An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium

An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium View ORCID Profile Claudia Müller-Sánchez , View ORCID Profile María Gertrudis Muñiz-Banciella , View ORCID Profile Manuel Reina , View ORCID Profile Francesc X. Soriano , View ORCID Profile Ofelia M. Martínez-Estrada doi: https://doi.org/10.1101/2025.05.23.655755 Claudia Müller-Sánchez 1 Celltec-UB, Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, University of Barcelona , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Claudia Müller-Sánchez For correspondence: cmuller{at}ub.edu ofeliamartinez{at}ub.edu María Gertrudis Muñiz-Banciella 1 Celltec-UB, Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, University of Barcelona , Barcelona, Spain 2 Institute of Biomedicine (IBUB), University of Barcelona , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for María Gertrudis Muñiz-Banciella Manuel Reina 1 Celltec-UB, Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, University of Barcelona , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Manuel Reina Francesc X. Soriano 1 Celltec-UB, Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, University of Barcelona , Barcelona, Spain 3 Institut de Neurociències, Universitat de Barcelona , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francesc X. Soriano Ofelia M. Martínez-Estrada 1 Celltec-UB, Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, University of Barcelona , Barcelona, Spain 2 Institute of Biomedicine (IBUB), University of Barcelona , Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ofelia M. Martínez-Estrada For correspondence: cmuller{at}ub.edu ofeliamartinez{at}ub.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Embryonic epicardium is a major source of cardiac fibroblasts (CFs), which play essential roles in heart development and response to heart injury. In this study, we developed a novel mouse model to identify distinct populations of epicardium-derived CFs. Our Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP model enables lineage tracing of WT1Cre-labeled (RFP + ) fibroblasts and the identification of cells actively expressing WT1 (GFP + ). Flow cytometry at early postnatal stages showed that RFP + cells form a heterogeneous stromal population, with 20.13% co-expressing GFP, indicating persistent WT1 expression in a subset. We successfully immortalized RFP + cardiac stromal cells, highly enriched in fibroblasts, by excluding other Wt1Cre-active cell types. Through culture condition optimization, we could selectively expand or differentiate specific fibroblast subpopulations, increasing the model’s utility. These immortalized cells, carrying an integrated WT1 reporter system, provide a robust in vitro platform to study fibroblast activation, differentiation, and plasticity under defined conditions. Summary blurb This study presents a novel in vitro mouse model for investigating the activation, identity, and functional properties of epicardium-derived cardiac fibroblasts. INTRODUCTION Cardiac fibroblasts (CFs) are essential non-myocyte cells that play a crucial role in heart development and homeostasis ( Tallquist, 2018 ). They are among the most abundant non-cardiomyocyte cell types in the postnatal heart and are responsible for extensive extracellular matrix (ECM) deposition. Beyond their structural role, CFs produce paracrine factors critical to heart development, myocardial growth, functional maturation, and blood vessel formation ( Acharya et al, 2012 ; Hortells et al, 2020 ; Ieda et al, 2009 ). They are also central to the formation of pathological fibrosis, a hallmark of various cardiovascular diseases, by promoting excessive ECM deposition and tissue remodeling ( Humeres & Frangogiannis, 2019 ). The understanding of CF biology has advanced significantly over the past decade ( Tallquist & Molkentin, 2017 ). Although several origins have been proposed, lineage tracing studies in mice have unequivocally demonstrated that adult resident CFs primarily arise from the embryonic epicardium and endocardium, with the epicardium being the predominant source ( Moore-Morris et al, 2014 ; Tallquist, 2018 ). Notably, the population of resident CFs of epicardial origin expands in response to adult cardiac fibrosis, such as that induced by pressure overload, fibroelastosis, or myocardial infarction ( Forte et al, 2020 ; Moore-Morris et al, 2014 ; Ruiz-Villalba et al, 2015 ; Zhang et al, 2017 ). Compared with other cell types, the absence of unique fibroblast markers, coupled with their inherent heterogeneity, has made the isolation of fibroblasts particularly challenging when using conventional techniques such as fluorescence-activated cell sorting (FACS) ( Tallquist & Molkentin, 2017 ). Various genetic mouse models have, therefore, been developed to facilitate fibroblast identification. Some models employ direct fibroblast reporter systems, such as the Pdgfra-GFP and Collagen1a1-GFP reporter lines ( Hamilton et al, 2003 ; Yata et al, 2003 ). Additionally, Cre transgenic mouse models have significantly improved the identification and characterization of CFs ( Acharya et al, 2011 ; Tallquist & Molkentin, 2017 ; Wessels et al, 2012 ; Zhang et al, 2008 ; Zhou et al, 2008 ). The WT1 gene encodes a zinc finger protein essential for heart development ( Hastie, 2017 ; Moore et al, 1999 ). WT1 is highly expressed in the epicardium and epicardial derived cells (EPDCs) during heart development, where it plays a critical role in their formation and function ( Martinez-Estrada et al, 2010 ; Moore et al, 1999 ; Velecela et al, 2019 ). Epicardial derivatives, such as CFs, also express WT1 both during heart development and following myocardial infarction ( Braitsch et al, 2013 ; Perez-Pomares et al, 2002 ; Zhou et al, 2011 ). These data, originally identified by immunostaining, have been validated by several transcriptomic studies( Deng et al, 2025 ; Feng et al, 2022 ). Both constitutive and inducible Wt1Cre mouse models have been generated and used to study fibroblast formation and expansion during development ( Wessels et al, 2012 ; Zhou et al, 2008 ), and these models have become essential tools for investigating resident fibroblasts of epicardial origin in heart diseases ( Forte et al, 2020 ; Moore-Morris et al, 2018 ; Ruiz-Villalba et al, 2015 ). In this study, we used a constitutive BAC Wt1Cre transgenic mouse model, which efficiently labels resident CFs, and generated a new Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model. This includes WT1 lineage cells that are RFP-positive (RFP + ), while cells actively expressing WT1 are GFP-positive (GFP + ). We subsequently established immortalized CFs from this model, which represent a valuable and versatile in vitro tool for dissecting the molecular mechanisms underlying fibroblast activation, differentiation, and plasticity. RESULTS Characterizing cardiac cells from the Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model Although several studies have used Wt1Cre mouse models to study fibroblast formation during cardiac development and disease states, none have combined lineage tracing with a Wt1 reporter model to identify both Wt1Cre progeny and Wt1-expressing cells simultaneously, which can be used to FACS-isolate different populations of Wt1Cre positive cells ( Forte et al, 2020 ; Moore-Morris et al, 2014 ; Ruiz-Villalba et al, 2015 ; Wessels et al, 2012 ). We aimed to generate such a model by crossbreeding Wt1Cre;ROSA26-tdRFP mice with Wt1 GFP/+ mice ( Fig. 1A ). Download figure Open in new tab Figure 1. Generation and characterization of freshly isolated cardiac cells from the Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model. (A) Overview of the protocol for enzymatic digestion and flow cytometry analysis of heart ventricles from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP. (B) Flow cytometry analysis of enzymatically digested P2 heart ventricles from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice reveals that a subset of RFP + cells retains sustained GFP expression. Littermate negative mice were used to establish gating parameters. (C, D) Representative flow cytometry plots for identification of RFP + subsets and corresponding quantification. Data are presented as mean ± s.e.m. (n = 4). In the Wt1 GFP/+ model, GFP is knocked into exon 1 and driven by the endogenous Wt1 regulatory sequences, providing a GFP reporter that faithfully reflects Wt1 expression ( Hosen et al, 2007 ; Velecela et al, 2019 ). In the new model, Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice contain RFP-positive (RFP + ) cells that exhibiting Cre recombinase activity, along with their progeny, while GFP-positive (GFP + ) cells express abundant WT1 levels. Flow cytometry analysis of ventricular cardiac cells at early postnatal stages showed that RFP + cells represented 13.75% of the total cardiac cell population. Of these, 20.13% were GFP + , indicating sustained WT1 expression in a cell subset ( Fig. 1A, B , Table 1 , 2 ). View this table: View inline View popup Download powerpoint Table 1. Percentage of RFP-positive cells relative to the total number of cells, determined by flow cytometry. Data are mean ± s.e.m. (n=4). View this table: View inline View popup Download powerpoint Table 2. Percentage of cells expressing the indicated markers within the RFP-positive cell population, determined by flow cytometry. Data are mean ± s.e.m. (n=4). To further characterize the RFP + population, we employed a panel of several antibodies. This showed that 25.83% of RFP + cells expressed CD31 and 27.25% expressed CD45, confirming the presence of endothelial and hematopoietic cells within the RFP + population, consistent with previous reports ( Fig. 1C, D , Table 2 ) ( Cano et al, 2016 ; Pogontke et al, 2022 ). Notably, 69.61% of the RFP + CD45 + cells and 76.34% of the RFP + CD31 + cells co-expressed GFP ( Table 3 ). We also included markers commonly used to identify the stromal cell populations GP38, CD90, and CD39. Among the RFP + cells, 73.25% were GP38 + , 18.03% were CD90 + , and 29.03% were CD39 + ( Fig. 1C, D , Table 2 ). Of note, the percentages of GFP + cells varied among these populations, accounting for 28.91% of GP38 + , 36.46% of CD90 + , and 71.04% of CD39 + cells ( Table 4 ). View this table: View inline View popup Download powerpoint Table 3. Percentage of GFP cells within the RFP+ CD31+ and RFP+ CD45+ cell population, determined by flow cytometry. Data are mean ± s.e.m. (n=4). View this table: View inline View popup Download powerpoint Table 4. Percentage of cells expressing the indicated markers within the RFP-positive cell population, determined by flow cytometry. Data are mean ± s.e.m. (n=4). Taken together, these results demonstrate that the Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model allows the identification of different populations of Wt1Cre-positive stromal cells based on GFP expression. Immortalization of cardiac cells in the Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model Most studies using CF cultures rely on selective attachment to isolate the cell population, often without additional enrichment strategies ( Garvin & Katwa, 2023 ). In this study, we FACS-isolated RFP + ventricular cardiac stromal cells from the Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model, excluding endothelial (RFP + , CD31 + , CD45 − ), hematopoietic (RFP + , CD31 − , CD45 + ), and high GFP-expressing cells, thereby including epicardial cells, among others ( Fig. 2A ). The sorted RFP + cells were cultured on poly-L-lysine-coated plates, where they exhibited a flattened, irregular morphology with abundant cytoplasmic protrusions ( Fig. 2B ). Next, the RFP + cells were immortalized using a viral vector containing the SV40 large T antigen (SV40 TAg), which is well-recognized for its robust cell immortalization capability. Following puromycin selection, the immortalized cells exhibited a more uniform morphology with abundant cytoplasmic protrusions ( Fig. 2C ). Immunofluorescence analysis confirmed uniform expression of vimentin, a key fibroblast-specific marker, in all cells, along with the presence of some patches of smooth muscle actin (SMA)-positive cells ( Fig. 2D, E ). Download figure Open in new tab Figure 2. Generation of immortalized cardiac cells from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice. (A) Workflow for the strategy used to generate and immortalize postnatal RFP + cardiac fibroblasts from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice excluding endothelial cells (RFP + ,CD31 + , CD45 - ), hematopoietic cells (RFP + CD31 - ,CD45 + ), and cells with the highest GFP expression (RFP + ,GFP ++ ). (B, C) Bright-field image of primary (B) and immortalized (C) cardiac fibroblasts. (D) Immunofluorescence staining for the intermediate filament vimentin, demonstrating abundant vimentin expression in immortalized cells. (E) Immunofluorescence staining for SMA (green) and phalloidin (red), exhibiting some patches of SMA-positive cells. Scale bars: 200 µm in (B, C); 50 µm in (D, E). These results validate the successful immortalization of FACS-sorted RFP + cardiac stromal cells. Characterization of cardiac RFP + immortalized cells derived from the Wt1 GFP/+ ;Wt1Cre;R26-tdRFP mouse model Generating immortalized cells allows the study of CF signaling, proliferation, and activation with less need to use animals. This can be achieved because the immortalized cells remain proliferative across multiple passages without showing signs of senescence. Next, we aimed to characterize the RFP-immortalized cells in detail. Flow cytometry confirmed uniform RFP expression across all cells, with 15.49% expressing GFP ( Fig. 3A , Table 5 ). When we included the antibodies used for in vivo analysis, the flow cytometry revealed that 97.40% of the RFP + immortalized cells expressed GP38, 63.12% expressed CD90, and 5.87% expressed CD39 ( Fig. 3A , Table 5 ). Consistent with in vivo observations, the proportion of GFP + cells varied among subpopulations: 10.14%, 11.61%, and 29.98% among the RFP + GP38 + , RFP + CD90 + , and RFP + CD39 + subpopulations, respectively ( Fig. 3B , Table 6 ). Given that none of these markers are specific to fibroblasts, and that previous studies have shown the Wt1Cre line can recombine in other cell types, we assessed the expression of genes enriched in other cardiac cell populations, including Des (smooth muscle cells), Rgs5 (pericytes), and Myh6 (cardiomyocytes) ( Fig. 3C ). We also examined the expression of genes highly expressed in the epicardium, such as Msln and Upk3b ( Fig. 3D ). The qRT-PCR analysis revealed that immortalized and sorted RFP + cells exhibited undetectable levels of these non-fibroblast cell markers and abundant Vim expression ( Fig. 3C, D ). Since the vast majority of the immortalized cells were GP38 + CD39 − , we FACS-sorted four distinct cell subpopulations based on CD90 and GFP expression: CD90 + GFP + , CD90 + GFP − , CD90 − GFP + , and CD90 − GFP − . Next, we decided to compare the potential contribution of each of these cell populations to the SMA-positive patches observed in the cell culture. Interestingly, qRT-PCR analysis revealed that the CD90 + GFP − subpopulation expressed significantly higher levels of Acta2 compared to the other subpopulations ( Fig. 3E, F ). View this table: View inline View popup Download powerpoint Table 5. Percentage of immortalized cells expressing the indicated markers within the RFP-positive cell population, determined by flow cytometry. Data are mean ± s.e.m. (n=3). View this table: View inline View popup Download powerpoint Table 6. Percentage of immortalized cells at expressing the indicated markers within the RFP-positive cell population, determined by flow cytometry. Data are mean ± s.e.m. (n=3). Download figure Open in new tab Figure 3. Characterization of immortalized cardiac fibroblasts from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mouse model. (A) Representative flow cytometry plots showing the identification of RFP + cardiac fibroblast subsets (B) Representative flow cytometry plots for identification of GFP + subsets. (C, D) qRT-PCR analysis of the indicated genes in immortalized cardiac cells from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice. Compared with total heart tissue or immortalized epicardial cells (Epi), immortalized fibroblasts exhibited undetectable expression of markers specific to smooth muscle cells, pericytes, cardiomyocytes, and epicardial cells. Data are presented as mean ± s.e.m. (n = 3); **P < 0.001, ***P < 0.0001 two-tailed student t-test. (E) Schematic representation of the FACS-based isolation of four fibroblast subpopulations defined by CD90 and GFP expression. (F) qRT–PCR analysis of Acta2 expression in these subsets revealed elevated Acta2 levels in the CD90 + GFP − population. Data are presented as mean ± s.e.m. (n = 6); ***P < 0.0001, One-way ANOVA followed by Tukey’s post-hoc test. Collectively, these findings demonstrate that the RFP + cells represent a heterogeneous population of CFs. Transforming growth factor-beta (TGF-β) modulates the fibrotic response in RFP + immortalized CFs Increased SMA expression is a widely used marker for identifying fibroblast activation ( Garvin & Katwa, 2023 ). After observing SMA-positive patches in our culture, we sought conditions to prevent this activation. Commercial fibroblast-specific media reduce serum concentration, which helps prevent spontaneous activation. As expected, CFs cultured in this medium exhibited lower Acta2 expression compared to those cultured in DMEM ( Fig. 4A ). Interestingly, this reduction in Acta2 expression was accompanied by an increase in the CD90 + GFP + and CD90 − GFP + populations, along with a decrease in the CD90 + GFP − and CD90 − GFP − populations ( Fig. 4B, C ). Download figure Open in new tab Figure 4. TGF-β induces a profibrotic phenotype in RFP + cardiac fibroblasts. (A) qRT–PCR analysis of Acta2 expression in RFP + cardiac fibroblasts maintained in DMEM or fibroblast medium (FM). Data are presented as mean ± s.e.m. (n = 4); ***P < 0.0001 two-tailed student t-test. (B) Representative flow-cytometry plots of RFP + cardiac fibroblasts cultured in FM. (C) Quantification of CD90/GFP fibroblast subsets (CD90 + GFP + , CD90 + GFP − , CD90 − GFP + , CD90 − GFP − ) in DMEM versus FM. Data are presented as mean ± s.e.m. (n = 3), **P < 0.001, ***P < 0.0001, two-tailed student t-test ( D, E ) Western blot and densitometric analysis of SMAD2 phosphorylation (pSMAD2) in cardiac fibroblasts treated with TGF-β, with total SMAD2 serving as the loading control. Data are presented as mean ± s.e.m. (n = 3); ***P < 0.0001, one-way ANOVA followed by Tukey’s post-hoc test. ( F) qRT– PCR analysis of the indicated genes in cardiac fibroblasts cultured with TGF-β for 3 days. Data are presented as mean ± s.e.m. (n = 3), *P < 0.05, **P < 0.001, two-tailed student t-test. After successfully controlling the activation of our cell culture, our next objective was to investigate the effect of TGF-β, a central regulator of tissue fibrosis, and assess whether our model could serve as an ideal system for studying cardiac fibrosis in vitro ( Deng et al, 2024 ). Western blot analysis revealed a robust increase in Smad2 phosphorylation in response to TGF-β, confirming the activation of the TGF-β-SMAD signaling cascade ( Fig. 4D, E ). To further characterize the fibrotic response of CFs to TGF-β, we examined the expression of two key fibrosis markers, Acta2 and Col1a1 . qRT-PCR analysis demonstrated a significant upregulation of Acta2 and Col1a1 expression in TGF-β-treated CFs, highlighting the induction of a fibrotic phenotype. In contrast, the expression of Vim , a pan-fibroblast marker, remained unchanged, suggesting that the observed changes were specific to a fibrotic phenotype ( Fig. 4F ). These findings collectively demonstrate that the immortalized CFs constitute a robust and reliable model for studying stimulus-induced fibrotic activation in CFs. DISCUSSION We developed a novel mouse model that enables distinct cardiac stromal cell populations, including resident fibroblasts, to be isolated based on WT1 reporter expression. The Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP model facilitates both the lineage tracing of cells labeled with Wt1Cre (RFP + ) and the identification of cells actively expressing WT1 (GFP + ). We generated an immortalized population highly enriched in different CF subtypes that retained RFP expression and expressed key fibroblast-specific markers, such as vimentin. The integrated GFP reporter enabled the isolation and functional analysis of distinct subpopulations within the Wt1Cre lineage. During cardiac development, a subset of epicardial cells undergoes epithelial-to-mesenchymal transition, giving rise to EPDCs. These cells invade the subepicardial matrix before migrating into the myocardium, where they differentiate into various cardiac lineages, including most CFs ( Gittenberger-de Groot et al, 1998 ; von Gise & Pu, 2012 ). During early postnatal (P) development, CFs undergo significant expansion via cell proliferation, which dramatically decreases after P7 ( Ieda et al, 2009 ; Wu et al, 2020 ). While several mechanisms that regulate the initial formation and differentiation of EPDCs have been elucidated, the molecular pathways controlling CF proliferation and terminal differentiation remain largely unknown ( Tallquist, 2020 ; von Gise & Pu, 2012 ). Given these limitations, the immortalized CFs generated in this study offer a valuable platform for screening candidate pathways involved in fibroblast function. Indeed, such analyses are typically difficult to perform using primary cultures because they contain limited numbers of cells in each identified subpopulation, hindering in-depth cellular and molecular studies. The utility of this model also extends beyond developmental studies, because the adult mammalian heart has a limited capacity for regeneration. Healing typically occurs through scar formation, in which resident fibroblasts of epicardial origin play a central role ( Tallquist, 2020 ). Although this response is essential for maintaining structural integrity and preventing ventricular rupture following injury, excessive or dysregulated activation can result in adverse cardiac remodeling, reduced contractile function, and ultimately, heart failure ( Moore-Morris & Evans, 2023 ; Quijada et al, 2019 ). Notably, gene expression profiling has revealed that proliferating fibroblasts following MI closely resemble neonatal fibroblasts underscoring the relevance of our model ( Kretzschmar et al, 2018 ). Understanding the regulatory mechanisms that govern the expansion and differentiation of CFs into specific subpopulations could help develop therapeutic strategies that mitigate the effects of pathological fibrosis while preserving essential wound healing processes. WT1 is a transcription factor essential for development and tissue homeostasis ( Hastie, 2017 ). Recent studies have highlighted its profibrotic role in lung fibrosis; however, WT1 deletion in the liver has also been shown to exacerbate fibrogenesis following injury ( Kendall et al, 2019 ; Sontake et al, 2018 ). In the heart, studies using a zebrafish model of myocardial injury have shown that wt1a:GFP + cells upregulate a fibrotic gene program in response to damage. After analyzing different scRNA-seq datasets in mammals, WT1 has been classified as an early transcriptional regulator following myocardial infarction ( Patrick et al, 2024 ; Sanchez-Iranzo et al, 2018 ). Despite these findings, the contribution of Wt1-expressing fibroblasts to the early stages of fibroblast activation and scar tissue formation after myocardial infarction remains unclear. Interestingly, CD90 + GFP − cells exhibited higher Acta2 expression than CD90 + GFP + cells. Based on this, we hypothesize that, similar to TCF21, WT1 functions as a repressor of fibroblast activation and acquisition of a myofibroblast phenotype ( Johansen et al, 2025 ). Currently, no effective pharmacological treatment for cardiac fibrosis exists, posing a major challenge in cardiovascular medicine. Our immortalized cells represent the first specific model of epicardial-origin CFs, offering a unique and valuable platform for studying the molecular mechanisms underlying cardiac fibrosis. Given their robustness and relevance, our model could prove highly effective for high-throughput drug screening, aiding in the identification of novel therapeutic agents that may reverse or alleviate cardiac fibrosis. Furthermore, the ability to genetically modify these cells facilitates the development of reporter-specific models, providing deeper insights into the cellular and molecular processes driving fibrosis in the heart and paving the way for more targeted, effective treatments. In summary, we have developed a versatile model for studying CFs of epicardial origin. By adjusting the culture conditions, specific fibroblast subpopulations can be selectively promoted to enhance the model’s flexibility. The choice of conditions should be guided by the physiologically relevant context and desired subpopulation, which will allow for not only screening GFP expression but also for identifying treatment combinations that can modulate the proportions of different cell subpopulations. In turn, this can provide deeper insights into the functional roles of these cells. MATERIAL AND METHODS Animal models The Wt1 GFP/+ , Wt1Cre (Tg(Wt1-cre)#Jbeb), and ROSA26-tdRFP mice have been described previously ( Hosen et al, 2007 ; Luche et al, 2007 ; Wessels et al, 2012 ). To generate Wt1Cre;ROSA26-tdRFP mice, male Wt1Cre mice were mated with ROSA26dtRFP females. To generate Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice, Wt1Cre;ROSA26-tdRFP males were mated with Wt1 GFP/+ females. The primers used for genotyping are listed in Table 7 . View this table: View inline View popup Download powerpoint Table 7. List of PCR primers used for genotyping All animal experiments were carried out in accordance with the regulations of the Animal Experimentation Ethics Committee (CEEA) of the University of Barcelona (ID C121CG2V6), thereby complying with current Spanish and European legislation. Flow cytometry analysis Flow cytometry analyses were performed on freshly isolated cells from the cardiac ventricles of mice or on immortalized cells. For the in vivo analyses, early postnatal heart ventricles from Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice and littermate negative controls were digested in 1 mg/mL collagenase type I solution (Worthington, 9001-12-1) for 30 minutes at 37°C, using a shaking block set to 1000 rpm (Eppendorf Thermomixer Compact). Collagenase activity was stopped by washing the cells with Dulbecco’s Modified Eagle Medium (DMEM) (Gibco,11960044) containing 10% fetal bovine serum (FBS; Capricorn scientific, FBS-12B). Subsequently, the cells were then pelleted by centrifugation at 300 ×g for 5 minutes and filtered through a 40 μm cell strainer (Corning, 352340) before being resuspended in Hanks’ Balanced Salt Solution (HBSS Ca 2+ , Mg 2+ ; Gibco, 14025092) containing 2% FBS (FACs buffer) ( Ramiro-Pareta et al, 2023 ; Torres-Cano et al, 2022 ). For antibody staining, freshly isolated or immortalized cells were incubated with primary monoclonal or isotype control antibodies at 4°C for 30 minutes ( Table 8 ) ( Ramiro-Pareta et al, 2023 ). Samples were washed twice and resuspended in FACs buffer. Flow cytometry analysis was carried out using a Cytek® Aurora 4L (Cytek) and data were analyzed using FlowJo™ software (v10.10). Cell gating of the corresponding fluorescent protein was carried out using samples negative for the fluorescent transgenic protein and isotype control antibodies. View this table: View inline View popup Download powerpoint Table 8. List of antibodies used for flow cytometry analysis Immortalization of RFP+ cardiac cells RFP + cells were isolated by FACS (FACSAria™ Fusion I, BD) from the enzymatically digested hearts of Wt1 GFP/+ ;Wt1Cre;ROSA26-tdRFP mice, excluding endothelial (RFP + , CD31 + , CD45 − ), hematopoietic (RFP + , CD31 − , CD45 + ), and epicardial (RFP + , GFP ++ ) cell populations. The sorted RFP + cells were subsequently cultured on 24-well plates coated with poly-L-lysine (Sigma, P7886) in completed cell culture medium (DMEM high-glucose; Gibco, 11960044) supplemented with L-glutamine (Corning, 25-005-CV), 100 µg/mL streptomycin, 100 U/mL penicillin (Gibco, 15140122), and 10% FBS (Capricorn Scientific, FBS-12B) until reaching approximately 70% confluence. To induce immortalization, cells were incubated overnight at 37°C with 1.2 µL of SV40 viral vector supernatant (GeneCopoeia, LP721-025). After 24 hours, the supernatant was removed and fresh culture medium was added. Cells were then cultured for an additional 21 days in presence of puromycin (Sigma, P8833). Non-transduced cells served as controls for the immortalization process. Once confluence was achieved, cells were trypsinized and designated as passage one (p1). Fibroblast medium and TGF-β treatment conditions Immortalized cells were maintained in complete DMEM culture medium and passaged every 4 to 5 days upon reaching 70% confluency. To assess the impact of fibroblast medium on cellular phenotype and marker expression stability, cells were cultured in a commercially available fibroblast medium (Promocell, C-23025) for three passages, followed by subsequent analyses. Cells between passages 6 and 16 were used for all experiments. To investigate the effect of TGF-β stimulation, cells were cultured at a density of 10,000 cells/cm 2 in fibroblast growth medium for 24 hours, followed by overnight incubation in Opti-MEM medium. TGF-β (10 ng/mL) was then applied for 1, 2 or 72 hours, after which samples were collected for marker analysis by Western blot or qPCR, respectively. Isolation of RNA and real-time PCR Total RNA from mixed cultures or FACS-sorted subpopulations was isolated using the PureLink RNA Mini Kit (Invitrogen, Cat. No. 12183025) according to the manufacturer’s instructions. The purified RNA was reverse transcribed into cDNA using UltraScript® Reverse Transcriptase (PCR Biosystems, PB30.11-10). Relative gene expression levels were quantified using SYBR Master Mix (Promega, A6002) and real-time PCR was performed on 96-well QuantStudio 3 (AppliedBiosystems) plates using the Connect analysis software (Thermo Fisher). The primers used are listed in Table 9 . View this table: View inline View popup Table 9. qRT-PCR primer list Immunofluorescence Immortalized cells were seeded on 12 mm glass coverslips (6,000 cells/cm 2 ) and cultured for 48 hours in complete DMEM culture medium or fibroblast growth medium. Cells were then fixed with 4% paraformaldehyde (Sigma, 158127) for 15 minutes, washed with phosphate buffered saline (PBS), permeabilized with 0.1% Triton X-100 (Sigma, 9036-19-5) for 5 minutes, washed again with PBS, and blocked with PBS supplemented with 1% bovine serum albumin (Sigma, 810664) for 1 hour at room temperature. After blocking, cells were incubated overnight at 4°C with primary antibodies against vimentin and SMA along with phalloidin, to stain filamentous actin ( Table 10 ). Then, the cells were incubated with fluorochrome-conjugated secondary antibodies ( Table 10 ) for 1 hour at 37°C. Cell nuclei were then stained with DAPI (Sigma, D9542). Immunofluorescence images were obtained using a fluorescence microscope (Leica Thunder). View this table: View inline View popup Download powerpoint Table 10. List of primary and secondary antibodies used for immunofluorescence. Western Blot For western blot analyses, control and cells treated with TGF-β (10 ng/mL for 1 and 2 hours) were lysed using SDS buffer (1.5 M Tris pH 6.8, 15% glycerol, 3% SDS, 7.5% β-mercaptoethanol and 0.0375% Bromophenol Blue). Lysates were separated by SDS-PAGE gel and transferred to PVDF membranes for immunoblotting. Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% bovine serum albumin (BSA) and immunoblots were incubated overnight at 4°C with antibodies against p-Smad2 and Smad2. Signal detection was performed using HRP-conjugated IgG secondary antibodies ( Table 11 ). Blot intensities were quantified using ImageJ software and one-way ANOVA followed by Tukey’s post hoc test was used to examine statistical significance. View this table: View inline View popup Download powerpoint Table 11. List of antibodies used for Western Blot analysis. Quantification and statistical analysis Data are presented as mean ± standard error of the mean (s.e.m). Statistical significance between two groups was determined using an unpaired, two-tailed Student’s t-test. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s post hoc test was applied. Statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software). Funding This work was supported by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación with grants to O.M.M.-E. (PID2023-148793NB-I00) and F.X.S. (PID2023-149249NB-I00). Conflict of interest The authors declare no conflicts of interest. Acknowledgments We are grateful for the assistance provided by the Centres Científics i Tecnològics (CCiTUB) of the University of Barcelona for the use of advanced microscopy, flow cytometry, and animal facilities. Funder Information Declared Ministerio de Ciencia e Innovación , PID2023-148793NB-I00 , PID2023-149249NB-I00 Footnotes In this revised version of the manuscript, we present additional experimental data that further support the validity of our cellular model for studying heart fibrosis. References ↵ Acharya A , Baek ST , Banfi S , Eskiocak B , Tallquist MD ( 2011 ) Efficient inducible Cremediated recombination in Tcf21 cell lineages in the heart and kidney . Genesis 49 : 870 – 877 OpenUrl CrossRef PubMed Web of Science ↵ Acharya A , Baek ST , Huang G , Eskiocak B , Goetsch S , Sung CY , Banfi S , Sauer MF , Olsen GS , Duffield JS , Olson EN , Tallquist MD ( 2012 ) The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors . Development 139 : 2139 – 2149 OpenUrl Abstract / FREE Full Text ↵ Braitsch CM , Kanisicak O , van Berlo JH , Molkentin JD , Yutzey KE ( 2013 ) Differential expression of embryonic epicardial progenitor markers and localization of cardiac fibrosis in adult ischemic injury and hypertensive heart disease . Journal of molecular and cellular cardiology 65 : 108 – 119 OpenUrl CrossRef PubMed ↵ Cano E , Carmona R , Ruiz-Villalba A , Rojas A , Chau YY , Wagner KD , Wagner N , Hastie ND , Munoz-Chapuli R , Perez-Pomares JM ( 2016 ) Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections . Proceedings of the National Academy of Sciences of the United States of America 113 : 656 – 661 OpenUrl Abstract / FREE Full Text ↵ Deng Y , He Y , Xu J , He H , Zhang M , Li G ( 2025 ) Cardiac fibroblasts regulate myocardium and coronary vasculature development in the murine heart via the collagen signaling pathway . eLife 13 ↵ Deng Z , Fan T , Xiao C , Tian H , Zheng Y , Li C , He J ( 2024 ) TGF-beta signaling in health, disease, and therapeutics . Signal transduction and targeted therapy 9 : 61 OpenUrl ↵ Feng W , Bais A , He H , Rios C , Jiang S , Xu J , Chang C , Kostka D , Li G ( 2022 ) Single-cell transcriptomic analysis identifies murine heart molecular features at embryonic and neonatal stages . Nature communications 13 : 7960 OpenUrl PubMed ↵ Forte E , Skelly DA , Chen M , Daigle S , Morelli KA , Hon O , Philip VM , Costa MW , Rosenthal NA , Furtado MB ( 2020 ) Dynamic Interstitial Cell Response during Myocardial Infarction Predicts Resilience to Rupture in Genetically Diverse Mice . Cell reports 30 : 3149 – 3163 e3146 OpenUrl PubMed ↵ Garvin AM , Katwa LC ( 2023 ) Primary cardiac fibroblast cell culture: methodological considerations for physiologically relevant conditions . American journal of physiology Heart and circulatory physiology 325 : H869 – H881 OpenUrl CrossRef PubMed ↵ Gittenberger-de Groot AC , Vrancken Peeters MP , Mentink MM , Gourdie RG , Poelmann RE ( 1998 ) Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions . Circulation research 82 : 1043 – 1052 OpenUrl Abstract / FREE Full Text ↵ Hamilton TG , Klinghoffer RA , Corrin PD , Soriano P ( 2003 ) Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms . Molecular and cellular biology 23 : 4013 – 4025 OpenUrl Abstract / FREE Full Text ↵ Hastie ND ( 2017 ) Wilms’ tumour 1 (WT1) in development, homeostasis and disease . Development 144 : 2862 – 2872 OpenUrl Abstract / FREE Full Text ↵ Hortells L , Valiente-Alandi I , Thomas ZM , Agnew EJ , Schnell DJ , York AJ , Vagnozzi RJ , Meyer EC , Molkentin JD , Yutzey KE ( 2020 ) A specialized population of Periostin-expressing cardiac fibroblasts contributes to postnatal cardiomyocyte maturation and innervation . Proceedings of the National Academy of Sciences of the United States of America 117 : 21469 – 21479 OpenUrl Abstract / FREE Full Text ↵ Hosen N , Shirakata T , Nishida S , Yanagihara M , Tsuboi A , Kawakami M , Oji Y , Oka Y , Okabe M , Tan B , Sugiyama H , Weissman IL ( 2007 ) The Wilms’ tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis . Leukemia 21 : 1783 – 1791 OpenUrl CrossRef PubMed Web of Science ↵ Humeres C , Frangogiannis NG ( 2019 ) Fibroblasts in the Infarcted, Remodeling, and Failing Heart . JACC Basic to translational science 4 : 449 – 467 OpenUrl PubMed ↵ Ieda M , Tsuchihashi T , Ivey KN , Ross RS , Hong TT , Shaw RM , Srivastava D ( 2009 ) Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling . Developmental cell 16 : 233 – 244 OpenUrl CrossRef PubMed Web of Science ↵ Johansen AKZ , Kasam RK , Vagnozzi RJ , Lin SJ , Gomez-Arroyo J , Shittu A , Bowers SLK , Kuwabara Y , Grimes KM , Warrick K , Sargent MA , Baldwin TA , Quaggin SE , Barski A , Molkentin JD ( 2025 ) Transcription Factor 21 Regulates Cardiac Myofibroblast Formation and Fibrosis . Circulation research 136 : 44 – 58 OpenUrl CrossRef PubMed ↵ Kendall TJ , Duff CM , Boulter L , Wilson DH , Freyer E , Aitken S , Forbes SJ , Iredale JP , Hastie ND ( 2019 ) Embryonic mesothelial-derived hepatic lineage of quiescent and heterogenous scar-orchestrating cells defined but suppressed by WT1 . Nature communications 10 : 4688 OpenUrl PubMed ↵ Kretzschmar K , Post Y , Bannier-Helaouet M , Mattiotti A , Drost J , Basak O , Li VSW , van den Born M , Gunst QD , Versteeg D , Kooijman L , van der Elst S , van Es JH , van Rooij E , van den Hoff MJB , Clevers H ( 2018 ) Profiling proliferative cells and their progeny in damaged murine hearts . Proceedings of the National Academy of Sciences of the United States of America 115 : E12245 – E12254 OpenUrl Abstract / FREE Full Text ↵ Luche H , Weber O , Nageswara Rao T , Blum C , Fehling HJ ( 2007 ) Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies . European journal of immunology 37 : 43 – 53 OpenUrl CrossRef PubMed Web of Science ↵ Martinez-Estrada OM , Lettice LA , Essafi A , Guadix JA , Slight J , Velecela V , Hall E , Reichmann J , Devenney PS , Hohenstein P , Hosen N , Hill RE , Munoz-Chapuli R , Hastie ND ( 2010 ) Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin . Nature genetics 42 : 89 – 93 OpenUrl CrossRef PubMed Web of Science ↵ Moore-Morris T , Cattaneo P , Guimaraes-Camboa N , Bogomolovas J , Cedenilla M , Banerjee I , Ricote M , Kisseleva T , Zhang L , Gu Y , Dalton ND , Peterson KL , Chen J , Puceat M , Evans SM ( 2018 ) Infarct Fibroblasts Do Not Derive From Bone Marrow Lineages . Circulation research 122 : 583 – 590 OpenUrl Abstract / FREE Full Text ↵ Moore-Morris T , Evans SM ( 2023 ) Cardiac Pericyte Diversity in Infarct Remodeling: Not Just Vascular Support Cells? Circulation 148 : 899 – 902 OpenUrl CrossRef PubMed ↵ Moore-Morris T , Guimaraes-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 ) Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis . The Journal of clinical investigation 124 : 2921 – 2934 OpenUrl CrossRef PubMed ↵ Moore AW , McInnes L , Kreidberg J , Hastie ND , Schedl A ( 1999 ) YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis . Development 126 : 1845 – 1857 OpenUrl Abstract / FREE Full Text ↵ Patrick R , Janbandhu V , Tallapragada V , Tan SSM , McKinna EE , Contreras O , Ghazanfar S , Humphreys DT , Murray NJ , Tran YTH , Hume RD , Chong JJH , Harvey RP ( 2024 ) Integration mapping of cardiac fibroblast single-cell transcriptomes elucidates cellular principles of fibrosis in diverse pathologies . Science advances 10 : eadk8501 OpenUrl CrossRef PubMed ↵ Perez-Pomares JM , Phelps A , Sedmerova M , Carmona R , Gonzalez-Iriarte M , Munoz-Chapuli R , Wessels A ( 2002 ) Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs) . Developmental biology 247 : 307 – 326 OpenUrl CrossRef PubMed Web of Science ↵ Pogontke C , Guadix JA , Sanchez-Tevar AM , Munoz-Chapuli R , Ruiz-Villalba A , Perez-Pomares JM ( 2022 ) Dynamic Epicardial Contribution to Cardiac Interstitial c-Kit and Sca1 Cellular Fractions . Frontiers in cell and developmental biology 10 : 864765 OpenUrl ↵ Quijada P , Misra A , Velasquez LS , Burke RM , Lighthouse JK , Mickelsen DM , Dirkx RA , Jr . ., Small EM ( 2019 ) Pre-existing fibroblasts of epicardial origin are the primary source of pathological fibrosis in cardiac ischemia and aging . Journal of molecular and cellular cardiology 129 : 92 – 104 OpenUrl CrossRef PubMed ↵ Ramiro-Pareta M , Muller-Sanchez C , Portella-Fortuny R , Soler-Botija C , Torres-Cano A , Esteve-Codina A , Bayes-Genis A , Reina M , Soriano FX , Montanez E , Martinez-Estrada OM ( 2023 ) Endothelial deletion of Wt1 disrupts coronary angiogenesis and myocardium development . Development 150 ↵ Ruiz-Villalba A , Simon AM , Pogontke C , Castillo MI , Abizanda G , Pelacho B , Sanchez-Dominguez R , Segovia JC , Prosper F , Perez-Pomares JM ( 2015 ) Interacting resident epicardium-derived fibroblasts and recruited bone marrow cells form myocardial infarction scar . Journal of the American College of Cardiology 65 : 2057 – 2066 OpenUrl FREE Full Text ↵ Sanchez-Iranzo H , Galardi-Castilla M , Sanz-Morejon A , Gonzalez-Rosa JM , Costa R , Ernst A , Sainz de Aja J , Langa X , Mercader N ( 2018 ) Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart . Proceedings of the National Academy of Sciences of the United States of America 115 : 4188 – 4193 OpenUrl Abstract / FREE Full Text ↵ Sontake V , Kasam RK , Sinner D , Korfhagen TR , Reddy GB , White ES , Jegga AG , Madala SK ( 2018 ) Wilms’ tumor 1 drives fibroproliferation and myofibroblast transformation in severe fibrotic lung disease . JCI insight 3 ↵ Tallquist MD ( 2018 ) Cardiac fibroblasts: from origin to injury . Current opinion in physiology 1 : 75 – 79 OpenUrl CrossRef PubMed ↵ Tallquist MD ( 2020 ) Cardiac Fibroblast Diversity . Annual review of physiology 82 : 63 – 78 OpenUrl CrossRef PubMed ↵ Tallquist MD , Molkentin JD ( 2017 ) Redefining the identity of cardiac fibroblasts . Nature reviews Cardiology 14 : 484 – 491 OpenUrl PubMed ↵ Torres-Cano A , Portella-Fortuny R , Muller-Sanchez C , Porras-Marfil S , Ramiro-Pareta M , Chau YY , Reina M , Soriano FX , Martinez-Estrada OM ( 2022 ) Deletion of Wt1 during early gonadogenesis leads to differences of sex development in male and female adult mice . PLoS genetics 18 : e1010240 OpenUrl ↵ Velecela V , Torres-Cano A , Garcia-Melero A , Ramiro-Pareta M , Muller-Sanchez C , Segarra-Mondejar M , Chau YY , Campos-Bonilla B , Reina M , Soriano FX , Hastie ND , Martinez FO , Martinez-Estrada OM ( 2019 ) Epicardial cell shape and maturation are regulated by Wt1 via transcriptional control of Bmp4 . Development 146 ↵ von Gise A , Pu WT ( 2012 ) Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease . Circulation research 110 : 1628 – 1645 OpenUrl Abstract / FREE Full Text ↵ Wessels A , van den Hoff MJ , Adamo RF , Phelps AL , Lockhart MM , Sauls K , Briggs LE , Norris RA , van Wijk B , Perez-Pomares JM , Dettman RW , Burch JB ( 2012 ) Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart . Developmental biology 366 : 111 – 124 OpenUrl CrossRef PubMed ↵ Wu R , Ma F , Tosevska A , Farrell C , Pellegrini M , Deb A ( 2020 ) Cardiac fibroblast proliferation rates and collagen expression mature early and are unaltered with advancing age . JCI insight 5 ↵ Yata Y , Scanga A , Gillan A , Yang L , Reif S , Breindl M , Brenner DA , Rippe RA ( 2003 ) DNase I-hypersensitive sites enhance alpha1(I) collagen gene expression in hepatic stellate cells . Hepatology 37 : 267 – 276 OpenUrl CrossRef PubMed Web of Science ↵ Zhang H , Huang X , Liu K , Tang J , He L , Pu W , Liu Q , Li Y , Tian X , Wang Y , Zhang L , Yu Y , Wang H , Hu R , Wang F , Chen T , Wang QD , Qiao Z , Zhang L , Lui KO , Zhou B ( 2017 ) Fibroblasts in an endocardial fibroelastosis disease model mainly originate from mesenchymal derivatives of epicardium . Cell research 27 : 1157 – 1177 OpenUrl CrossRef PubMed ↵ Zhang LP , Nie Q , Kang JB , Wang B , Cai CL , Li JG , Qi WJ ( 2008 ) [Efficacy of whole body gamma-knife radiotherapy combined with thermochemotherapy on locally advanced pancreatic cancer] . Ai Zheng 27 : 1204 – 1207 OpenUrl PubMed ↵ Zhou B , Honor LB , He H , Ma Q , Oh JH , Butterfield C , Lin RZ , Melero-Martin JM , Dolmatova E , Duffy HS , Gise A , Zhou P , Hu YW , Wang G , Zhang B , Wang L , Hall JL , Moses MA , McGowan FX , Pu WT ( 2011 ) Adult mouse epicardium modulates myocardial injury by secreting paracrine factors . The Journal of clinical investigation 121 : 1894 – 1904 OpenUrl CrossRef PubMed Web of Science ↵ Zhou B , Ma Q , Rajagopal S , Wu SM , Domian I , Rivera-Feliciano J , Jiang D , von Gise A , Ikeda S , Chien KR , Pu WT ( 2008 ) Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart . Nature 454 : 109 – 113 OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted August 05, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium Claudia Müller-Sánchez , María Gertrudis Muñiz-Banciella , Manuel Reina , Francesc X. Soriano , Ofelia M. Martínez-Estrada bioRxiv 2025.05.23.655755; doi: https://doi.org/10.1101/2025.05.23.655755 Share This Article: Copy Citation Tools An innovative in vitro model for studying the biology of cardiac fibroblasts originating from the epicardium Claudia Müller-Sánchez , María Gertrudis Muñiz-Banciella , Manuel Reina , Francesc X. Soriano , Ofelia M. Martínez-Estrada bioRxiv 2025.05.23.655755; doi: https://doi.org/10.1101/2025.05.23.655755 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41968) Biophysics (21460) Cancer Biology (18603) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19909) Epidemiology (2067) Evolutionary Biology (24326) Genetics (15614) Genomics (22513) Immunology (17741) Microbiology (40423) Molecular Biology (17193) Neuroscience (88645) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4825) Physiology (7647) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)