Modelling cardiac fibrosis using three-dimensional cardiac microtissues derived from human embryonic stem cells
Cardiac fibrosis is the most common pathway of many cardiac diseases. To date, there has been no suitable in vitro cardiac fibrosis model that could sufficiently mimic the complex environment of the human heart. Here, a three-dimensional (3D) cardiac sphere platform of contractile cardiac microtissue, composed of human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) and mesenchymal stem cells (MSCs), is presented to better recapitulate the human heart.
We hypothesized that MSCs would develop an in vitro fibrotic reaction in response to treatment with transforming growth factor-β1 (TGF-β1), a primary inducer of cardiac fibrosis. The addition of MSCs improved sarcomeric organization, electrophysiological properties, and the expression of cardiac-specific genes, suggesting their physiological relevance in the generation of human cardiac microtissue model in vitro. MSCs could also generate fibroblasts within 3D cardiac microtissues and, subsequently, these fibroblasts were transdifferentiated into myofibroblasts by the exogenous addition of TGF-β1. Cardiac microtissues displayed fibrotic features such as the deposition of collagen, the presence of numerous apoptotic CMs and the dissolution of mitochondrial networks. Furthermore, treatment with pro-fibrotic substances demonstrated that this model could reproduce key molecular and cellular fibrotic events.
This highlights the potential of our 3D cardiac microtissues as a valuable tool for manifesting and evaluating the pro-fibrotic effects of various agents, thereby representing an important step forward towards an in vitro system for the prediction of drug-induced cardiac fibrosis and the study of the pathological changes in human cardiac fibrosis.
KeywordsCardiac fibrosis Cardiac sphere Cardiac microtissue Cardiomyocyte Mesenchymal stem cell
Cardiac differentiation medium
Cardiac muscle troponin T
Differentially expressed gene
Field potential duration
Gene set enrichment analysis
Human embryonic stem cell
Human induced pluripotent stem cell
Human pluripotent stem cell
Mesenchymal stem cell
Principal component 1
Principal component 2
Transforming growth factor β1
α-smooth muscle actin
Cardiac fibrosis is a common feature of most myocardial pathologies, including ischaemic cardiomyopathy, inherited cardiomyopathy mutations, metabolic syndrome, diabetes, and ageing [1, 2]. Increased mechanical stress or myocardial injury can trigger cardiac fibrosis, which might contribute to contractile and diastolic dysfunctions and subsequent sudden death . Cardiac fibrosis is characterized by the excess accumulation of extracellular matrix (ECM) components, such as collagen and fibronectin, with the consequent pathological remodelling of ECM, followed by transforming fibroblasts into myofibroblasts [4, 5]. These myofibroblasts have high fibrotic activity marked by the expression of α-smooth muscle actin (α-SMA), resulting in myocardial fibrosis and stiffening, which eventually impairs cardiac function [6, 7].
The exploration of the pathogenesis and therapy development for cardiac fibrosis is hampered by the lack of appropriate experimental models that fully recapitulate human cardiac fibrosis. An improvement of in vitro cell or tissue models may open up new possibilities for disease modelling, drug discovery, and regenerative medicine by providing fast and controllable platforms with high availability and relatively low cost compared to animal models. As yet, few in vitro models of cardiac fibrosis have been developed [8, 9, 10, 11, 12]. Most are composed of cardiac cells derived from neonatal mouse and rat hearts and thus have less relevance to human pathophysiology. In addition, most current models of cardiac tissue contain only cardiomyocytes (CMs) and lack other key cell types found in the human heart . Therefore, there is a need for an in vitro human cardiac fibrosis model that possesses the physiologically relevant cell combination and can mimic the three-dimensional (3D) nature of native cardiac tissue.
Due to the limited amount of human primary CMs available, CMs derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have emerged as the most appropriate cell source for modelling the human heart in vitro, with obvious advantages of multiple functionalities and increased throughput [14, 15]. Though hPSC-derived CMs or cardiac progenitor cells represent immature phenotypes in their morphological and other physiological and biochemical properties , there is a clear incentive to use hPSC-derived CMs as a foundation to generate 3D human cardiac tissue that can eventually be tailored to patient-specific models of normal and diseased cardiac tissues. However, to date, there are no suitable in vitro cardiac fibrosis model based on hPSC-derived 3D cardiac tissue to study the pathological changes in human cardiac fibrosis and to evaluate novel treatments.
In this study, we developed an in vitro model of cardiac fibrosis through hESC-derived 3D cardiac microtissues, composed of CMs and mesenchymal stem cells (MSCs) differentiated from the same hESC line. In the normal adult heart, CMs represent only 30% of the total cell number and the remaining 70% consists of various types of cells, among which cardiac mesenchymal cells are in the majority . Cardiac mesenchymal cells can provide a major precursor population to generate fibroblasts, which mediate scar formation via fibroblast–myofibroblast transition during fibrosis [18, 19, 20, 21, 22, 23]. Therefore, to engineer a physiologically relevant in vitro cardiac microtissues model, we used MSCs to provide fibroblasts, which can be differentiated towards myofibroblasts after stimulation with pro-fibrotic agents. The molecular and cellular properties of these 3D cardiac microtissues were characterized and the pro-fibrotic consequences were observed after induction with transforming growth factor-β1 (TGF-β1) or other pro-fibrotic mediators. We believe that our disease model using 3D cardiac microtissues may be a suitable in vitro model for studying fibrotic changes in human heart tissue and can potentially contribute to the development of more physiologically relevant preclinical drug discovery platforms.
Differentiation and characterization of CMs from hESCs
Generation of 3D human cardiac microtissues
In this study, we attempted to simulate the pathological characteristics of cardiac fibrosis by introducing the 3D culture method. When 50,000 hESC-derived single CMs were seeded on round bottom 96-well ultra-low attachment plates, self-aggregation occurred slowly, resulting in uniform-sized cell spheroids (approximately 500 μm in diameter) (Additional file 2: Figure S1A). The created cardiac spheroids were beating at regular intervals and were maintained for more than 2 months (Additional file 3: Movie 2). TGF-β1 signaling has been shown to play important roles in mediating fibrotic responses by regulating ECM remodelling and excessive collagen deposition, which eventually results in cardiac hypertrophy and fibrosis . Therefore, we tested whether cardiac fibrotic phenotypes can be reproduced by treating cardiac spheroids with TGF-β1. However, TGF-β1 treatment for 3 weeks did not show any marked effect on collagen deposition in the cardiac spheroids (Additional file 2: Figure S1B).
Characterization of 3D human cardiac microtissues
TGF-β1-induced fibrosis in CM-MSC cardiac microtissues
Drug-induced fibrosis in CM-MSC cardiac microtissues
The goal of engineering a 3D cardiac tissue model is to provide new opportunities for in vitro cardiac modelling with physiologically relevant cell types and microenvironment. The human 3D cardiac microtissue described in this study is expected to improve our knowledge about clinically relevant information regarding cardiac fibrosis and disclose advanced features of cardiac fibrosis. It is now widely believed that cultures of single cell types are very simplistic models, because they do not mimic the complexity and heterogeneity of human tissues. The normal heart is composed of several different cell types, such as CMs, endothelial cells, smooth muscle cells and fibroblasts, which play a role in the development of the heart and its normal functions . Even within the in vitro environment, non-CMs influence the improvement of CM phenotypes and tissue architecture only in 3D cultures [40, 41]. These results are consistent with observations that most cell types do not exhibit the full spectrum of tissue-specific functionality in 2D cultures, since they do not maintain their phenotypes and patterns of gene expression precisely in the different environments compared with native tissue . Therefore, to mimic the native cardiac tissue, our in vitro 3D co-culture strategy that includes relevant cell types appears most appropriate.
In this study, we have established for the first time a human 3D cardiac microtissue containing hESC-derived CMs in combination with hESC-derived MSCs. During heart development, fibroblasts, which are important cells that produce fibrotic ECM proteins , arise from multipotent progenitor cells, especially MSCs [19, 20, 21, 23, 44]. The cellular origins of cardiac fibroblasts are varied, including epithelial-mesenchymal transition (EMT) of epicardium cells, endothelial-mesenchymal transition (endo-MT) of epithelial cells, and MSCs . Fibroblasts are connective tissue cells of mesenchymal origin that synthesize and secrete the main components of ECM, such as interstitial collagen and fibronectin [46, 47]. Fibroblasts also play an important role in fibrotic tissue formation by conversion into myofibroblasts under pathological conditions . However, there is currently no standardized protocol for the reliable differentiation of hPSCs into cardiac fibroblasts, whereas, the differentiation methods of hPSCs into MSCs are well established [49, 50]. It has been also reported that MSCs can transdifferentiate into fibroblasts in vitro and in vivo . Cardiac MSCs play an important role in preserving normal cardiac functions, as well as in cardiac remodelling at various pathological stages, by responding early to myocardial infarction . Therefore we developed 3D cardiac microtissues consisting of CMs in combination with MSCs as a source of fibroblasts for developing the in vitro cardiac fibrosis model. The generation of cardiac spheroids with hESC-derived CMs and MSCs has an advantage in terms of the spheroid formation time. As shown in Fig. 3a, aggregates started to form only 1 day after seeding CMs mixed with 20% MSCs, and compact and circular spheroids were successfully generated, whereas loose aggregates with poor cell-cell contacts were formed when CM was cultured only in 3D. Consistent with our findings, Ong et al. have shown that multicellular cardiac spheres are generated from hiPSC-CMs only when they are mixed with human adult ventricular cardiac fibroblasts and human umbilical vein endothelial cells . Furthermore, increased expression of sets of genes associated with ECM organization and cell adhesion (Fig. 4d), which are known to be essential for sphere formation , in CM-MSC cardiac spherical microtissues allows more rapid sphere formation in comparison to CM spheroids.
DDR2 is known to be selectively expressed in cardiac fibroblasts [45, 46, 55], although it appears to be also expressed in pericytes/vascular smooth muscle cells . As shown in Fig. 4b, all of vimentin-positive MSCs were not co-expressed with DDR2, whereas some cells within CM-MSC cardiac spheroids were double-positive for vimentin and DDR2, suggesting a transdifferentiation of MSCs into fibroblasts within CM-MSC cardiac spheroids. Our findings are supported by a report that CD44-positive MSCs served as a major precursor pool for fibroblasts that mediate scar formation and wound healing after myocardial infarction . In addition, bone marrow-derived MSCs [57, 58] and perivascular-resident MSCs [59, 60], as well as cardiac fibroblasts, which are known to be direct sources of myofibroblasts and MSCs, also exhibit myofibroblastic phenotypes in vitro in response to TGFβ treatment .
As in previous cardiac fibrosis models, mouse and rat neonatal CMs are commonly used for developing engineered cardiac tissue because terminally differentiated adult CMs cannot proliferate sufficiently in vitro [8, 10, 12]. In this study, CMs derived from hESCs were well characterized by the expression of cardiac function-related proteins (Fig. 1a-e). Particularly, CMs were connected to each other by the intercalated disc structure by using immunofluorescence staining of Cx43, the most abundant cardiac gap junction protein (Fig. 1e). Cx43 expression was observed as dots in the intercellular connected regions, in which the ends of myofibrils were connected, consistent with a previous study . hPSC-derived CMs have been widely used to perform drug and toxicity testing in vitro [63, 64]. Furthermore, CMs differentiated from hiPSCs generated from patients with genetic cardiac diseases also allow for the generation of CMs carrying the genetic background of a given patient, creating highly tailored models for cardiac diseases in vitro . Recently, a cardiac model of multicellular systems with several cell types, including CMs, endothelial cells, and fibroblasts, was developed to engineer a physiologically relevant cardiac tissue model using a 3D culture system, various biomaterials and bioprinting technology [10, 13, 53, 66, 67, 68, 69]. These multicellular spheroids and 3D models will be applied for developing cardiac fibrosis models by introducing external fibrotic signals. Moreover, when developing in vitro cardiac fibrosis models for personalized drug efficacy and toxicity testing, the iPSC technology applied in this study enables the creation of in vitro disease models in which various cell types with the same genetic background can be assembled. Therefore, disease models using patient-specific cells can be used as platforms to explore new therapies by providing individually optimized treatments.
In this work, we established a 3D in vitro model of cardiac fibrosis using 3D cardiac microtissues by treatment with TGF-β1, which is a potent stimulator that promotes the differentiation and proliferation of myofibroblasts during the course of this disease . Our in vitro model, long-term treatment of TGF-β1 induces irregular beating patterns (Fig. 5a), an increase in the accumulation of fibrillar collagen deposition (Fig. 5b), elevated expression of α-SMA, conversion to myofibroblasts (Fig. 5f), increase in the rate of apoptosis of CMs (Fig. 5c, d), and disruption of the mitochondrial network as shown by transformation of filamentous into punctate mitochondria (Fig. 6c), consistent with the pathological changes during cardiac fibrosis [70, 71]. However, we did not detect increased proliferation of MSC or transdifferentiated myofibroblasts in response to TGF-β1 treatment (data not shown), which may have been due to the inclusion of serum-free medium in the cardiac sphere culture conditions. Moreover, we examined whether 3D cardiac microtissues could be used to detect and monitor cardiac fibrosis using known pro-fibrotic mediators. After a 2-week exposure to pro-fibrotic mediators, including bisphenol A, aldosterone and metoprolol, 3D cardiac microtissues display fibrotic features (Fig. 7a-g), such as an up-regulation of pro-fibrotic and TGF-β1-responsive genes, collagen secretion and deposition; increased α-SMA expression; and disorganization of the mitochondrial network, leading to mitochondrial fragmentation, without significant differences compared to TGF-β1-treated cardiac microtissues. These results suggested that our 3D cardiac model can be applied to identify thus far unknown fibrotic compounds and to provide a platform for studying mechanisms of cardiac fibrosis. However, the expression levels of TGF-β1 responsive genes varied among pro-fibrotic drugs (Fig. 7a), suggesting that there may be different detailed molecular mechanisms for the activation of the TGF-β1 pathway. The link between pro-fibrotic drugs and the TGF- β1 pathway is very complex since different molecular mechanisms are involved. Therefore, this research model could provide the basics for studying the development of cardiac fibrosis in response to the different drugs.
However, we recognize the limitations of this in vitro model in recapitulating human diseases, as it lacks adequate blood flow and other physiological factors, such as endothelial and inflammatory cells, which are found in the native heart. There is also a need to apply natural and synthetic biomaterials mimicking native ECMs, as biomaterials play a major role in constructing 3D tissue models via supporting cell attachment and alignment and providing tissue-relevant stiffness . Therefore, the further introduction of various cell types present in the heart, application of microfluidics, and use of tissue engineering strategies can be exploited in the development of physiologically relevant in vitro human heart tissue.
In this study, we report a simplified cardiac microtissue comprising CMs and MSCs derived from hESCs to study cardiac fibrosis in a 3D spheroid platform. Our study highlights the importance of increasing the cellular complexity by adding the appropriate amount of CD44-positive MSCs which can contribute to cardiac fibrosis by generating fibroblasts to better model human cardiac tissues in vitro. Because our cardiac tissue model can be adapted to mimic various aspects of cardiac fibrosis, it cannot only be used to provide further insights into the mechanisms underlying cardiac fibrosis but can also potentially contribute to the development of in vitro assay systems for testing pro-fibrotic compounds and new anti-fibrotic therapies.
H9 hESCs were obtained from the WiCell Research Institute (Madison, WI, USA) and maintained as described previously . This study using human embryonic stem cell lines was approved by the Public Institutional Bioethics Committee designated by the Ministry of Health and Welfare (MoHW) (Seoul, Republic of Korea; IRB no. P01–201409-ES-01).
Differentiation of hESCs into human CMs
Human cardiomyocytes were differentiated as described previously . hESCs were transferred to Matrigel (BD Bioscience, San Jose, CA)-coated plates with mTESR medium (Stem Cell Technologies, Vancouver, Canada) without feeder cells; then, cells were grown to 90% confluency. For mesoderm induction, hESCs were treated with 6 μM CHIR99021 (Tocris, Bristol, UK) for 2 days in Cardiac Differentiation Medium (CDM) consisting of RPMI1640 supplemented with 50 μg/ml L-ascorbic acids (Sigma-Aldrich, St. Louis, MO, USA) and 500 μg/ml recombinant albumin (Sigma-Aldrich). Cardiac specification was performed by treating with 2 μM of Wnt-C59 (Selleck Chemicals, Houston, TX, USA) for another 2 days in CDMs. Subsequently, the cells were replaced with CDM medium once every two days until beating CMs were observed. Differentiated CMs were maintained in RPMI-B27 medium consisting of RPMI1640 supplemented with 50 μg/ml L-ascorbic acids and 1XB27 (Thermo Fisher Scientific, Waltham, MA, USA).
Differentiation of hESC into human MSCs
Human MSCs were differentiated from hESCs as described previously . hESCs were transferred to Matrigel-coated plates with mTESR medium (Stem Cell Technologies) without feeder cells; then, cells were grown to 50% confluency. For mesenchymal differentiation, medium was changed to MEM-alpha supplemented with 10% FBS and 5 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA) once every two days. Cells were transferred to a gelatine-coated dish every week.
Generation of CM-MSC cardiac microtissues
The differentiated CM and MSC cells were dissociated into single cells by 0.25% trypsin treatment, and the number of cells was counted with a haemocytometer. The CMs and MSCs were mixed at a ratio of 4:1, and 50,000 cells per well were seeded in a 96 well ultra-low attachment plate with RPMI-B27 media supplemented with 10% FBS. After the sphere formation was completed within 2–3 days, the medium was replaced with RPMI-B27 and the medium was replaced every 2–3 days. To develop the in vitro fibrosis model, 5 ng/ml TGF-β1 or 10 μM concentration of each cardiotoxic chemical, including Bisphenol A, Aldosterone, and Metoprolol (all reagents from Sigma-Aldrich) were treated independently with the indicated concentration of drugs as described in previous reports [36, 37, 38] and the samples were harvested for analysis after 2 weeks.
To verify the CM and MSC differentiation efficiency, FACS analysis was performed as described previously . The differentiated CMs and MSCs were treated with 0.25% trypsin, dissociated into single cells, and incubated in dPBS containing 2 mM EDTA and 2% FBS for 10 min. After that, antibodies to the surface markers of each cell (Additional file 9: Table S1) were diluted 1:50 and reacted for 30 min. After washing twice with FACS buffer, FACS analysis was performed using Accuri C6 flow cytometry (BD Biosciences) and analysed using FlowJo V10 software (TreeStar, USA).
Processing of cardiac microtissue, immunostaining and MT staining
Cardiac microtissues were fixed by 4% paraformaldehyde (PFA), cryo-protected in 10%~ 30% sucrose solution and frizzed after embedding in optimal-cutting-temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) as described previously . Frozen section were prepared by cutting at a thickness of 10–15 μm using a cryostat microtome. The frozen sections were permeablized with 0.1% Triton X-100 for immune-staining, blocked with 3% BSA, and incubated with the primary antibody (Additional file 9: Table S1). For immunofluorescence detection, cells were incubated with a fluorescence-conjugated secondary antibody. For immunoperoxidase detection, the sections were incubated with the biotinylated secondary antibody and then followed using the VECTASTAIN elite ABC kit (Cat. NO. PK-6100, vector laboratories) and DAB substrate kit (Cat. NO. SK-4100, Vector Laboratories, Burlingame, CA, USA). Staining of collagen fibres was performed using Masson’s Trichrome staining kit (Cat no. 25088–1, Polysciences, Inc. Warrington, USA).
Quantitative RT-PCR (qPCR)
To compare mRNA expression levels, total RNA was extracted according to the manufacturer’s protocol using the easyBLUE RNA extraction kit (iNtRON Biotechnology, Inc., Republic of Korea). cDNA was synthesized using the SuperScript™ IV First-strand Synthesis System (Cat.NO.1891050, Thermo Fisher Scientific), and the cDNA was used as a template for real-time qPCR. PCR reactions were performed three times independently using Fast SYBR ™ Green Master Mix (4,385,612, Applied Biosystems, Foster City, CA, USA). The sequence of the gene-specific primers are presented in the Additional file 10: Table S2.
Transcriptome analysis by microarray
For global transcriptome analysis, cDNA microarray was performed using an Agilent Human GE (V2) 4 X 44 K chip as described previously . Data were normalized by using GeneSpring software, and differentially expressed genes (DEGs) were analysed by MultiExperiment Viewer software (MeV version 4.9.0; http://mev.tm4.org/). Pathway enrichment analysis was performed by Reactome FI plugin of the Cytoscape software (Version 3.2.0, www.cytoscape.org), and gene set enrichment analysis was performed by GSEA 2.2.4 (http://software.broadinstitute.org/gsea/index.jsp). The H (hallmark genesets) subsets of MSigDB (50 gene sets), C5_BP (GO biological process, 4436 gene sets) and C5_CC (GO cellular component, 580 gene sets) were used in this study .
Beating rate analysis of CM-MSC microtissue by tracing of microscopic video files
Beating rates of CM-MSC microtissue were recorded on a microscope (IX83, Olympus, Japan) with an on-stage incubator (Live Cell Instrument, Seoul, South Korea) at 37 °C and 5% CO2 to maintain the physiological condition. The plot z-axis profile of each region of interest (ROI) was calculated by Image J software (https://imagej.nih.gov/ij/) program .
Field potential recordings using a multi-electrode array (MEA) system
The hESC-derived CMs were plated on a Matrigel-coated 12-well MEA plate (4 × 104 cell per well) (AXION, Atlanta, GA, USA) in RPMI-B27 media supplemented with 5% FBS. At 24 h after seeding, the medium was replaced with RPMI-B27. Field potentials were recorded at day 6 after seeding using Axion BioSystems’ Maestro MEA systems set as 37 °C and perfused with 5% CO2, 20% O2, and 75% N2. Field potential signals were recoded with Axion BioSystems’ Integrated Studio (AxIS) software version 2.3 and analysed with the Axion Cardiac Data Plotting Tool.
Statistical analysis and graph drawing
Whether treatments produce significantly different results was evaluated by statistical analysis. The unpaired t-test (Figs. 1c, 2c, 3b, e, 4e, 5d, and e) and one-way ANOVA followed by Dunnett’s multiple comparisons test (Fig. 7a, d, and e) were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, Inc., USA).
This work was supported by a grant from the Technology Innovation Program (No. 10063334) funded by the Ministry of Trade, Industry & Energy (MI, Korea), the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT and Future Planning (2018R1C1B6008256 and NRF-2018M3A9H3023077), and the KRIBB Research Initiative Program. The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional files.
MOL designed the studies, performed the experiments, analysed the data and prepared the manuscript. KBJ and JSJ performed the experiments and analysed the data. SAH, KSM, and JWS performed and analysed the MEA experiments. SHK and MYS planned the project, analysed the data, wrote and critically revised the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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