCs, particularly those in the atrioventricular groove, remained in an epicardial location and colocalized with fat tissue (Figure 1B). Co-immunostaining of RFP and adipocyte marker perilipin (PLIN) in tissue sections from four hearts (12 sections each) verified that among 3 560 PLIN+ fat cells counted, 23.4% ± 5.1% were RFP+ cells (Figure 1C).To further confirm this finding, we employed an adenovirus that expresses Cre under the epicardium-specific promoter Msln to selectively lineage-trace the epicardium7. Ad:Msln-Cre transduction of cultured epicardial cells activated expression of GFP from a Cre-dependent Rosa26mTmG/+ reporter (Supplementary information, Figure S1B). As Msln is specifically expressed in epicardium of the developing heart (Supplementary information, Figure S1C), we performed ultrasound-guided embryonic heart injection of Msln-Cre virus at E12.5. Examination of E15.5 hearts confirmed selective labeling of the epicardium (Supplementary information, Figure S1D). We next collected P8w hearts and found that a subset of Msln-Cre-labeled EPDCs co-expressed PLIN, demonstrating contribution of epicardial progenitors to fat cells during normal heart development (Figure 1D). Altogether, the above data showed that embryonic epicardial progenitors contribute to epicardial fat. We refer to this process as epicardium-to-fat transition (EFT).From this intriguing observation in heart development, we next asked whether adult epicardial progenitors contribute to epicardial fat cells. We first performed adult epicardial lineage tracing by tamoxifen injection in an 8-week-old adult Wt1-CreER; Rosa26RFP/+ mice. A significant portion of WT1+ epicardial progenitors in the atrioventricular groove were labeled after 4-6 weeks (Supplementary information, Figure S1E). However, we did not detect any RFP+ epicardial fat cells (Supplementary information, Figure S1F), suggesting that adult epicardial progenitors do not actively generate epicardial fat during normal heart homeostasis. This was not simply due to limited tamoxifen-induced labeling by Wt1-CreER, as a significant portion of epicardial cells were labeled in the vicinity of epicardial adipose tissue (Supplementary information, Figure S1E and S1F). In addition, labeling of renal cells in the kidneys of the same mice was high (Supplementary information, Figure S1E). Taken together, the above data suggested that while embryonic epicardial cells contribute to epicardial adipose tissue, there is minimal EFT in normal adult heart.Our previous work shows that postnatal epicardial cells are a dynamic progenitor population that reactivates fetal properties after injury7,9,10. To directly address the lineage conversion of epicardial progenitors to fat cells after cardiac injury, we first lineage-tagged epicardium by tamoxifen induction of Wt1-CreER; Rosa26mTmG/+ hearts, and then performed myocardial infarction (MI) by ligation of the left anterior descending coronary artery (Figure 1E). Since WT1 is specifically expressed in epicardium but not fat cells (Supplementary information, Figure S1G), epicardium was specifically prelabeled before MI (Supplementary information, Figure S1H). Three weeks after MI, we collected the injured hearts with attached fat tissue on their surface, and found that a subset of epicardial fat cells near the border zone of infarcted myocardium was genetically labeled (Figure 1F). We confirmed that these labeled cells were adipocytes by co-staining of PLIN and GFP (Figure 1G). Adult epicardial cells contributed to 9.3% ± 3.7% of fat cells in epicardial adipose tissue in the peri-infarct region (n = 4, Figure 1G). We verified this result by serial sections from Wt1-CreER; Rosa26RFP/+ MI hearts (Supplementary information, Figure S1I). To show that cell labeling was not due to CreER activity in the absence of the inducing agent under MI stress conditions, we examined Wt1-CreER; Rosa26mTmG/+ MI hearts that were not treated with tamoxifen and found no GFP+ epicardial fat cells. MI also induced proliferation of adipocytes after MI, suggesting that adipocyte expansion as well as EFT contribute to expansion of epicardial fat after MI (Supplementary information, Figure S1J).To verify this finding, we injected Msln-Cre virus into adult Rosa26mTmG/+ heart before MI and confirmed epicardial labeling in pre-MI hearts (Supplementary information, Figure S1K). We then performed MI on these Msln-Cre-injected mice, and collected the hearts 4 weeks afterwards. We found that a subset of cells in epicardial fat tissue were expressing GFP, suggesting that labeled epicardial cells contribute to adipocytes in injured heart (Figure 1H). By co-immunostaining of PLIN and GFP, we confirmed GFP+PLIN+ fat cells in MI heart (Figure 1I), suggesting that these epicardial fat cells were derived from Msln-Cre-prelabeled epicardial progenitors.EPDCs have been reported to differentiate into multiple cell types, most recently adipocytes11. To ask whether single EPDC is multipotent, we performed clonal assays on EPDCs isolated from MI hearts and expanded clonally from single cells. We FACS sorted EPDCs from Wt1-CreER; Rosa26mTmG/+ hearts 1 week after MI and generated single cell clonal outgrowths. FACS analysis of these clones confirmed that they phenotypically resemble mesenchymal stem cells (Supplementary information, Figure S1L-S1P). When cultured in different conditions, the clonal outgrowths differentiated into adipocyte, chondrocyte, and osteoblast lineages (Supplementary information, Figure S1Q), demonstrating their multipotency.By genetic lineage tracing, our study revealed that epicardial progenitors contribute to fat cells during development. This EFT potential is quiescent in adult heart, but is reactivated after MI. Our work suggested that the EFT might be a cardiac response to severe injury such as MI, and that EFT could be a new signature of cardiovascular diseases. A recent study showed that mesothelium, including epicardium, contributes to visceral fat11. Our study of the epicardium confirmed this result and extended it to homeostasis and diseases of the adult heart. EFT makes limited contributions to epicardial adipose tissue during heart homeostasis. However, this potential is unlocked under stress such as MI, which reactivates the developmental EFT program and leads to injury-stimulated EFT in vivo. Understanding of the molecular regulatory mechanisms of EFT will provide insights into the treatment of cardiovascular diseases and regenerative medicine1,12,13.This work was supported by the Ministry of Science and Technology of China (2012CB945102 and 2013CB945302), the National Natural Science Foundation of China (91339104, 31271552, 31222038), the Chinese Academy of Sciences (Hundred Talents Program; KSCX2-EW-R-09), Organization Department of the CPC Central Committee Bajian Talents Program, Shanghai Pujiang Program (11PJ1411400) and Basic Research Key Project (14JC1407400). WTP was funded by NIH (2 R01 HL094683).(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality world-wide. Whether endogenous repair and regenerative ability could be augmented by drug administration is an important issue for generation of novel therapeutic approach. Recently it was reported that in mice pretreated with thymosin beta 4 (TB4) and subsequently subjected to experimental MI, a subset of epicardial cells differentiated into cardiomyocytes. In clinical settings, epicardial priming with TB4 prior to MI is impractical. Here we tested if TB4 treatment after MI could reprogram epicardium into cardiomyocytes and augment the epicardium's injury response. Using epicardium genetic lineage trace line Wt1CreERT2/+ and double reporter line Rosa26mTmG/+, we found post-MI TB4 treatment significantly increased the thickness of epicardium and coronary capillary density. However, epicardium-derived cells did not adopt cardiomyocyte fate, nor did they migrate into myocardium to become coronary endothelial cells. Our result thus indicates that TB4 treatment after MI does not alter epicardial cell fate to include the cardiomyocyte lineage, providing both cautions and insights for the full exploration of the potential benefits of TB4 in the clinical settings. This article is part of a Special Issue entitled ‘Possible Editorial’.Highlights► Unlike TB4 treatment prior to MI, TB4 treatment after MI does not reprogram epicardial cells into cardiomyocytes. ► Epicardial cells treated with TB4 after MI adopt mesenchymal fibroblast fate. ► TB4 stimulates epicardial cells proliferation in vivo.