Author + information
- Received January 18, 2017
- Revision received May 30, 2017
- Accepted June 2, 2017
- Published online July 31, 2017.
- Elisa Yaniz-Galende, PhDa,
- Maguelonne Roux, MSca,
- Sophie Nadaud, PhDa,
- Nathalie Mougenot, PhDb,
- Marion Bouvet, PhDa,
- Olivier Claude, PhDa,
- Guillaume Lebreton, MDa,
- Catherine Blanc, PhDa,
- Florence Pinet, PhDc,
- Fabrice Atassi, BSca,
- Claire Perret, MSca,
- France Dierick, PhDa,
- Sébastien Dussaud, PhDa,
- Pascal Leprince, MD, PhDa,
- David-Alexandre Trégouët, PhDa,
- Giovanna Marazzi, MDa,
- David Sassoon, PhDa and
- Jean-Sébastien Hulot, MD, PhDa,∗ ()
- aSorbonne-Universités, Université Pierre-et-Marie-Curie (UPMC), INSERM UMRS_1166, Institute of Cardiometabolism and Nutrition, Paris, France
- bSorbonne-Universités, UPMC, Plate forme d'Expérimentation Coeur Muscles Vaisseaux, Paris, France
- cINSERM U1167, Université de Lille, Institut Pasteur de Lille, Fédération Hospitalo-Universitaire Remodeling in Valvulopathy and Heart Failure, Lille, France
- ↵∗Address for correspondence:
Dr. Jean-Sébastien Hulot, UMR_S 1166 ICAN Faculté de Médecine Pitié-Salpêtrière, 3ème étage, 91 Boulevard de l'hôpital, 75013 Paris, France.
Background Pw1 gene expression is a marker of adult stem cells in a wide range of tissues. PW1-expressing cells are detected in the heart but are not well characterized.
Objectives The authors characterized cardiac PW1-expressing cells and their cell fate potentials in normal hearts and during cardiac remodeling following myocardial infarction (MI).
Methods A human cardiac sample was obtained from a patient presenting with reduced left ventricular (LV) function following a recent MI. The authors used the PW1nLacZ+/− reporter mouse to identify, track, isolate, and characterize PW1-expressing cells in the LV myocardium in normal and ischemic conditions 7 days after complete ligature of the left anterior descending coronary artery.
Results In both human and mouse ischemic hearts, PW1 expression was found in cells that were mainly located in the infarct and border zones. Isolated cardiac resident PW1+ cells form colonies and have the potential to differentiate into multiple cardiac and mesenchymal lineages, with preferential differentiation into fibroblast-like cells but not into cardiomyocytes. Lineage-tracing experiments revealed that PW1+ cells differentiated into fibroblasts post-MI. Although the expression of c-Kit and PW1 showed little overlap in normal hearts, a marked increase in cells coexpressing both markers was observed in ischemic hearts (0.1 ± 0.0% in control vs. 5.7 ± 1.2% in MI; p < 0.001). In contrast to the small proportion of c-Kit+/PW1− cells that showed cardiogenic potential, c-Kit+/PW1+ cells were fibrogenic.
Conclusions This study demonstrated the existence of a novel population of resident adult cardiac stem cells expressing PW1+ and their involvement in fibrotic remodeling after MI.
Ischemic heart failure (HF) remains a leading cause of mortality and morbidity worldwide (1–3). Following myocardial infarction (MI), the heart progressively replaces lost cardiomyocytes (CMs) with fibrous noncontractile scar tissue. In contrast to other organs, the heart displays minimal regeneration following injury that severely compromises its function and further contributes to HF. In many tissues (e.g., skin, intestine, skeletal muscle), tissue repair relies upon resident adult stem cells, defined as endogenous stem cells that can self-renew and replace lost damaged cells. The existence of adult cardiac muscle stem cells (CSCs) and their role in response to ischemic injury has been controversial (4–6). Several putative CSCs have been identified in the adult heart based upon stem cell capacity, morphology, and expression of different surface and transcription markers (4–6). Among the subsets of CSCs, the c-Kit+ CSCs display a capacity to self-renew, differentiate into CMs, and contribute to cardiac regeneration in the ischemic heart, although this contribution appears to be minimal (7–10). Other resident CSC in embryonic, neonatal, and adult mammalian hearts have been identified by different surface markers (including stem cell antigen [Sca]-1 or platelet-derived growth factor [PDGF] receptor alpha [PDGFRα]) (11,12).
Pw1 (also known as paternally expressed gene Peg3) has been shown to be expressed in stem cells in all adult tissues thus far examined (13,14); however, the existence and cell fate of PW1+ cells in the normal and post-MI adult heart had not been examined previously. We demonstrate here the existence of a cardiac population of cells expressing PW1 in both human and mouse hearts. We used the PW1 reporter transgenic mouse, PW1nLacZ, which expresses the nuclear beta-galactosidase (β-gal) in the context of the PW1 gene (14), to isolate and characterize the PW1+ cells in normal and ischemic hearts.
MI by left anterior descending coronary artery ligation
MI was performed in male 8-week-old C57BL/6 or PW1-reporter (PW1nLacZ) mice by left anterior descending coronary artery permanent ligation. Mice were analyzed 7 days after left anterior descending coronary artery permanent ligation.
Small cell suspensions were prepared from total heart upon atria removal from 8-week-old PW1nLacZ mice. Ventricular tissue was enzymatically digested with collagenase II and dissociated. To detect β-gal–reported activity, cells were incubated at 37°C for 1 h using the fluorescent substrate 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG). The different populations were gated, analyzed, and sorted in a fluorescence-activated cell sorting (FACS) cytometer.
We used 300 ng of total RNA extracted from freshly isolated cells to perform library preparation with the SureSelect Strand-Specific RNA kit (Agilent Technologies, Santa Clara, California) per the manufacturer’s instructions.
We used the Cutadapt program (15) to trim sequenced bases with low quality (<28) and restricted downstream analyses to reads with length >90 bp. Selected reads were mapped to a murine reference transcriptome that was generated by the RSEM package (16) from the full mouse reference genome and the gtf transcript annotations file from Ensembl (17). Analyses were conducted under the R environment (version 3.2.2).
Data were expressed as mean ± SEM. When comparing more than 2 groups, quantitative data were analyzed using 1-way analysis of variance and pair-wise comparisons with Tukey’s test for multiple comparisons. The Mann-Whitney U test was used for comparing continuous variables between 2 groups. All p values <0.05 were considered significant.
A detailed description of all experimental procedures is provided in the Online Appendix.
Because PW1+ cells undergo a pronounced increase in number following injury in multiple tissues (18,19), we first investigated PW1 expression in human hearts following MI. We performed immunofluorescence analyses on a cardiac explant from a patient presenting with reduced left ventricular (LV) function following a recent MI (Figures 1A to 1D). We observed PW1 staining in cells located in the infarct zones (Figure 1B), whereas PW1+ cells were barely detectable in the myocardium distal to the infarct site (Figure 1C). PW1 was not expressed in CMs. A nuclear and punctiform PW1 staining was identified in PW1-expressing cells (Figure 1D). PW1 protein expression was also confirmed by western blot in human normal and ischemic hearts (Figure 1E).
To further characterize these newly identified PW1+ cardiac cells, we assessed Pw1 expression in normal mouse LV myocardium using immunofluorescence. We found that Pw1 was expressed in the adult myocardium by cells located in the epicardium and interstitial space (Figures 1F and 1G). Using a PW1nLacZ reporter mouse (14), we observed a similar pattern of expression (Figures 1H and 1I). In these mice, we confirmed that β-gal and PW1 staining colocalized as reported previously in other tissues (14) (Online Figure 1). Previous studies have demonstrated that PW1+ cells can be successfully isolated by FACS from PW1nLacZ-reporter mice using C12FDG, a fluorescent substrate for β-gal activity (14,20). Therefore, to further characterize the PW1 expressing population, we isolated C12FDG+ cells (referred as PW1+ cells) from the heart of PW1nLacZ-reporter mice (14). Small cells were separated from CMs by differential centrifugation and PW1+ cells were then sorted based upon their β-gal activity. After isolation by FACS, β-gal reporter expression corresponded to endogenous PW1 expression in freshly isolated and cultured cells (Online Figures 2A to 2C). Representative FACS plots illustrated the gating strategy used to define PW1+ cells that were estimated to represent 3.4 ± 0.4% of the non-CM subset in normal left ventricles (Figure 1J).
We next analyzed the expression of classical markers of mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) in PW1+ cells (Figures 1K and 1L). A large proportion (88.3 ± 2.9%) of PW1+ cells did not express CD45, whereas most (83.8 ± 4.3%) were positive for CD105 (Figure 1L). PW1+ cells showed Sca-1 and PDGFRα expression in 58.3 ± 7.6% and 53.4 ± 7.3% of the cells, respectively. Similarly, 32.6 ± 5.3% and 31.7 ± 5.8% of PW1+ cells expressed CD90 and CD44 markers, respectively, whereas only 0.3% of PW1+ cells expressed c-Kit (Figures 1K and 1L).
Cardiac PW1+ cells form colonies
To determine whether cardiac PW1+ cells are clonogenic and to determine their cell fate potentials, freshly isolated C12FDG− and C12FDG+ cells from PW1nLacZ-reporter mouse hearts were sorted and subsequently cultured for 14 days to test their capacity to form colony-forming units (Figures 2A and 2B). In contrast to C12FDG− cells, PW1+ cells were able to form colonies consisting of cells with a flat morphology and a high nucleus/cytoplasm ratio (Figures 2C to 2E). Primary colony-forming unit fibroblast colonies were manually picked up, reseeded, and the resultant secondary colonies still maintained the same morphology. Freshly isolated colonies were placed onto adherent conditions and induced to differentiate under multiple conditions that favor specific cell fate outcomes (i.e., PDGF, basic fibroblast growth factor, and ascorbic acid/dexamethasone for smooth muscle cells [SMCs], fibroblast, and CMs, respectively). We found that PW1+ colonies gave rise to cells expressing alpha smooth muscle actin (SMA) and fibroblast-specific protein 1 (FSP1) proteins depending on stimulation conditions after 2 weeks in culture (Figures 2A and 2F to 2H). However, we were unable to derive CM from the isolated colony-forming unit fibroblast colonies as assessed by the lack of any alpha-sarcomeric actinin expressing cells after 2 weeks in differentiation media.
The expression of CD44 and CD90 suggested that the PW1+ population partially shares a surface marker profile with MSCs. We therefore tested MSC cell fate potential in the cardiac PW1+ population, compared directly the outcomes with bone marrow (BM)-derived MSCs (21) (Online Figure 3). Following exposure to conditions that stimulate specific MSC lineages (Figure 2I), we found that PW1+ cells can differentiate into Oil Red O+ adipocytes, alkaline phosphatase+ osteoblasts, and Alcian blue+ chondrocytes after 2–3 weeks in culture (Figures 2J to 2M). Taken together, these data suggested that cardiac PW1+ cells constitute an adult cardiac stem cell population with colony-forming potential, able to give rise to multiple cardiovascular and mesenchymal lineages.
Transcriptomic profiling of PW1+ cells
To better characterize the cardiac PW1+ cells, we performed an RNA sequencing-based profile of cardiac PW1+ cells isolated from normal left ventricle, comparing these results with mouse embryonic stem cells (mESCs), BM-MSCs, and CMs. Principal component analysis coupled with hierarchical clustering revealed that cardiac PW1+ cells are distinct from BM-MSCs, mESCs, and CMs (Figures 3A and 3B). Functional enrichment analysis of 100 genes (Figure 3C, Online Table 1) that contributed the most to discriminate PW1+ cells from other cell types (third principal component shown on axis 3 in Figure 3A) identified a significant enrichment of genes involved in developmental processes including 21 in tissue development (p = 0.0007) and 10 in skeletal muscle development (p = 0.04) (Figure 3C) compared with the other cell types tested (axis 3; Figure 3A). These latter 10 genes were Fgfr1, Has2, Col27a1, Ext1, P3h1, Tiparp, Twist1, Csrnp1, Thbs3, and Sfrp2, encoding for proteins with molecular functions such as receptor, catalytic, and signal transducer activities.
Because cardiac PW1+ cells showed a marked capacity to differentiate into fibroblasts in vitro, we used our transcriptomic datasets to assess a set of 21 genes encoding for collagen, extracellular matrix proteins, and growth factors typically expressed in cardiac fibroblasts. These pro-fibrotic genes were expressed at higher levels in PW1+ cells versus other studied cell types (Figure 3D). Overall, the expression of PW1 identified a population of resident cardiac cells bearing a molecular signature of cells that guide tissue development and remodeling.
PW1+ cells in ischemic myocardium
Using immunofluorescence staining on ischemic hearts from wild-type mice (1 week post-MI), we observed PW1+ cells in the infarcted area (Figure 4A), as seen in humans. We globally found a higher number of cells expressing PW1 in ischemic mice hearts, with a 3-fold increase in the total number of PW1+ cells in ischemic hearts compared with controls (Figure 4B). After inducing MIs on PW1nLacZ-reporter mice and isolating C12FDG+ cardiac cells by FACS 2 and 7 days post-MI, we found a progressive increase in the number of PW1+ cells that was significant and maximal 7 days after MI where these cells represented more than 10% of non-CMs (Online Figure 4). These estimates were concordant with those obtained using immunofluorescence staining, albeit slightly different as the PW1+ cells detection sensitivity might differ between the 2 approaches.
We next performed cell-cycle analysis and found that PW1+ cells showed a higher proliferative state after MI (Online Figures 5A to 5C). Whereas in nonischemic myocardium FACS cell-cycle analysis showed that most PW1+ cells were quiescent, a higher proportion of PW1+ cells entered the cell cycle after MI (0.3 ± 0.1% in sham vs. 1.0 ± 0.2% in MI in S phase [p <0.05] and 2.5 ± 0.1% in sham vs. 4.2 ± 0.5% in MI in G2/M phase [p < 0.05]). Additionally, PW1+ cells in the infarct zone showed DNA synthesis as detected by bromodeoxyuridine incorporation (Online Figure 5D).
Cardiac PW1+ cells following MI
Previous reports in other tissues have shown that PW1 is down-regulated during stem cell differentiations (20,22) and that the PW1nLacZ-reporter mouse model allows for short-term lineage tracing of cells derived from β-gal+ cells (on the basis of the stability [produrance] of the β-gal reporter). FSP1 (encoded by S100a4), a fibroblast marker not expressed in CMs, has been shown to increase after MI (23–25). Indeed, we found a significant increase of S100a4 expression in the infarcted and border areas of ischemic hearts versus sham hearts (Online Figure 6A). Because PW1+ cells displayed a fibrogenic cell fate in vitro, we examined β-gal activity and FSP1 expression in vivo following MI (Figures 4C and 4D). β-gal expression was detected in histological sections in the epicardium and interstitium and through the infarct and border zones of ischemic hearts from PW1nLacZ-reporter mice (Figure 4D). Importantly, we detected numerous cells in the infarct and border zones that coexpressed β-gal and FSP1 (Figure 4D). These cells were in close vicinity to cells expressing only 1 of these markers, consistent with the scenario of differentiation of PW1+ cells into fibroblasts. Specifically, 21.4 ± 2.2% of FSP1+ cells were β-gal positive in the ischemic heart (Figure 4E). In contrast, no β-gal+/FSP1+ cells were detected in control hearts (Online Figure 7) or in β-gal+ cells in the epicardium of ischemic hearts. Reciprocally, we found an increase in the expression of S100a4 in PW1+ cells sorted from ischemic hearts 7 days after MI as compared with sham heart (Online Figure 6B).
Cells coexpressing PW1 and c-Kit following MI
Our data in normal hearts showed that PW1+ cells expressed multiple CPC and MSC markers, leading us to analyze expression of these markers in C12FDG+ cells isolated from hearts 1 week post-MI. As compared with controls (Figures 1K and 1L), we observed an increase in Sca-1 and CD44 expression and a decrease in CD90 and CD105 expression; expression of other markers remained stable (Figures 5A to 5C). The expression of c-Kit was the most markedly changed in C12FDG+ cells after MI (Figure 5B). We therefore investigated PW1 and c-Kit coexpression in C12FDG− cells and C12FDG+ cells from PW1nLacZ-reporter mouse in normal and ischemic hearts. There was a 3-fold increase in the C12FDG+ population following MI compared to controls (12.3 ± 2.0% of non-CMs in MI vs. 3.4 ± 0.4% in controls; p < 0.01) (Figure 5D). By gating the cells for C12FDG and c-Kit expression, we defined 3 populations according to PW1 and c-Kit expression (Figure 5E). Fraction I corresponded to c-Kit+/PW1− cells and represented 0.3 ± 0.1% of nonmyocytes cells in normal hearts versus 0.9 ± 0.1% in ischemic hearts (p < 0.001). Fraction II was defined as cells coexpressing both markers (PW1+/c-Kit+); fraction III was defined as PW1+/c-Kit− cells. Although PW1 and c-Kit expression were nearly mutually exclusive in cells isolated from normal hearts, there was a cell population coexpressing both c-Kit and PW1 (fraction II) following MI (57-fold increase for fraction II [0.1 ± 0.0% in control vs. 5.7 ± 1.2% in MI; p < 0.001] and 2-fold increase in fraction III [3.3 ± 0.4% in control vs. 7.3 ± 3.5% in MI; p < 0.01]) (Figures 5E and 5F). Therefore, although PW1+/c-Kit− cells were the most abundant fraction in normal hearts, a large proportion of PW1+/c-Kit+ cells appeared following MI (Figure 5G).
PW1 expression and differentiation capacity of c-Kit+ cells
The ability of isolated cardiac-derived PW1-expressing cells to differentiate in vitro into several cardiac lineages led us to test the potential of the fractions to give rise to CM-, smooth muscle-, and fibroblast-like cells. After 1 week in culture under the same stimulatory factors (Figure 6A), only fraction I (c-Kit+/PW1−) generated alpha-sarcomeric actinin-expressing cells, suggesting a lack of cardiomyocyte differentiation capacity in cells expressing PW1 (Figure 6B). The 3 fractions showed potential to generate SMC-like cells as indicated by the presence of α-SMA+-expressing cells derived from all fractions (Figure 6B). In contrast, fibroblast-like cells identified by FSP1 expression were obtained from fractions II and III, but not fraction I. While no differences were detected in the proportion of generated fibroblasts between fractions II and III (45.1 ± 1.7% vs. 44.5 ± 1.5%, respectively; p = NS), a higher proportion of α-SMA+ cells were generated from fraction II cells (expressing both PW1 and c-Kit) versus fraction III cells expressing PW1 only (38.0 ± 1.9% vs. 13.0 ± 1.2%, respectively; p < 0.001) (Figure 6C). These data suggested that PW1-expressing cells display a pronounced fibrogenic potential. Compared with the small proportion of c-Kit+/PW1− cells that showed cardiogenic potential, the abundant fraction of PW1+/c-Kit+ in post-MI hearts adopted a fibrogenic fate.
Our results support that PW1 expression identifies a resident cardiac endogenous stem cell population (Central Illustration). In healthy tissue, the cardiac PW1 stem cell population was distinct from previously reported cardiac progenitor populations, as shown by a partial overlap of typical stem cell marker expression (including Sca-1, PDGFRα, and c-Kit), presented a predominant epicardial and interstitial localization, had colony-forming capacity, and possessed multilineage differentiation capability in vitro. Our data further supported that PW1 identified an adult CSC population that preferentially generated matrix-forming stromal cells as shown by high expression of pro-fibrotic genes and by differentiation into fibroblasts both in vitro and in vivo as shown by cell fate–tracing experiments in ischemic hearts (Central Illustration). This result also fits with the presence of PW1+ cells in the infarct zones of human ischemic hearts, an area that experiences the most intense fibrotic remodeling after MI. Furthermore, PW1+ colonies did not give rise to cardiomyocytes in vitro. Finally, we found that PW1 and c-Kit expression were almost mutually exclusive in cells isolated from normal hearts but that a marked increase occurred in a cell population coexpressing both markers following MI. Strikingly, the c-Kit+/PW1− cells showed cardiogenic potential but represent a minor proportion of the total c-Kit cells. Conversely, c-Kit+/PW1+ cells were much more abundant in ischemic hearts and did not generate CMs but gave rise to fibroblast-like cells.
Our data extended the recent finding that PW1 is a pan-stem cell marker in adult tissues (13,14,19,22) and suggested that PW1 indicates a CSC population different from previously reported CPCs. The cardiac PW1+ cells showed expression of mouse MSC surface markers (such as presence of CD90, CD44, CD105, and PDGFRα, and absence of CD45), and can differentiate into typical MSC lineages, therefore reaching the commonly used definition of MSC (26). MSC-like cardiac progenitor cells have previously been reported in the adult heart (11). These cells were mainly characterized by PDGFRα expression, a marker that only partially overlaps with PW1 expression in both healthy and ischemic hearts. Derived from proepicardial progenitors, these MSCs are usually found in the epicardium and the adjacent myocardial interstitium, locations where cardiac PW1+ cells were also found in healthy hearts. However, the transcriptome profile of PW1+ cells isolated from healthy hearts showed significant differences with the 1 from BM-MSCs, the putative cell origin of solid organ MSCs (27). Interestingly, our transcriptomic analyses identified a higher expression of a set of genes involved in tissue and skeletal muscle development in PW1+ cells compared with mESCs, BM-MSCs, or CMs. This aligned with previous reports of PW1+ cells as skeletal muscle resident progenitors (20). However, in our experiments, we found that the cardiac PW1+ cells lacked cardiogenic potential, a capacity reported in MSCs (28).
Alternatively, resident MSCs have been proposed as a source of scar-forming myofibroblasts in heart fibrosis after MI (29). Interestingly, in this latter study, resident MSCs were identified as CD44+CD45− and were shown to accumulate in the infarct, expressing both stem cell and canonical stem cell fibroblast markers. Whether these cells express PW1 was not determined. More recently, another type of MSC-like cells identified by Gli1 expression has been reported as a source of myofibroblasts that play a central role in organ fibrosis after injury, including after MI (30). Taken together, these data indicate that resident MSCs represent a heterogeneous cell population that can be divided into subpopulations. It is likely that cardiac PW1+ cells represent a distinct pool of MSC-like cells, but further experiments are needed to understand the level of overlap among these different MSC-like cell fractions.
The observations reported here were consistent with the proposal that resident MSC-like cells contribute to the adaptive pathophysiological response to tissue injury (31). We found that a substantial fraction (∼20%) of FSP1+ cells originated from PW1+ cells in ischemic hearts. Reciprocally, we found an increase in the expression of S100a4 (encoding for FSP1) in PW1+ cells from ischemic hearts as well as a high expression of pro-fibrotic markers (including collagen genes Col1a1, Col1a2, Col6a1, and metalloproteinases) in PW1+ cells from normal hearts. Therefore, our data did not contradict the existence of other mechanisms such as endothelial-to-mesenchymal transition (24) or a direct contribution of resident stromal cells (32) but rather proposed an additional source of fibroblasts in cardiac fibrosis. We recently reported that resident lung PW1+ cells participate in the neomuscularization of pulmonary arteries in a mouse experimental model of pulmonary hypertension (19), providing another example of the involvement of resident PW1+ cells in pathological remodeling.
We observed that post-MI, PW1+ cells accumulated in the infarct and border zones of ischemic hearts. This increase in PW1+ cells was associated with an increase in their proliferative state. Most cardiac PW1+ cells were CD45−, suggesting a local mobilization of resident cardiac PW1+ cells, but we cannot rule out circulating cell recruitment. However, the existence of PW1+ circulating cells has not been reported thus far and previous BM transplant experiments did not show a significant recruitment of circulating cells to support the role of PW1+ cells in pulmonary artery neomuscularization (19). Another possibility might be that PW1+ cells arise de novo from PW1− cells involved in post-MI healing. Previous studies have revealed that ischemic injury leads to the proliferation and migration of epicardial-derived cells into the damaged myocardium (5). PW1 expression has been shown to increase in response to several stress stimuli, including hypoxia, in different organs (19,33,34). However, PW1+ cells isolated from post-MI hearts displayed similar characteristics to PW1+ cells isolated from healthy hearts, including a similar expression of cell surface markers and differentiation potential into nonmyocyte mesodermal lineages.
Finally, our data supported the recent proposal that at least 2 populations of cardiac cells express c-Kit (35), a key marker for resident cardiac cells with cardiomyogenic capacity (7,8); however, recent lineage-tracing experiments showed a largely vasculogenic and adventitial lineage predisposition (9,10). During development, c-Kit is expressed in early first heart field progenitors that give rise exclusively to CMs and SMCs. However, the reminiscence of these cardiogenic progenitors in the adult heart is uncertain. Different studies have shown c-Kit expression localized to the proepicardial progenitors that also express MSC-associated markers that have mesodermal lineage differentiation potential. Our results showed that c-Kit+ cells isolated from ischemic hearts can be separated based upon PW1 expression. After MI, a large majority of c-Kit+ cells express PW1 (constituting ∼5.7% of the total non-CM pool of ischemic hearts). As opposed to the few c-Kit+ cells that do not express PW1 (quantified as ∼0.9% of nonmyocyte cardiac cells after MI), the PW1+/c-Kit+ cells were unable to generate CMs. Rather, these cells displayed differentiation potential into SMCs and fibroblasts. These data therefore supported that most c-Kit+ cells are unable to generate cardiomyocytes and display MSC-like characteristics as seen in cardiac PW1+ cells. Conversely, the c-Kit+ cells not expressing PW1, as identified in this study, might represent a CPC population. Intriguingly, our results further suggested a gradient in myogenic potential following c-Kit expression because PW1+/c-Kit- cells displayed a lower capacity to generate SMCs compared with PW1+/c-Kit+ cells. Further experiments, such as transcriptomic profiling, will be needed to understand the underlying molecular mechanisms explaining the loss of cardiogenic potential in c-Kit+ cells expressing PW1.
This study did not explore the angiogenic potential of cardiac PW1+ cells. Whether PW1+ cells can also contribute to vessel formation deserves further investigation. Additionally, we explored cardiac PW1+ cells in a murine MI model with permanent occlusion of the coronary artery but not in the context of myocardial ischemia-reperfusion injury.
Taken together, these data demonstrated the existence of a population of resident adult cardiac stem cells expressing PW1+ and their involvement in fibrotic remodeling after MI. Our results also supported the existence of PW1+ cells in human ischemic hearts. Whether these cells could represent a source of new therapeutic strategies for ischemic HF deserves further investigation.
COMPETENCY IN MEDICAL KNOWLEDGE: In patients with myocardial infarction, defunct cardiac myocytes are progressively replaced by fibrous, noncontractile tissue. Resident adult stem cells expressing PW1+ are involved in fibrotic remodeling and may play a role in muscle repair and recovery of myocardial contractile function.
TRANSLATIONAL OUTLOOK: Targeting adult cardiac stem cells expressing PW1 could represent a therapeutic opportunity to limit adverse myocardial remodeling after myocardial infarction.
RNA sequencing was performed with the help of Plateforme P3S at Sorbonne Universités, Paris, France.
For a supplemental Methods section as well as figures and tables, please see the online version of this article.
This work was supported by grants from the Fondation Leducq (13CVD01, CardioStemNet project), the Institute of Cardiometabolism and Nutrition (ANR-10-IAHU-05), the ANR REVIVE (Laboratoire d’Excellence) (Dr. Sassoon), the Fondation pour la Recherche Médicale and GENMED Laboratory of Excellence on Medical Genomics (ANR-10-LABX-0013) (Dr. Roux), and the Agence Nationale de la Recherche (ANR-15-CE14-0020-01) (Dr. Nadaud). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- smooth muscle actin
- bone marrow
- 5-dodecanoylaminofluorescein di-β-D-galactopyranoside
- cardiac progenitor cell
- cardiac muscle stem cells
- fluorescence-activated cell sorting
- fibroblast specific protein 1
- heart failure
- left ventricular
- mouse embryonic stem cell
- myocardial infarction
- mesenchymal stem cells
- platelet-derived growth factor
- stem cell antigen
- smooth muscle cell
- Received January 18, 2017.
- Revision received May 30, 2017.
- Accepted June 2, 2017.
- 2017 American College of Cardiology Foundation
- Roger V.L.,
- Go A.S.,
- Lloyd-Jones D.M.,
- et al.
- Seferovic P.M.,
- Stoerk S.,
- Filippatos G.,
- et al.
- Lin Z.,
- Pu W.T.
- Oh H.,
- Bradfute S.B.,
- Gallardo T.D.,
- et al.
- Besson V.,
- Smeriglio P.,
- Wegener A.,
- et al.
- Dierick F.,
- Hery T.,
- Hoareau-Coudert B.,
- et al.
- Pannerec A.,
- Formicola L.,
- Besson V.,
- Marazzi G.,
- Sassoon D.A.
- Besson V.,
- Kyryachenko S.,
- Janich P.,
- Benitah S.A.,
- Marazzi G.,
- Sassoon D.
- Zeisberg E.M.,
- Kalluri R.
- Karantalis V.,
- Hare J.M.
- Kim J.,
- Braun T.
- Yamaguchi A.,
- Taniguchi M.,
- Hori O.,
- et al.
- Keith M.C.,
- Bolli R.