Stem cell antigen-1 cell-derived fibroblasts are crucial for cardiac fibrosis during heart failure

doi:10.21203/rs.3.rs-3087476/v1

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Research Article

Stem cell antigen-1 cell-derived fibroblasts are crucial for cardiac fibrosis during heart failure

https://doi.org/10.21203/rs.3.rs-3087476/v1

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Journal Publication

published 23 Sep, 2023

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Aims

Mesenchymal stem cells (MSCs) present in the heart cannot differentiate into cardiomyocytes, but may play a role in pathological conditions. Therefore, the aim of this study was to scrutinise the role and mechanism of MSC differentiation in vivo during heart failure.

Methods and Results

We performed single-cell RNA sequencing of total non-cardiomyocytes from murine and adult human hearts. By analysing the transcriptomes of single cells, we illustrated the dynamics of the cell landscape during the progression of heart hypertrophy, including those of stem cell antigen-1 (Sca1)⁺ stem/progenitor cells and fibroblasts. By combining genetic lineage tracing and bone marrow transplantation models, we demonstrated that non-bone marrow-derived Sca1⁺ cells give rise to fibroblasts. Interestingly, partial depletion of Sca1⁺ stem cells alleviated the severity of myocardial fibrosis and led to a significant improvement in cardiac function in Sca1-CreER^T2;Rosa26-eGFP-DTA mice. Similar non-cardiomyocyte cell composition and heterogeneity were observed in human patients with heart failure. Mechanistically, our study revealed that Sca1⁺ cells can transform into fibroblasts and affect the severity of fibrosis through the Wnt4-Pdgfra pathway

Conclusions

Our study describes the cellular landscape of hypertrophic hearts and reveals that fibroblasts derived from Sca1⁺ cells with a non-bone marrow source of largely account for cardiac fibrosis. These findings provide novel insights into the pathogenesis of cardiac fibrosis and have potential therapeutic implications for heart failure.

Graphical abstract:

Sca1+ cells

single-cell RNA sequencing

genetic lineage tracing

transverse aortic constriction

myocardial fibrosis

Heart failure currently results in high rates of morbidity and mortality [1–2], and myocardial hypertrophy and cardiac fibrosis are critical steps in its progression [3–5]. Cardiac fibroblasts are not only a major cell population in the heart that play a critical role in maintaining normal cardiac function, but also the effective cells as they become activated and proliferative contributing to myocardial hypertrophy under different conditions [3–4, 6]. The heterogeneity of cardiac fibroblasts means they may contribute to cardiac fibrosis in various ways [7–8]. However, a clear definition of cardiac fibroblasts has yet to be established, and it remains to be determined whether these cells proliferate from existing cells or differentiate from stem cells [9–10]. The exact role of cardiac stem/progenitor cells in the progression to heart failure also remains uncertain.

Although the existence of adult cardiac stem cells has been denied, it does not mean that all efforts in cardiovascular stem cell research areas are futile. Other resident stem cells, like mesenchymal stem cells (MSCs), adipose-derived stem cells and endothelial progenitor cells have been reported to differentiate into different types of mature cells (non-cardiomyocytes) under specific conditions, and these cells warrant further exploration [11–12]. It has been reported that these stem cells play a role in cardiovascular endothelium repair, cardiovascular remodeling, angiogenesis, inflammatory responses, and other pathological disease processes [13–14]. However, it is uncertain which sources of MSCs involved in cardiac fibrosis, and the inherent heterogeneity of these cell populations is poorly understood [15]. Thus, more endeavor and research are required to identify potential therapeutic targets for cardiac fibrosis.

Stem cell antigen-1 (Sca1) has been described as a marker of cardiac stem/progenitor cells [16–18]. Endogenous Sca1⁺ cells have been reported to differentiate into other cell types, such as smooth muscle cells [19], endothelial cells, and fibroblasts [18, 20], under certain conditions. However, the role of Sca1⁺ cells, classified as MSCs in some studies [21–22], in cardiac fibrosis, remains to be established. In this study, we generated a Sca1-CreER^T2;R26-tdTomato mouse model to investigate endogenous Sca1⁺ cells under both physiological and pathological conditions using single-cell sequencing and genetic lineage tracing techniques. Additionally, Sca1-CreER^T2;R26-DTA and Sca1-CreER^T2;Pdgfra^flox/flox;R26-tdTomato mice were utilized to reveal the specific role of Sca1⁺ cardiac cells in vivo through diphtheria toxin subunit A (DTA)-induced cell ablation. Our findings suggest that Sca1⁺ cells can differentiate into fibroblasts both in vitro and in vivo; bone marrow (BM) transplantation (BMT) confirmed that these cells originated from a non-BM source. Further studies revealed that cardiac fibrosis is regulated by Pdgfra via the Wnt4 pathway.

2.1 Animals

All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the Zhejiang University School of Medicine and performed according to the National Institutes of Health guidelines in the Guide for the Use and Care of Laboratory Animals. In this study, we used the following mouse strains: Sca1-CreER^T2, Rosa26-tdTomato (JAX: 007909), Rosa26-eGFP-DTA (JAX: 006331) and C57BL/6/J (JAX: 000664), which were purchased from Shanghai Biomodel Organism Co., Ltd (Shanghai, China). Sca1-CreER^T2;R26-tdTomato mice were obtained by crossing Sca1-CreER^T2 with R26-tdTomato mice. Sca1-CreER^T2;R26-DTA/tdTomato mice were obtained by crossing Sca1-CreER^T2;R26-tdTomato and R26-eGFP-DTA mice. Sca1-CreER^T2;Pdgfrα^flox/flox;R26-tdTomato mice were generated by crossing the Pdgfrα^flox/flox and Sca1-CreER^T2;R26-tdTomato mice with further appropriate genotyping. All generated or purchased mice were housed for at least one week before use. The mice were maintained under a 12 h light/dark cycle, given free access to water and food and monitored daily for health; detailed methods of the generation and breeding of mice for genetic tracing are available in the online Supplementary Data.

2.2 Anaesthetics and analgesia

Mice were anaesthetized before TAC surgery by intraperitoneal injection of ketamine (100 mg kg^− 1 Imalgene®) and xylazine (10 mg kg^− 1 Rompun® 2%, Bayer Health Care). Buprenorphine (50 µg kg^− 1, Buprecare®, Axcience) was injected subcutaneously 6 h after surgery and twice per day until 3 days post-operation. For transthoracic echocardiography animals were anaesthetized with isoflurane vaporized (2.5% for induction, 1.0% for maintenance) in oxygen. At the end of the study, animals were euthanized by barbiturate over-dose (100 mg kg^− 1 intraperitoneal, Sodium Thiopental, Rotexmedica).

2.3 Animal models and echocardiography

All mice (at 10–12 weeks old) were subjected to a sham or transverse aortic constriction (TAC) operation, as previously described [23–24]. Subsequently, mice were euthanized (as described in 2.2) and blood and hearts were collected for histological, protein, RNA and immunofluorescence analyses. For full details, see Supplementary Methods.

In vivo cardiac echocardiography was performed using transthoracic ultrasonography with a Vevo 770 high-resolution ECHO system equipped with a 35 MHz transducer. Mice were sedated with vaporized isoflurane (2.5% for induction and 1.0% for maintenance) in oxygen, and the inhalational flow of isoflurane was adjusted to maintain their heart rate at 450–550 beats/min. Left ventricular function was assessed by M-mode scanning of the left ventricular chamber, and left ventricular size (end-diastolic and end-systolic dimensions), wall thickness (posterior wall thickness), left ventricular fractional shortening (FS), and left ventricular ejection fraction (EF) were measured for at least three beats from each projection and averaged. All mice underwent echocardiography at 0 and 5 weeks after TAC surgery. The investigators were blinded to the experimental conditions.

2.4 Human tissue

The collection of human heart tissue was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (approval no. IIT20210018B-R2). All patients authorized their rights for tissue collection with legal consent and signature. All procedures were performed in accordance with local law and the principles of the Declaration of Helsinki. In this study, we included two types of human left ventricular samples: control samples (nonfailing, non-transplantable hearts), and heart failure samples (end-stage dilated cardiomyopathy). Control heart tissue was collected from healthy organ donors and the left ventricles of patients with heart failure were collected from patients undergoing heart transplantation. All patients with heart failure were diagnosed with dilated cardiomyopathy with EF < 30% (systolic heart failure). All samples were frozen for RNA-sequencing analysis or fixed in formalin and embedded in paraffin for histological and immunofluorescence analyses.

2.5 Processing of single-cell RNA-sequencing data

Single-cell RNA-sequencing (scRNA-seq) data were collected from the GEO database (scRNA-seq data of total mouse non-cardiomyocytes from GSE198833 and scRNA-seq data of total human non-cardiomyocytes from GSE222144). The data were normalised using the batchelor package [25]. Doublets were filtered using the DoubletFinder R package [26]. Dimensionality reduction, cluster identification on ‘merged’ or ‘integrated’ data, and differential gene expression analysis were performed with the Seurat package [27]. Detailed methods of bioinformatics analysis, including sc-RNA-seq and bulk RNA-sequencing (bulk RNA-seq) analyses are described in the Supplementary Methods.

2.6 Statistical analysis

GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA) was used to draw statistical images and perform comparative analyses in Fig. 1k, 3d, 3e, 3g, 3h, 3l, 3m, 3n, 3o, 5f, 5g, 6b, 6d, 6g, 6j, 6k, 7b, 7d, 7e, 7f, and Supplementary Fig. 7c-d, S7g-h, S8f-g, S9d-f, S9h, S9j, S10d-f. Number (n) refers to the number of mice or patients used in the experiments, as indicated in the relevant figure legends. Data are expressed as mean ± standard deviation. For animal and human data, the normality of the data was analysed using the D’Agostino Pearson omnibus test or the Shapiro Wilk test. Data that passed the normality and lognormality tests were analysed using the two-tailed unpaired Student’s t-test. Data which did not pass the normality and lognormality tests were analysed using the Mann Whitney U test. A P-value of < 0.05 was considered statistically significant. Images which most accurately matched the quantitative analysis and had a high image quality are shown. Each independent experiment was performed at least four times.

3.1 ScRNA-seq analyses of human hearts

To characterise non-cardiomyocytes in human hearts, we first generated a scRNA-seq dataset by integrating two groups of human heart samples (Control: normal heart; HF: Heart failure) and obtained single-cell transcriptomes for 22,537 cells after strict quality control. We defined nine major cell types: fibroblasts (DCN⁺, PDGFRA⁺), endothelial cells (CDH5⁺), cardiomyocytes (ACTA1⁺), T/NK cells (CD3G⁺, NKG7⁺), macrophages/monocytes/dendrtic cells (CD68⁺), B cells (CD79A⁺), neuronal cells (PLP1⁺), neutrophils (S100A8⁺), and smooth muscle cells (ACTA2⁺), based on their respective molecular signatures (Fig. 1a, Supplementary Figure S1a-c). Furthermore, as shown in Fig. 1b, we found that fibroblasts were the largest group of the non-cardiomyocyte cell types. The proportion of fibroblasts was obviously higher in the heart failure group (56.49%) than in the control group (42%).

Given the above results and the vital role of fibroblasts in fibrosis [3, 6], in the subsequent experiments, fibroblasts from the two groups (10,424 cells) were extracted and partitioned into distinct subtypes. The dispersion of the fibroblast clusters in the Uniform Manifold Approximation and Projection (UMAP) plots indicated a high degree of fibroblast heterogeneity (Supplementary Figure S1d); fibroblasts from both groups were partitioned into 13 distinct subtypes (Fig. 1c, Supplementary Figure S2a-c). Moreover, we found that the expression levels of fibroblast genes (POSTN, COLLAGEN 1A1, COLLAGEN 1A2, THBS4, VIMENTIN, and FN1) were significantly higher in the heart failure group than the control group (Supplementary Figure S2d). Additionally, as shown in the volcano plot in Supplementary Figure S2e, expression of the active fibroblast marker POSTN was increased in the heart failure group compared with the control group (Supplementary Figure S2e).

Surprisingly, cluster 3 showed elevated expression levels of multiple progenitor cell markers, including CD34, PI16, CD248 and NT5E, which indicate multi-lineage differentiation [24, 28–29] (Fig. 1c). In addition, gene ontology (GO) enrichment analysis showed that vasculature development and differentiation were highly enriched in cluster 3, indicating that this sub-cluster of fibroblasts may exhibit specific functions during cardiac homeostasis and after injury (Fig. 1d). Therefore, we investigated the potential developmental relationships among these subclusters of fibroblasts. Pseudotime(s) were visualised using principal curves representing the trajectories of fibroblast differentiation across the steady-state atlas, with cluster 3 set as the root (Fig. 1e). Fibroblasts were ordered along a trajectory, and cells in different states were identified (Fig. 1e-f). Using the Monocle R package [30–31], we defined the different cellular states along the trajectory. Interestingly, different subgroups of fibroblasts arose and were ordered from a certain point; a split view of the trajectory analysis of each cluster is shown in Supplementary Figure S1e. Further trajectory analysis clearly showed that the expression of specific MSC markers (including CD34, ENG, NT5E, and PI16) significantly changed with the state, corresponding to the pseudotime trajectory (Fig. 1g-j).

We further measured the expression of these biomarkers at the RNA level using quantitative PCR (Fig. 1k, primer sequences used for qPCR including human and mice are listed in Supplementary Table S1) and found that the expression levels of VIMENTIN, DDR2, POSTN, PDGFRA, COLLAGEN 1A, COLLAGEN 3A, and FN1 were significantly higher in the heart failure group than in the control group. In addition, immunofluorescence staining showed increased expression of VIMENTIN, DDR2, and PDGFRA in the heart failure group compared with the control group (Fig. 1l, Supplementary Figure S3a-b).

3.2 ScRNA-seq analyses of murine hearts.

To delineate the heterogeneity of mouse fibroblasts during the development of heart failure, we collected scRNA-seq datasets of total non-cardiomyocytes from a public repository (GSE198833). After applying strict quality control filters, we obtained single-cell transcriptomes for 12,157 cells by integrating two groups (sham and TAC), and graph-based clustering was performed to classify these cells according to their gene expression profiles using UMAP plots (Supplementary Figure S4a). We defined 7 major cell types: fibroblasts (Dcn⁺, Pdgfra⁺, Ddr2⁺), lymphocytes (Cd3g⁺, Cd79a⁺), neutrophils (S100a8⁺), endothelial cells (Cdh5⁺, Pecam1⁺), neuronal cells (Plp1⁺), smooth muscle cells (Acta2⁺, Myh11⁺) and Mac_Mo_DC cells (Cd68⁺, Adgre1⁺), based on established canonical markers (Supplementary Figure S4a-d). Compared with the sham group, we found that the proportion of fibroblasts increased following TAC (sham: 53.74%; TAC: 79.99%) (Fig. 2a). We distinguished these fibroblasts according to characteristic biomarkers above and displayed the distribution of each subpopulation. Based on transcriptome characteristics, fibroblasts were further analysed to investigate their heterogeneous features, and 10 subclusters were identified between the sham and TAC groups (Fig. 2b, Supplementary Figure S4e and S5a). The dispersion of the fibroblast clusters in the UMAP plots indicated a high degree of fibroblast heterogeneity (Fig. 2b). Similar to the previous human results, we also found a group of fibroblast subsets with high expression of multiple progenitor cell markers, including Ly6a, Cd34, Cd248, Thy1 and Pi16 (Fig. 2c). These fibroblast subpopulations mentioned above showed an elevated capacity for extracellular matrix organisation, extracellular structure organisation and ossification, which are all involved in fibrosis (Fig. 2d).

In addition, GO enrichment analysis showed that extracellular structure organisation, positive regulation of blood vessel development, and vasculogenesis were highly enriched in cluster 7 (Supplementary Figure S5b), indicating that this subcluster of fibroblasts may perform specific functions during heart failure. We then conducted pseudotime trajectory analyses for each subcluster of fibroblasts, as for human samples, visualising the pseudotime(s) using principal curves representing trajectories of fibroblast differentiation across a steady-state atlas with cluster 7 set as the root. Fibroblasts were ordered along a trajectory, and cells in different states were identified (Fig. 2e, Supplementary Figure S5d). Surprisingly, further trajectory analysis clearly showed that the expression of specific MSC markers, including Cd34, Ly6a, and Pi16, significantly changed with the state, corresponding to the pseudotime trajectory (Fig. 2f-h, Supplementary Figure S5c and S5e).

Combining the human and mouse data, we identified fibroblast subsets in the heart with high expression levels of MSC markers and a progenitor cell-like phenotype, which can transdifferentiate into multiple cell types, especially fibroblasts, and perform specific functions in cardiac fibrosis. Furthermore, we performed TAC procedures in C57BL/6 mice to establish a heart failure model; mice were analysed after five weeks (Fig. 2i). Immunostaining of Vimentin, Ddr2, Postn and Pdgfra revealed a significant increase in fibroblasts during the development of murine heart failure (Fig. 2j, Supplementary Figure S6a-c). Given the high proportion and heterogeneity of fibroblasts in both human and murine hearts as revealed by scRNA-seq analyses and the comprehensive differentiation profile of MSC markers such as Sca1 shown by trajectory analyses, we next explored the role of Sca1⁺ cells in heart failure.

3.3 Sca1⁺ cells can differentiate towards fibroblasts in vivo

As shown in Supplementary Figure S6d-e, by analysing the correlation between human and mouse single-cell datasets, we found that the cellular composition of mouse and human hearts was very similar, as was the high expression of MSC markers (CD34, PI16, CD248) in fibroblast subsets. Thus, in order to further investigate the specific fibroblast subset expressing MSC markers in injury-induced cardiac fibrosis and the relationship between this fibroblast subset and other fibroblasts in vivo, we used an inducible genetic stem/progenitor cell lineage tracing mouse model (Sca1-CreER^T2;Rosa26-tdTomato) (Fig. 3a). After genotyping (primer sequences used for genotyping in this study are listed in Supplementary Table S2), mice were treated with five doses of tamoxifen (0.15 mg/g body weight) over the course of one week to induce tdTomato (tdT) labelling of Sca1⁺ cells. We performed TAC surgery approximately two weeks after the last tamoxifen dose avoiding the potential side effect of tamoxifen (Fig. 3b), and five weeks postoperatively echocardiography was performed on each mouse to comprehensively evaluate cardiac function. Echocardiography results demonstrated poor average cardiac function in mice that underwent TAC surgery (Fig. 3c), as demonstrated by a lower EF (P = 0.0001) and left ventricular FS (P = 0.0001) than the sham group (Fig. 3d-e). In addition, histology confirmed that the volume of the mouse heart and the thickness of the myocardial wall were significantly greater in mice from the TAC group than in those from the sham group (Fig. 3f). TdT⁺ positive cells increased in TAC heart samples, and the expression of characteristic fibroblast markers such as VIMENTIN, DDR2, POSTN, and PDGFRA also significantly increased compared with those from the sham group. There was also greater co-staining of tdT⁺ with these fibroblast markers in TAC heart samples than in samples from the sham group (Fig. 3i-j, Supplementary Figure S7a-b), which was statistically significant (Fig. 3g-h, Supplementary Figure S7c-d).

Ablation of target cells by inducing intracellular expression of DTA is a well-established mouse model [32]. To further clarify the role of Sca1⁺ cells in myocardial remodelling in response to TAC injury, we used the DTA system to deplete Sca1⁺ cells to determine their role in myocardial fibrosis. We generated Sca1-CreER^T2; R26-eGFP-DTA mice which were then pulsed with tamoxifen to induce recombination for DTA expression in Sca1⁺ cells. TAC was performed in Sca1-CreER^T2; R26-eGFP-DTA mice treated with tamoxifen. As shown in Fig. 3k, depletion of Sca1⁺ cells significantly affected heart function, as Sca1-CreER^T2;R26-DTA mice had better EF and FS than Sca1-CreER^T2;R26-tdTomato mice (Fig. 3l-m). We also observed a reduction in the expression of fibroblast markers along with tdT⁺ in the cardiac cells (Fig. 3p-q, Supplementary Figure S7e-f), which was statistically significant (Fig. 3n-o, Supplementary Figure S7g-h). This suggests that Sca1⁺ cells have the ability to differentiate into fibroblasts in vivo during cardiac fibrosis, and that depletion of Sca1⁺ cells not only affects their transformation, but also the degree of cardiac fibrosis.

3.4 Sca1 ⁺ cells which differentiate into fibroblasts to accelerate fibrosis after injury have a non-bone marrow source

We investigated the exact source of these special clusters of fibroblasts and whether they originated from BM or a non-BM source. Therefore, we generated two types of chimeric mice using a bone marrow transplantation (BMT) model. One was BMT^Sca1→WT, where bone marrow cells from Sca1-CreER^T2;R26-tdTomato mice were transferred to irradiated wild-type C57BL/6J mice via a tail vein injection. The other was BMT^WT→Sca1, which reversed this combination (Fig. 4a). After a recovery period of approximately two weeks, all chimeric mice were treated with tamoxifen by oral gavage and subjected to myocardial remodelling by transverse aortic constriction (TAC). Successful bone marrow reconstruction was indirectly confirmed by a control group of recipient mice all died within one week of irradiation without BMT.

After 5 weeks, heart samples were collected from all chimeric mice and immunostaining was performed to observe the extent of myocardial fibrosis. In the BMT^Sca1→WT chimeric mouse model, almost no tdT⁺ cells were detected, let alone co-expressing cells among the abundant cardiac fibroblasts. However, in the BMT^WT→Sca1 chimeric mouse model, a large proportion of tdT⁺ cells co-stained with fibroblast markers such as VIMENTIN, DDR2, POSTN, and PDGFRA (Fig. 4b-e). This suggests that this cluster of cells participates in fibroblast regeneration. Thus, our data demonstrate that the crucial group of Sca1⁺ fibroblasts that respond to myocardial fibrosis is derived from a non-BM source.

3.5 Sca1⁺ cells can differentiate towards fibroblasts in vitro

We then investigated whether Sca1⁺ cells could differentiate into fibroblasts in vitro. We harvested hearts from Sca1-CreER^T2;R26-tdTomato mice that had completed tamoxifen treatment, sorted the Sca1⁺ cells, and cultured them in complete stem cell culture medium. Next, we treated them with connective tissue growth factor (CTGF), which is sufficient to differentiate MSCs into fibroblasts [33]. Immunofluorescence staining showed that tdT-positive cells co-stained for Vimentin, Ddr2, Pdgfra, and Postn, indicating that Sca1⁺ cells can differentiate into fibroblasts in vitro (Fig. 5a-d, Supplementary Figure S8a-d). Furthermore, quantitative PCR analysis showed that the expression levels of characteristic fibroblast markers significantly increased after treatment with CTGF or transforming growth factor-β for 3, and 5 days in vitro (Fig. 5f, Supplementary Figure S8f), which was consistent with the results of western blotting (Fig. 5e, 5g, Supplementary Figure S8e and S8g). Collectively, these results confirmed that Sca1⁺ cells have the potential to differentiate into fibroblasts at the molecular level.

3.6 Role of Wnt4 activation in the differentiation of Sca1⁺ cells

As a result of the above data, we explored in detail how Sca1⁺ cells differentiate into fibroblasts. Therefore, we performed bulk RNA-seq analyses on control and CTGF-treated cardiac fibroblasts from Sca1⁺ lineage-tracing mice. Using differential gene expression profiling (Fig. 6a, Supplementary Figure S9a), we examined genes related to the Wnt signalling pathway and identified Wnt4, which has recently been associated with the development and differentiation of many cell types [19]. We then knocked down Wnt4 in Sca1⁺ cardiac fibroblasts using small interfering RNA (siRNA). The primer sequences used are listed in Supplementary Table S3 and the verification results are shown in Supplementary Figure S9c. Knocking down Wnt4 led to a significant reduction in the expression of fibroblast markers such as VIMENTIN, DDR2, POSTN, and PDGFRA (Fig. 6b-d). Considering the expression of PDGFRA following FB-inducing factors treatment (Fig. 5e-g, Fig. 6c-d, Supplementary Figure S8e-g), we suggest that Sca1⁺ cells can differentiate into PDGFRA-expressing cells involved in fibrosis, possibly via Wnt4.

Tentatively, we next knocked down Pdgfra in Sca1⁺ cardiac fibroblasts using siRNA, and cultured these cells with or without CTGF, and added the groups of Pdgfra deficient by siRNA. Surprisingly, bulk RNA-seq analyses revealed an updated series of Wnt pathway-related differentially expressed genes, including Wnt4 (Fig. 6e). The genes that were differentially expressed among the four groups are shown in Supplementary Figure S9b. The verification results, including those of other primary differentially expressed genes are shown in Supplementary Figure S9d. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, which revealed a tendency for cardiac remodelling, are shown in Fig. 6f. Furthermore, RNA expression levels of the fibroblast markers Ddr2, Collagen I, Collagen3a, Postn, Cd248, and Fn1 were significantly reduced when Pdgfra was knocked down (Fig. 6g). To confirm this result, overexpression of Pdgfra using plasma was performed, the primer sequences are shown in Supplementary Figure S9g. The protein expression levels of the fibroblast markers VIMENTIN, POSTN, and PDGFRA were downregulated or upregulated, in accordance with the inhibition or overexpression of Pdgfra (Fig. 6h-i), the statistical results were demonstrated in Fig. 6j-k, which were to a great extent consistent with our hypothesis. In addition to western blotting, we used Cell Counting Kit-8 and a lactate dehydrogenase (LDH) kit to observe the effect on Sca1⁺ cellular activity in vitro. These demonstrated that higher expression of Pdgfra impaired cellular activity and caused increased LDH release (Supplementary Figure S9e-f).

We also investigated WNT4 expression in human samples and found that it was significantly increased in patients with heart failure (Supplementary Figure S9h-j, P = 0.0001 in Supplementary Figure S9h, P = 0.003 in Supplementary Figure S9j), which was consistent with our findings in mice. We therefore hypothesised that Pdgfra plays a crucial role in the differentiation of Sca1⁺ cells into fibroblasts via Wnt4.

3.7 Sca1⁺ cells play a role in myocardial fibrosis significantly related to PDGFRα

To test this hypothesis, we generated a new genetic lineage tracing system, Sca1-CreER^T2 Pdgfra^flox/flox; Rosa26-tdTomato mice, to observe the effect of conditional Pdgfra knockdown on myocardial fibrosis in vivo. We followed the same experimental design as previously described (Fig. 7a) and collected heart samples and blood from mice after evaluating their cardiac function using echocardiography. The serum isolated from blood samples was used to test the brain natriuretic peptide (BNP) concentration in peripheral circulating blood and found that BNP levels decreased significantly in Sca1-CreER^T2 Pdgfra^flox/flox;R26-tdTomato mice compared with Sca1-CreER^T2;R26-tdTomato mice after TAC surgery (P = 0.0024) (Fig. 7b). The results of echocardiography showed that Sca1-CreER^T2 Pdgfra^flox/flox;R26-tdTomato mice had a significant improvement in left ventricular ejection fraction (EF) (P = 0.002) and left ventricular fractional shortening (FS) (P = 0.0001) compared with Sca1-CreER^T2;R26-tdTomato mice after TAC surgery (Fig. 7c-e). In agreement with previous research, we observed a sharp reduction in the ratio of tdT⁺ cells, as well as significantly lower expression of fibroblast markers such as VIMENTIN, DDR2, POSTN, and PDGFRA in conditional Pdgfra knock down mice compared with Sca1-CreER^T2;R26-tdTomato mice, as evidenced by immunofluorescence staining (Fig. 7f-g, Supplementary Figure S10a-f) and confirmed by histology (Fig. 7h). Based on these findings, we confirmed that Sca1⁺ cells can transform into fibroblasts and affect the severity of fibrosis through the Wnt4-Pdgfra pathway.

Cardiac fibroblasts have been recognised for their critical role in the response to various forms of injury [34–35]. Pathological cardiac remodelling and fibrosis are driven by fibroblast accumulation and excessive deposition of extracellular matrix, which may lead to heart failure [7, 34]. However, the exact origins of cardiac fibroblasts have not been fully elucidated, with possibilities including bone marrow, mature tissue fibroblasts, and stem cells [10, 36–37]. Several recent reports have indicated that mesenchymal stem cells (MSCs) or Sca1⁺ cells can differentiate into a variety of cells that may participate in cardiac remodelling [37–39], however, little is known about the direct contribution of Sca1⁺ stem/progenitor cells to fibrosis during heart failure. In this study, we investigated stem/progenitor cell-derived fibroblasts that could differentiate into an active population of fibroblasts using scRNA-seq and genetic cell lineage tracing techniques in human and mouse models. First, a landscape of cells from the hearts of patients with cardiac fibrosis and heart failure was established, identifying a major population of fibroblasts, possibly derived from stem/progenitor cells. Second, we used a mouse model of left ventricular pressure overload to demonstrate that Sca1⁺ cells are an important source of fibroblasts leading to cardiac fibrosis. Third, the partial depletion of Sca1⁺ cells resulted in a significant reduction in fibrosis and heart failure in animal models. Finally, we depicted the mechanisms by which Wnt4-PDGFRaαsignalling pathways play a part in this process. Taken together, our findings suggest that MSCs/Sca1⁺ cells are key cell populations responsible for cardiac fibrosis, implicating them as potential targets for future therapeutic strategies.

Recently, there have been several reports showing the nature and functions of fibroblasts during heart remodelling or heart failure, in which different types of cells have been described, including fibroblasts expressing MSC markers [40]. As the meaning of MSC is now highly controversial, the biomarkers of MSC are not specific. It is suggested that certain MSC marker could express in different organs or diseases, and play a different role [41]. It is unknown whether these fibroblasts are derived from MSCs or are merely expressing MSC markers [2]. In this study, we combined several models and techniques to address this issue. We identified several subpopulations of fibroblasts in human heart tissue using scRNA-seq analyses, such as THBS4-fibroblasts, POSTN-fibroblasts, CD248-fibroblasts, and PDGFRa-fibroblasts. The expression of these fibroblast markers was higher in the heart failure group than in the control group. Additionally, we identified clusters of fibroblasts that not only highly expressed these characteristic fibroblast markers, but also highly expressed multiple MSC markers, such as Ly6a (Sca1), Pi16 and CD34. We conducted pseudotime trajectory analyses of each subcluster of fibroblasts in both human and mouse datasets and found that Cd34, Ly6a, and Pi16 positive cells were roots that expanded into mature fibroblasts. Finally, Sca1 expression was eventually lost during Sca1⁺ cell differentiation into fibroblasts both in vitro and in vivo. Based on these findings, we hypothesised that Sca1⁺ cells are fibroblast progenitors that differentiate into active fibroblasts and participate in cardiac fibrosis.

Sca1 is reported to be expressed in various tissues, including fat deposits, adventitial and endothelial cells of arteries, bone marrow, skeletal muscle, heart, and other tissue/organ systems [16, 42]. It is known that Sca1⁺ cells can also differentiate into endothelial when vessel injury [20, 43] and smooth muscle cells after severe vessel injury [44]. We therefore investigated whether other cell types derived from Sca1⁺ cells play a role in cardiac fibrosis using a pressure overload model. When used vascular endothelial growth factor receptor (VEGFR) inhibitor to inhibit the growth of endothelial, there were still a high expression of fibroblasts and co-stained with tdT⁺ cells. And in our mouse model, there was a poor expression of smooth muscle cells (Supplementary Figure S11). We combined genetic cell lineage tracing and scRNA-seq analysis to trace the fate of Sca1⁺ cells and found that Sca1⁺ cells mainly differentiated into fibroblasts and, to a much lesser extent, endothelial and smooth muscle cells. Briefly, cardiac fibrosis was not affected by inhibiting endothelial differentiation in our mouse model of pressure overload to left ventricle. These results indicate that fibroblasts, rather than endothelial cells or smooth muscle cells, derived from Sca1⁺ cells contribute to cardiac fibrosis in this animal model.

As described above, Sca1⁺ cells are present in a variety of organs and tissues, of which bone marrow contains abundant stem cells [45–46]. Some studies have indicated that BM stem cells can differentiate into fibroblasts and participate in wound healing and fibrosis [47–49]. To explore the potential involvement of BM stem cells, two correlative types of chimeric mice were created through BMT. We confirmed that non-BM sources of Sca1⁺ cells play a key role in cardiac fibrosis, implying that heart tissue-resident Sca1⁺ stem/progenitor cells directly differentiate into fibroblasts to induce fibrosis.

Different signalling pathways leading to Sca1⁺ stem/progenitor cell differentiation have been identified [22]. For example, AKT/mTOR-dependent glycolysis is critical for endothelial differentiation [50], whereas smooth muscle cell differentiation from Sca1⁺ cells seem to involve the YAP signalling pathway [44]. In our study, we first examined the differential gene, Wnt4, among the groups when knocking down the expression of PDGFRA. After Wnt4 activation, a series of differentially expressed genes lead to PDGFRa activation, which plays a major role in the regulating of Sca1⁺ cell differentiation into fibroblasts. When Wnt4 was knocked down in Sca1⁺ cells, the expression levels of VIMENTIN, DDR2, POSTN, and PDGFRA were significantly reduced. Considering the impact of PDGFRa, we propose that Sca1⁺ cells differentiate into fibroblasts using the Wnt4 signalling pathway via PDGFRa. This result has important implications because Pdgfra knockdown in vivo alleviated the severity of fibrosis in our mouse model. Thus, these results provide a potential target for the treatment of myocardial fibrosis.

For limitations of this study, firstly, we did not explore deeply enough in mechanism, and examined the Wnt4-Pdgfra signalling pathway only in vitro. Secondly, we were concerned primarily fibroblasts that differentiated from Sca1⁺ cells, and might be lacking of deep exploration in the differentiation of other cell types. Thirdly, Sca1 expressed only in mice and not on human [51], which inevitably limited our results to extrapolate to human, therefore, it is necessary a further exploration on other MSC and cardiac progenitor/stem cell biomarkers such as c-Kit, Gli1 [43]. Fourthly, the method of cardiac function evaluation was limited.

In summary, our study provides evidence that Sca1⁺ cardiac resident stem/progenitor cells can differentiate into fibroblasts both in vitro and in vivo under specific conditions, and that they are of non-bone marrow origin. Mechanistically, we demonstrated that Wnt4-PDGFRa signalling pathways are critical for this process. Depletion of Sca1⁺ cells markedly reduced myocardial fibrosis in a mouse model, as did knockdown of Pdgfra of Sca1⁺ cells. Thus, the combination of genetic and biological evidence obtained in this study may provide new insights into cardiac stem cells and contribute to the development of therapeutic approaches for the treatment of cardiac fibrosis.

Supplementary material

Supplementary material is available at Cellular and Molecular Life Science online.

Acknowledgements

We are grateful to Ms. Chao Bi and Ms. Xiaoli Hong at the Core Facilities, Zhejiang University School of Medicine for technical assistance in confocal microscopy.

Funding

This work is supported by National Natural Science Foundation of China (82030008, 82270409, 31830039), Science and technology Project of Zhejiang Province (2023C03087).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation was performed by Tingting Tao, Yan Guo, Xuyang Wang. Data collection was performed by Peng Teng, Haige Zhao, Liang Ma. Analysis was performed by Luping Du. The first draft of the manuscript was written by Tingting Tao, Luping Du, Yanhua Hu, Qingbo Xu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

The single-cell RNA sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE198833 and GSE222144. The data underlying this article will be shared on reasonable request to the corresponding author.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (approval no. IIT20210018B-R2).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors affirm that human research participants provided informed consent for publication of the images in Figures 1L and Supplementary Figure 3A-B.

Emmons-Bell S, Johnson C, Roth G Prevalence, incidence and survival of heart failure: a systematic review. Heart. 2022 Aug 11;108(17):1351–1360. doi: 10.1136/heartjnl-2021-320131
Hall C, Gehmlich K, Denning C, Pavlovic D (2021 Feb) Complex Relationship Between Cardiac Fibroblasts and Cardiomyocytes in Health and Disease. J Am Heart Assoc 10(5):e019338. 10.1161/JAHA.120.019338Epub 2021 Feb 15
Travers JG, Kamal FA, Robbins J, Yutzey KE, Blaxall BC (2016 Mar) Cardiac Fibrosis: The Fibroblast Awakens. Circ Res 18(6):1021–1040. 10.1161/CIRCRESAHA.115.306565
Frangogiannis NG (2021) Cardiac fibrosis. Cardiovasc Res. May 25;117(6):1450–1488. doi: 10.1093/cvr/cvaa324
Frieler RA, Mortensen RM (2015 Mar) Immune cell and other noncardiomyocyte regulation of cardiac hypertrophy and remodeling. Circulation 17(11):1019–1030. 10.1161/CIRCULATIONAHA.114.008788
Tallquist MD (2020 Feb) Cardiac Fibroblast Diversity. Annu Rev Physiol 10:82:63–78. 10.1146/annurev-physiol-021119-034527
Moore-Morris T, Guimarães-Camboa N, Banerjee I, Zambon AC, Kisseleva T, Velayoudon A, Stallcup WB, Gu Y, Dalton ND, Cedenilla M, Gomez-Amaro R, Zhou B, Brenner DA, Peterson KL, Chen J, Evans SM (2014 Jul) Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest 124(7):2921–2934. 10.1172/JCI74783Epub 2014 Jun 17
Czubryt MP, Hale TM (2021 Sep) Cardiac fibrosis: Pathobiology and therapeutic targets. Cell Signal 85:110066. 10.1016/j.cellsig.2021.110066Epub 2021 Jun 17
Moore-Morris T, Guimarães-Camboa N, Yutzey KE, Pucéat M, Evans SM (2015 Aug) Cardiac fibroblasts: from development to heart failure. J Mol Med (Berl) 93(8):823–830. 10.1007/s00109-015-1314-yEpub 2015 Jul 14
Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, Gustafsson E, Chandraker A, Yuan X, Pu WT, Roberts AB, Neilson EG, Sayegh MH, Izumo S, Kalluri R (2007 Aug) Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 13(8):952–961. 10.1038/nm1613Epub 2007 Jul 29
Zhang L, Issa Bhaloo S, Chen T, Zhou B, Xu Q (2018) Role of Resident Stem Cells in Vessel Formation and Arteriosclerosis. Circ Res. 25(11):1608–1624. 10.1161/CIRCRESAHA.118.313058
Planat-Benard V, Silvestre JS, Cousin B, André M, Nibbelink M, Tamarat R, Clergue M, Manneville C, Saillan-Barreau C, Duriez M, Tedgui A, Levy B, Pénicaud L, Casteilla L (2004 Feb) Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 10(5):656–663. 10.1161/01.CIR.0000114522.38265.61Epub 2004 Jan 20
Liu Q, Huang X, Zhang H, Tian X, He L, Yang R, Yan Y, Wang QD, Gillich A, Zhou B (2015 Aug) c-kit(+) cells adopt vascular endothelial but not epithelial cell fates during lung maintenance and repair. Nat Med 21(8):866–868. 10.1038/nm.3888Epub 2015 Jul 13
van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD (2014 May) c-kit + cells minimally contribute cardiomyocytes to the heart. Nature 15(7500):337–341. 10.1038/nature13309Epub 2014 May 7
Gong H, Wang T, Xu Q (2021) Resident stem cells in the heart. Med Rev 1(1):10–13. https://doi.org/10.1515/mr-2021-0003
Holmes C, Stanford WL (2007 Jun) Concise review: stem cell antigen-1: expression, function, and enigma. Stem Cells. 25(6):1339–1347. 10.1634/stemcells.2006-0644
Uchida S, De Gaspari P, Kostin S, Jenniches K, Kilic A, Izumiya Y, Shiojima I, Grosse Kreymborg K, Renz H, Walsh K, Braun T (2013 Oct) Sca1-derived cells are a source of myocardial renewal in the murine adult heart. Stem Cell Reports 24(5):397–410. 10.1016/j.stemcr.2013.09.004
Tang J, Li Y, Huang X, He L, Zhang L, Wang H, Yu W, Pu W, Tian X, Nie Y, Hu S, Wang QD, Lui KO, Zhou B (2018 Dec) Fate Mapping of Sca1 + Cardiac Progenitor Cells in the Adult Mouse Heart. Circulation 18(25):2967–2969. 10.1161/CIRCULATIONAHA.118.036210
Jolly AJ, Lu S, Strand KA, Dubner AM, Mutryn MF, Nemenoff RA, Majesky MW, Moulton KS, Weiser-Evans MCM (2022 May) Heterogeneous subpopulations of adventitial progenitor cells regulate vascular homeostasis and pathological vascular remodelling. Cardiovasc Res 6(6):1452–1465. 10.1093/cvr/cvab174
Vagnozzi RJ, Sargent MA, Lin SJ, Palpant NJ, Murry CE, Molkentin JD (2018) Genetic Lineage Tracing of Sca-1 + Cells Reveals Endothelial but Not Myogenic Contribution to the Murine Heart. Circulation. Dec 18;138(25):2931–2939. doi: 10.1161/CIRCULATIONAHA.118.035210. Erratum in: Circulation. 2018 Oct 9;138(15):e424
Houlihan DD, Mabuchi Y, Morikawa S, Niibe K, Araki D, Suzuki S, Okano H, Matsuzaki Y (2012 Dec) Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α. Nat Protoc 7(12):2103–2111. 10.1038/nprot.2012.125Epub 2012 Nov 15
Roulis M, Kaklamanos A, Schernthanner M, Bielecki P, Zhao J, Kaffe E, Frommelt LS, Qu R, Knapp MS, Henriques A, Chalkidi N, Koliaraki V, Jiao J, Brewer JR, Bacher M, Blackburn HN, Zhao X, Breyer RM, Aidinis V, Jain D, Su B, Herschman HR, Kluger Y, Kollias G, Flavell RA (2020 Apr) Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche. Nature 580(7804):524–529. 10.1038/s41586-020-2166-3Epub 2020 Apr 1
Hu P, Zhang D, Swenson L, Chakrabarti G, Abel ED, Litwin SE (2003 Sep) Minimally invasive aortic banding in mice: effects of altered cardiomyocyte insulin signaling during pressure overload. Am J Physiol Heart Circ Physiol 285(3):H1261–1269. 10.1152/ajpheart.00108.2003Epub 2003 May 8
Du L, Sun X, Gong H, Wang T, Jiang L, Huang C, Xu X, Li Z, Xu H, Ma L, Li W, Chen T, Xu Q (2023 Feb) Single cell and lineage tracing studies reveal the impact of CD34 + cells on myocardial fibrosis during heart failure. Stem Cell Res Ther 20(1):33. 10.1186/s13287-023-03256-0
Haghverdi L, Lun ATL, Morgan MD, Marioni JC (2018 Jun) Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors. Nat Biotechnol 36(5):421–427. 10.1038/nbt.4091Epub 2018 Apr 2
McGinnis CS, Murrow LM, Gartner ZJ, DoubletFinder (2019 Apr) Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Syst 24(4):329–337e4. 10.1016/j.cels.2019.03.003Epub 2019 Apr 3
Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, Hao Y, Stoeckius M, Smibert P, Satija R (2019 Jun) Comprehensive Integration of Single-Cell Data. Cell 13(7):1888–1902e21. 10.1016/j.cell.2019.05.031Epub 2019 Jun 6
Buechler MB, Pradhan RN, Krishnamurty AT, Cox C, Calviello AK, Wang AW, Yang YA, Tam L, Caothien R, Roose-Girma M, Modrusan Z, Arron JR, Bourgon R, Müller S, Turley SJ (2021 May) Cross-tissue organization of the fibroblast lineage. Nature 593(7860):575–579. 10.1038/s41586-021-03549-5Epub 2021 May 12
Pu X, Zhu P, Zhou X, He Y, Wu H, Du L, Gong H, Sun X, Chen T, Zhu J, Xu Q, Zhang H (2022) CD34 + cell atlas of main organs implicates its impact on fibrosis. Cell Mol Life Sci. Oct 31;79(11):576. doi: 10.1007/s00018-022-04606-6
Qiu X, Hill A, Packer J, Lin D, Ma YA, Trapnell C (2017 Mar) Single-cell mRNA quantification and differential analysis with Census. Nat Methods 14(3):309–315. 10.1038/nmeth.4150Epub 2017 Jan 23
Trapnell C, Cacchiarelli D, Grimsby J, Pokharel P, Li S, Morse M, Lennon NJ, Livak KJ, Mikkelsen TS, Rinn JL (2014 Apr) The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol 32(4):381–386. 10.1038/nbt.2859Epub 2014 Mar 23
Plummer NW, Ungewitter EK, Smith KG, Yao HH, Jensen P (2017 Oct) A new mouse line for cell ablation by diphtheria toxin subunit A controlled by a Cre-dependent FLEx switch. Genesis 55(10). 10.1002/dvg.23067Epub 2017 Sep 19
Lee CH, Shah B, Moioli EK, Mao JJ (2010) Sep;120(9):3340–3349 CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J Clin Invest. doi: 10.1172/JCI43230. Epub 2010 Aug 2. Erratum in: J Clin Invest. 2015 Oct 1;125(10):3992
Maruyama K, Imanaka-Yoshida K The Pathogenesis of Cardiac Fibrosis: A Review of Recent Progress. Int J Mol Sci 2022 Feb 27;23(5):2617. doi: 10.3390/ijms23052617
Han M, Liu Z, Liu L, Huang X, Wang H, Pu W, Wang E, Liu X, Li Y, He L, Li X, Wu J, Qiu L, Shen R, Wang QD, Ji Y, Ardehali R, Shu Q, Lui KO, Wang L, Zhou B (2023 Apr) Dual genetic tracing reveals a unique fibroblast subpopulation modulating cardiac fibrosis. Nat Genet 55(4):665–678. 10.1038/s41588-023-01337-7
van Amerongen MJ, Bou-Gharios G, Popa E, van Ark J, Petersen AH, van Dam GM, van Luyn MJ, Harmsen MC (2008 Feb) Bone marrow-derived myofibroblasts contribute functionally to scar formation after myocardial infarction. J Pathol 214(3):377–386. 10.1002/path.2281
Tallquist MD, Molkentin JD (2017 Aug) Redefining the identity of cardiac fibroblasts. Nat Rev Cardiol 14(8):484–491. 10.1038/nrcardio.2017.57Epub 2017 Apr 24
Tang J, Zhu H, Liu S, Wang H, Huang X, Yan Y, Wang L, Zhou B Sca1 marks a reserve endothelial progenitor population that preferentially expand after injury. Cell Discov 2021 Sep 28;7(1):88. doi: 10.1038/s41421-021-00303-z
Hu X, Xu Y, Zhong Z, Wu Y, Zhao J, Wang Y, Cheng H, Kong M, Zhang F, Chen Q, Sun J, Li Q, Jin J, Li Q, Chen L, Wang C, Zhan H, Fan Y, Yang Q, Yu L, Wu R, Liang J, Zhu J, Wang Y, Jin Y, Lin Y, Yang F, Jia L, Zhu W, Chen J, Yu H, Zhang J, Wang J (2016 Mar) A Large-Scale Investigation of Hypoxia-Preconditioned Allogeneic Mesenchymal Stem Cells for Myocardial Repair in Nonhuman Primates: Paracrine Activity Without Remuscularization. Circ Res 18(6):970–983. 10.1161/CIRCRESAHA.115.307516Epub 2016 Jan 19
Plikus MV, Wang X, Sinha S, Forte E, Thompson SM, Herzog EL, Driskell RR, Rosenthal N, Biernaskie J, Horsley V Fibroblasts: Origins, definitions, and functions in health and disease. Cell 2021 Jul 22;184(15):3852–3872. doi: 10.1016/j.cell.2021.06.024
Soliman H, Theret M, Scott W, Hill L, Underhill TM, Hinz B, Rossi FMV Multipotent stromal cells: One name, multiple identities. Cell Stem Cell 2021 Oct 7;28(10):1690–1707. doi: 10.1016/j.stem.2021.09.001
Torsney E, Xu Q (2011 Feb) Resident vascular progenitor cells. J Mol Cell Cardiol 50(2):304–311. 10.1016/j.yjmcc.2010.09.006
Ni Z, Lyu L, Gong H, Du L, Wen Z, Jiang H, Yang H, Hu Y, Zhang B, Xu Q, Guo X, Chen T (2023) Multilineage commitment of Sca-1 + cells in reshaping vein grafts. Theranostics 13(7):2154–2175. 10.7150/thno.77735
Tang J, Wang H, Huang X, Li F, Zhu H, Li Y, He L, Zhang H, Pu W, Liu K, Zhao H, Bentzon JF, Yu Y, Ji Y, Nie Y, Tian X, Zhang L, Gao D, Zhou B (2020) Arterial Sca1⁺ Vascular Stem Cells Generate De Novo Smooth Muscle for Artery Repair and Regeneration. Cell Stem Cell. Jan 2;26(1):81–96.e4. doi: 10.1016/j.stem.2019.11.010. Epub 2019 Dec 27
Ito CY, Li CY, Bernstein A, Dick JE, Stanford WL Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice. Blood 2003 Jan 15;101(2):517–523. doi: 10.1182/blood-2002-06-1918. Epub 2002 Aug 29.
Morcos MNF, Schoedel KB, Hoppe A, Behrendt R, Basak O, Clevers HC, Roers A, Gerbaulet A (2017 Jun) SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells. Stem Cell Reports 6(6):1472–1478 Epub 2017 May 11
assone NM, Li B, Patel MS, Devine MY, Firmiss PR, Gould AD, Kochan KS, Stubbee RA, Bowen DK, Dettman RW, Gong EM (2019) Stem cell antigen/Ly6a protects against bladder fibrosis in mice. Am J Physiol Renal Physiol. Dec 1;317(6):F1503-F1512. doi: 10.1152/ajprenal.00160.2019. Epub 2019 Sep 18
Walter DH, Haendeler J, Reinhold J, Rochwalsky U, Seeger F, Honold J, Hoffmann J, Urbich C, Lehmann R, Arenzana-Seisdesdos F, Aicher A, Heeschen C, Fichtlscherer S, Zeiher AM, Dimmeler S (2005 Nov) Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease. Circ Res 25(11):1142–1151. 10.1161/01.RES.0000193596.94936.2cEpub 2005 Oct 27
Zhou H, Bian ZY, Zong J, Deng W, Yan L, Shen DF, Guo H, Dai J, Yuan Y, Zhang R, Lin YF, Hu X, Li H, Tang QZ (2012 Sep) Stem cell antigen 1 protects against cardiac hypertrophy and fibrosis after pressure overload. Hypertension 60(3):802–809. 10.1161/HYPERTENSIONAHA.112.198895Epub 2012 Jul 30
Deng J, Ni Z, Gu W, Chen Q, Nowak WN, Chen T, Issa Bhaloo S, Zhang Z, Hu Y, Zhou B, Zhang L, Xu Q (2020 Dec) Single-cell gene profiling and lineage tracing analyses revealed novel mechanisms of endothelial repair by progenitors. Cell Mol Life Sci 77(24):5299–5320. 10.1007/s00018-020-03480-4Epub 2020 Mar 13
van de Rijn M, Heimfeld S, Spangrude GJ, Weissman IL (1989 Jun) Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci U S A 86(12):4634–4638. 10.1073/pnas.86.12.4634

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Non-bone marrow-derived Sca1⁺ cells differentiate into ﬁbroblasts involved in cardiac fibrosis via Wnt4-PDGFRα pathway.
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Stem cell antigen-1 cell-derived fibroblasts are crucial for cardiac fibrosis during heart failure

Status:

Journal Publication

Version 1

Abstract

Aims

Methods and Results

Conclusions

Graphical abstract:

Figures

1. Introduction

2. Materials and Methods

2.1 Animals

2.2 Anaesthetics and analgesia

2.3 Animal models and echocardiography

2.4 Human tissue

2.5 Processing of single-cell RNA-sequencing data

2.6 Statistical analysis

3. Results

3.1 ScRNA-seq analyses of human hearts

3.2 ScRNA-seq analyses of murine hearts.

3.3 Sca1⁺ cells can differentiate towards fibroblasts in vivo

3.5 Sca1⁺ cells can differentiate towards fibroblasts in vitro

3.6 Role of Wnt4 activation in the differentiation of Sca1⁺ cells

3.7 Sca1⁺ cells play a role in myocardial fibrosis significantly related to PDGFRα

4. Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Stem cell antigen-1 cell-derived fibroblasts are crucial for cardiac fibrosis during heart failure

Status:

Journal Publication

Version 1

Abstract

Aims

Methods and Results

Conclusions

Graphical abstract:

Figures

1. Introduction

2. Materials and Methods

2.1 Animals

2.2 Anaesthetics and analgesia

2.3 Animal models and echocardiography

2.4 Human tissue

2.5 Processing of single-cell RNA-sequencing data

2.6 Statistical analysis

3. Results

3.1 ScRNA-seq analyses of human hearts

3.2 ScRNA-seq analyses of murine hearts.

3.3 Sca1+ cells can differentiate towards fibroblasts in vivo

3.5 Sca1+ cells can differentiate towards fibroblasts in vitro

3.6 Role of Wnt4 activation in the differentiation of Sca1+ cells

3.7 Sca1+ cells play a role in myocardial fibrosis significantly related to PDGFRα

4. Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

3.3 Sca1⁺ cells can differentiate towards fibroblasts in vivo

3.5 Sca1⁺ cells can differentiate towards fibroblasts in vitro

3.6 Role of Wnt4 activation in the differentiation of Sca1⁺ cells

3.7 Sca1⁺ cells play a role in myocardial fibrosis significantly related to PDGFRα