The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for use in cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signalling is sufficient for epicardial induction from six different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA sequencing. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.
The epicardium is the outermost mesothelium layer of the heart, and contributes both multi-lineage descendants and trophic signals to the myocardium and coronary vessels1,2. During cardiogenesis, epicardial cells undergo an epithelial-to-mesenchymal transition (EMT) and invade the myocardium to form epicardial-derived cells (EPDCs) including cardiac fibroblasts, coronary smooth muscle cells and at least a subset of endothelial cells3,4. In addition, mounting evidence points to the epicardium as having a paracrine role in modulating injury responses as well as being a source of progenitor cells in facilitating neovascularization and myocardial repair5,6. Particularly, epicardial cells were shown to contribute to the cardiomyocyte lineage, highlighting their cardiogenic potential in cardiac regeneration and repair7. Therefore, understanding the molecular mechanisms that control the specification and self-renewal of epicardial lineages from naive progenitor cells is of fundamental importance to elucidate the regulatory mechanisms underlying both human heart development and cardiovascular diseases.
The advent of human pluripotent stem cells (hPSCs) significantly advanced the study of early human embryonic development, disease modelling and cell-based therapy strategies8. Recently, there have been substantial advances in elucidating the regulatory mechanisms that control cardiomyocyte9,
To address these issues, we generated self-renewing epicardial cells from hPSCs and investigated key signalling pathways during epicardial development and regeneration. We generated a WT1-2A-eGFP knock-in human embryonic stem cell (hESC) line and showed that temporal modulation of canonical Wnt signalling via small molecules is sufficient for epicardial induction from hPSCs under chemically defined, xeno-free conditions, and that a TGF-β-signalling-inhibitor treatment allows long-term self-renewal of hPSC-derived epicardial cells. In addition, these hPSC-derived epicardial cells retain many characteristics of primary epicardial cells, including formation of a polarized epithelial sheet, expression of the key epicardial genes WT1, TBX18 and ALDH1A2, and the ability to form smooth muscle cells and cardiac fibroblasts both in vitro and in vivo. RNA sequencing (RNA-seq) expression data and gene-set enrichment analysis (GSEA) confirm a similarity of hPSC-derived epicardial cells to primary human epicardial cells. These findings improve our understanding of differentiation and self-renewal mechanisms of the epicardium, and have implications for stimulating epicardial regeneration via small molecules or cell-based therapies.
Albumin-free conditions to generate cardiac progenitors
We previously demonstrated that temporal modulation of canonical Wnt signalling in RPMI basal medium (known as the GSK3 inhibitor, WNT inhibitor (GiWi) protocol) is sufficient to generate functional cardiomyocytes (CMs) from hPSCs21. We found that ISL1+NKX2.5+FLK-1+ cardiac progenitors are generated as intermediates during the GiWi protocol (Supplementary Fig. 1A). When re-passaged on gelatin-coated plates in LaSR basal medium14 on day 6, these hPSC-derived cardiac progenitors expressed progenitor markers including ISL1, NKX2.5 and FLK-1, as well as the proliferation marker Ki67 (Supplementary Fig. 1B). Molecular analysis of cardiac progenitor differentiation from hPSCs revealed dynamic changes in gene expression, with downregulation of the pluripotency markers OCT4 and NANOG, and induction of the primitive-streak-like gene T (refs 14,22) in the first 24 h after CHIR99021 (CHIR) addition (Supplementary Fig. 1C). Expression of the cardiac progenitor markers ISL1, NKX2-5 and FLK-1 was first detected between days 3 and 5, and was significantly upregulated at day 6 (Supplementary Fig. 1C).
Wnt/β-catenin signalling regulates epicardial specification
Pro-epicardium arises from ISL1+NKX2.5+ second-heart-field progenitors in vivo 1,23. To identify signalling mechanisms regulating cardiac progenitor specification to epicardial cells, we treated day 6 cardiac progenitors with different small molecules and protein modulators of developmental signalling pathways for 48 h (days 7 to 9) (Fig. 1a and Supplementary Table 1). hPSC-derived cardiac progenitors formed more than 85% WT1+ putative epicardial cells following CHIR treatment (Fig. 1b,c and Supplementary Movie 1), demonstrating that Wnt signalling induction between days 7 and 9 is sufficient to generate epicardial cells from cardiac progenitor cells in the absence of other exogenous signalling. In the absence of CHIR, robust beating sheets of cTnT+ cardiomyocytes were observed (Fig. 1b,c and Supplementary Movie 2), suggesting that the activation status of canonical Wnt signalling at day 7 toggles epicardial versus cardiomyocyte differentiation. Interestingly, untreated and bone morphogenetic protein 4 (BMP4)-, dorsomorphin (DM)- and retinoic acid (RA)-treated cells also yielded about 10% WT1+ cells that were distinct from cTnT+ cells (Fig. 1b,c and Supplementary Fig. 2A). Compared to the untreated condition, BMP4 did not significantly increase the WT1+ cell population up to 100 ng ml−1 when applied from days 7–9 (Supplementary Fig. 2B). To exclude the potential risk of using low-quality BMP4 protein, we tested the function of BMP4 used in our study by inducing brachyury expression according to a previous report24. As expected, 5 ng ml−1 BMP4 treatment for 2 days in mTeSR1 resulted in robust expression of brachyury (Supplementary Fig. 2C). In the presence of CHIR, BMP4 treatment did not generate cardiomyocytes, but instead yielded an unknown population at the expense of WT1+ cells. Inhibition of BMP4 signalling via DM resulted in a similar purity of WT1+ cells as CHIR treatment alone (Fig. 1b and Supplementary Fig. 2A), suggesting that BMP4 signalling is dispensable at this stage of epicardial development.
Construction of WT1-2A-eGFP knock-in reporter hPSCs
WT1 is required for the development of epicardium25 and for the formation of cardiovascular progenitor cells26. To better monitor the epicardial cell differentiation process and purify hPSC-derived epicardial cells in vitro, we engineered the ES03 cell line via CRISPR/Cas9-catalysed homology-directed repair (HDR) and generated a homozygous WT1-2A-eGFP knock-in reporter cell line (Fig. 2a). Two 2-kilobase homologous arm sequences located just upstream and downstream of the WT1 stop codon were inserted into the Oct4-2A-eGFP donor plasmid27 and replaced the Oct4 homologous arms. We then introduced the 2A-eGFP sequence into the target sites by transfecting hESCs with the WT1-2A-eGFP donor plasmid and the Cas9/sgRNA plasmids. After puromycin (Puro) selection, PCR genotyping and sequencing showed that approximately 50% (21 out of 44) of the clones were targeted in one (heterozygous) and approximately 25% (12 out of 44) in both alleles (Fig. 2b), similar to a previous report28. The homozygous clones were then subjected to TAT-Cre recombinase treatment and the PGK-Puro cassette was excised from WT1-2A-eGFP (Fig. 2c). WT1-2A-eGFP-targeted hPSCs after Cre-mediated excision of the PGK-Puro cassette were subjected to CHIR treatment; eGFP was detected at day 10 and elevated at day 12 (Fig. 2d). Dual immunostaining with anti-WT1 and anti-GFP antibodies detected expression of eGFP in WT1+ cells (Fig. 2e), demonstrating the successful generation of a WT1 reporter cell line for potential cell tracking or purification.
Chemically defined conditions to generate epicardial cells
We next optimized the concentration of CHIR and initial seeding density of cardiac progenitors at day 6 in LaSR basal medium, and found that 3 μM CHIR with an initial density of 0.06 million cells cm−2 yielded more than 95% WT1+ cells (Supplementary Fig. 3A–D), whereas the control (no CHIR) resulted in less than 10% WT1-2A-eGFP cells. However, LaSR basal medium, which contains bovine serum albumin, adds xenogenic components to the medium, which would not be desirable for the generation of epicardial cells that meet clinical requirements. In order to develop a xeno-free protocol, we systematically screened four commercially available basal media supplemented with 1 μg ml−1 human recombinant insulin and 100 μg ml−1 ascorbic acid (vitamin C), as these two factors were shown to improve the culture of cardiac cell lineages29,
Epicardial cell differentiation is β-catenin dependent
Selectivity is a concern when using chemical inhibitors of signalling pathways. Therefore, we tested other GSK3 inhibitors including BIO-acetoxime and CHIR98014 in the GiWiGi protocol, and found that 0.3 μM CHIR98014 and BIO-acetoxime generated WT1+ cells as effectively as 3 μM CHIR99021 (Supplementary Fig. 4A). In addition, we treated day 6 cardiac progenitors with Wnt3a of concentrations up to 500 ng ml−1, and found that Wnt3a significantly increased the WT1+ cell population compared with the no-Wnt3a control, although Wnt3a was less effective than small-molecule GSK3 inhibitors in generating WT1+ cells (Supplementary Fig. 4B). To further investigate the role of β-catenin in the GiWiGi epicardial differentiation, we employed an inducible pluripotent stem cell (iPSC) line (19-9-11 ischcat-1) expressing β-catenin shRNA under the control of a tet-regulated inducible promoter described in earlier work10. Upon doxycycline (dox) treatment, the shRNA efficiently downregulated β-catenin expression10. We also showed that the induction of NKX2.5+ISL1+ cardiac progenitors from hPSCs is β-catenin dependent10. In this study, we therefore focused on the examination of the stage-specific roles of β-catenin during the differentiation of epicardial cells from cardiac progenitors stimulated by GSK3 inhibition. We found that β-catenin knockdown at day 6 yielded significantly fewer WT1+ cells, instead generating robust beating sheets of cTnT+ cardiomyocytes at the expense of WT1+ cells (Supplementary Fig. 4C,D and Supplementary Movie 3). This finding is consistent with reports that Wnt/β-catenin inhibition is necessary for cardiomyocyte formation from cardiac progenitors both in vitro and in vivo 10,18,32,33, and further supports the notion that Wnt/β-catenin signalling regulates epicardial versus cardiomyocyte specification from cardiac progenitors. The inhibitory effects of β-catenin knockdown on WT1+ cell generation gradually diminished after day 6, with no inhibition after day 9 (Supplementary Fig. 4C,D and Supplementary Movies 3,4).
Molecular characterization of hPSC-derived epicardial cells
Pro-epicardial cells are marked by the expression of TBX18, WT1 and TCF2134,
Long-term expansion of hPSC-derived WT1+ epicardial cells
Primary mouse epicardial cells have been cultured for more than 3 years34, but epicardial cells isolated from the adult human heart rapidly undergo EMT in culture20. Similar to primary human epicardial cells, hPSC-derived WT1+ epicardial cells only retained their morphology for approximately 2 weeks in culture. Although, in the short term, hPSC-derived WT1+ epicardial cells retained the epicardial cobblestone-like morphology, basic fibroblast growth factor (bFGF)- and TGF-β1-treated cells adopted a fibroid spindle or fusiform-shaped appearance typical of cultured fibroblasts and smooth muscle cells, respectively (Fig. 4a,b). The expression of calponin and smooth muscle myosin heavy chain (SMMHC) in TGF-β1 + bFGF-induced cultures further support their smooth-muscle-cell identity, and vimentin (VIM) and CD90 expression support their fibroblast identity (Supplementary Fig. 7). We also cultured WT1+TBX18+ epicardial cells in endothelial cell medium, but did not detect expression of the endothelial markers CD31 and VE-cadherin (Supplementary Fig. 7). The expression of WT1 and ZO1 significantly decreased in both bFGF- and TGF-β1-treated samples, indicating the transition from epithelial towards mesenchymal-like cells (Fig. 4b). In addition, CDH1 (E-cadherin) expression decreased whereas CDH2 (N-cadherin) and SNAIL2 expression increased in all treated samples compared with untreated controls, suggesting that bFGF and TGF-β1 induced an EMT in hPSC-derived epicardial cells (Fig. 4c,d).
During long-term in vitro culture, hPSC-derived WT1+ epicardial cells spontaneously underwent an EMT and lost WT1 expression after several passages, even without exogenous TGF-β1 or bFGF treament (Fig. 5a). To identify signalling mechanisms regulating hPSC-derived epicardial cell self-renewal, we applied small molecules (Supplementary Table 1) that affect pathways that regulate cell proliferation, to study their effects on WT1+ cell self-renewal. TGF-β receptor 1 antibody (α-TGF-βI) and α-TGF-β pan-specific antibody (α-TGF-β Pan) partially increased the proliferation of epicardial cells and maintained their epithelial phenotype (Fig. 5a,b). A83-01, an inhibitor of TGF-β signalling, enabled expansion of hPSC-derived epicardial cells that retained polarized epithelial morphology and WT1 expression to a significantly greater extent than the untreated control (Fig. 5a,b). To reduce the likelihood that WT1+ cell self-renewal was an off-target effect of A83-01, we tested additional TGF-β inhibitors (RepSox, SB505124 and SB431542) and found that they also promoted the proliferation of hPSC-derived epicardial cells to a similar extent as A83-01 (Fig. 5b,c). Following A83-01 or SB431542 addition, hPSC-derived epicardial cells were capable of at least 25 population doublings, generating more than 10 million cells from a single hPSC-derived epicardial cell clone (Fig. 5c). After 48 days of expansion, the TGF-β-inhibitor-treated cells expressed significantly higher levels of WT1 and Ki67 than the untreated control cells (Fig. 5d). Epicardial cells generated from 19-9-11 iPSCs were also expandable after A83-01 treatment (Supplementary Fig. 8A), presenting a cobblestone morphology and expressing high levels of ALDH1A2, WT1 and TBX18 (Supplementary Fig. 8B–D). In addition, epicardial cells expanded for 48 days retained the potential to differentiate into SMMHC+calponin+ smooth muscle cells and VIM+CD90+ fibroblasts following TGF-β1 and bFGF treatment, respectively (Supplementary Fig. 8D). The day 50 epicardial cells retained normal karyotypes after long-term culture in medium containing A83-01 (Supplementary Fig. 9). These findings improve our understanding of self-renewal mechanisms of the epicardium and have implications for generating large quantities of hPSC-derived epicardial cells for research or cell-based therapy applications.
hPSC-derived epicardial cells were similar to primary cells
To further confirm the identity of hPSC-derived epicardial cells, RNA from four different hPSC-derived epicardial cell differentiations at two different expansion time points (days 12 and 48) and primary epicardial cells of four different donors were subjected to RNA-seq analysis. Hierarchical clustering analysis of RNA-seq expression data of hPSCs38,
Epicardial cells can undergo an EMT and give rise to cardiac fibroblasts and smooth muscle cells after transplantation into chicken embryos19 or infarcted mouse hearts20. To examine the ability of hPSC-derived epicardial cells to invade the myocardium and undergo an EMT in vivo, cardiac-fibroblast-derived extracellular matrix (CF-ECM) patches seeded with passage 5 eGFP-labelled hPSC-derived epicardial cells (Fig. 6c) were transferred to the heart surface (Supplementary Fig. 10C) of a mouse myocardial infarction model. eGFP+ cells were detected predominantly within the CF-ECM scaffold and in the epicardium beneath the scaffold before day 6, and scattered within the mid-myocardium after 12 days (Supplementary Fig. 10D,E), suggesting that epicardial cells invaded the myocardium. In addition, the hPSC-derived cells underwent an EMT and differentiated into αSMA+calponin+ smooth-muscle-like cells (Fig. 6d,e and Supplementary Fig. 10F,G) and VIM+ fibroblast-like cells (Supplementary Fig. 10F) in vivo. We did not observe any tumour formation 12 days after hPSC-derived epicardial cell transplantation into the mouse heart compared with the patch-only control (Supplementary Fig. 10H). These findings demonstrate that hPSC-derived epicardial cells can invade the myocardium and form EPDCs after infarction, underscoring their potential for cell-based therapeutic heart regeneration.
While differentiation of hPSCs to epicardial cells by applying complex mixtures of growth factors from different signalling pathways has been reported18,19, it is largely unknown which developmental signalling pathways are necessary and sufficient to specify epicardial cell fate and to control their self-renewal, limiting their large-scale production for clinical applications. This study reports the generation of a WT1-2A-eGFP knock-in stem cell line, and demonstrates efficient and robust generation of epicardial cells from multiple hPSC lines solely via stage-specific manipulation of Wnt/β-catenin signalling under chemically defined, albumin-free, xeno-free conditions. These hPSC-derived epicardial cells retain many characteristics of primary epicardial cells, including formation of an epithelial sheet, expression of the key epicardial proteins WT1, TBX18 and ALDH1A2, and the ability to generate fibroblast and vascular smooth muscle lineages both in vitro and in vivo. Their identity was further confirmed by RNA-seq expression data and GSEA at a global level. Using inducible knockdown hPSC lines, we showed that β-catenin is essential for epicardial cell induction from hPSC-derived cardiac progenitors under the GiWiGi protocol. Given the essential roles of β-catenin during cardiac progenitor induction from hPSCs10,43, we conclude that β-catenin is required at multiple stages of hPSC differentiation to epicardial cells via small-molecule modulation of canonical Wnt signalling. Differences between our study and previous reports18,19, including the initial starting cardiac progenitor cells and the exposure windows for signalling modulators, may account for their conclusion that BMP4 signalling, but not Wnt alone, is required for robust epicardial differentiation.
This study also demonstrates long-term self-renewal of hPSC-derived epicardial cells via TGF-β-inhibitor treatment in a chemically defined medium. For cell-based therapeutic applications, it is highly desirable to generate homogeneous committed progenitors that can expand in culture and differentiate into various tissue-specific cells of interest, avoiding the contamination of unwanted cell lineages, especially tumorigenic hPSCs44. We showed that TGF-β- inhibitor treatment is sufficient for the self-renewal of hPSC-derived epicardial cells, in contrast to primary mouse epicardial cells, which can self-renew in the absence of a TGF-β inhibitor34. It will also be interesting to test whether or not TGF-β-inhibitor treatment can promote the self-renewal of human primary epicardial cells in vitro. Recent work has demonstrated that epicardial cell lineages improved the performance of the scarred myocardium by preservation of cardiac function and attenuation of ventricular remodelling after transplantation into a myocardial infarction model45. More recently, it has been reported that the epicardium of the zebrafish heart is required for muscle regeneration and can itself regenerate following Sonic hedgehog (Shh) treatment46. Our results suggest that TGF-β inhibitors may impact heart regeneration following injection into the epicardium in vivo, similarly to strategies used to test the effect of TGF-β inhibitors on scar formation after glaucoma surgery in rabbits47.
In summary, our findings support a model (Fig. 7) of human epicardial development in which small-molecule-mediated exogenous modulation of Wnt/β-catenin signalling is sufficient for the specification of epicardial cells from hPSCs. This finding is consistent with the report that DKK1 and DKK2 double-null mice increase epicardial specification and display a hypercellular epicardium48. This completely defined, xeno-free epicardial differentiation platform is compatible with current good manufacturing practice (cGMP) standards and can be employed to efficiently derive self-renewing epicardial cell lineages from hPSCs, which can thereby provide insights into mechanisms of heart development, maturation and response to cardiac injury. Moreover, we show that hPSC-derived epicardial cells can invade the myocardium in an infarcted mouse model, suggesting potential applications in cell-based heart regeneration. Our results also point to TGF-β signalling as a regulator of epicardial cell self-renewal and differentiation, indicating the potential of TGF-β signalling modulators in heart regeneration.
Construction of donor plasmid and sgRNA
Human codon-optimized Streptococcus pyogenes wild-type Cas9 (pCas9-2A-eGFP) was obtained from Addgene (plasmid no. 44719) and chimeric guide RNA expression cassette was cloned into this Cas9-2A-eGFP plasmid with two BbsI restriction sites for rapid sgRNA cloning. Two sgRNAs targeting at or near WT1 stop codon (1: AACTCCAGCTGGCGCTTTGAGGG and 2: GGACACTGAACGGTCCCCGAGGG) were used. To generate the WT1-2A-eGFP donor plasmid, DNA fragments of about 2 kb in length were PCR amplified from the genomic DNA before and after the stop codon of WT1 and were cloned into the OCT4-2A-eGFP donor plasmid27 (Addgene no. 31938), replacing the OCT4 homologous arms.
Maintenance of hPSCs and TAT-Cre treatment of WT1 knock-in hPSCs
Transgene and vector-free hPSCs were maintained on Matrigel (Corning) or SyntheMax (BD Biosciences) coated plates in mTeSR1 or E8 medium (STEMCELL Technologies), according to previously published methods49. To remove the PGK-Puro cassette from the WT1-2A-eGFP cells, targeted homozygous clones were treated with 2 μM TAT Cre recombinase (Excellgen, EG-1001) for 6 h in E8 medium. After two days, cells were singularized with Accutase and seeded into a Matrigel-coated 96-well plate at a density of 100 to 150 cells per well. After two weeks, the cells were subjected to PCR genotyping.
hESCs were pre-treated with 10 μM ROCK inhibitor (Y27632) for 3 to 4 h before electroporation. Cells were digested by Accutase (Innovative Cell Technologies) at 37 °C for 8 min and 2.5–3 million single cells were electroporated with 3 μg gRNA1, 3 μg gRNA2 and 6 μg WT1-2A-eGFP donor plasmids in 200 μl cold PBS−/− using the Gene Pulser Xcell System (Bio-Rad) at 320 V, 200 μF and 1,000 Ω (time constant should be around 15 ms) in a 0.4 cm cuvette. Two electroporations were performed and 5–6 million cells were subsequently plated onto a Matrigel-coated 10 cm dish in 10 ml mTeSR1 with 10 μM Y27632. 24 h later, and every day afterwards, the medium was changed with fresh mTeSR1. Three days after electroporation, 1 μg ml−1 puromycin was added into the mTeSR1 for selection for about two weeks. Single cell clones were then picked into wells of a Matrigel-coated 96-well plate and subjected to PCR genotyping after 4 to 7 days.
Cardiac progenitor induction via modulation of canonical Wnt signalling
As described previously, hPSCs were singularized with Accutase (Innovative Cell Technologies) at 37 °C for 5 min once they achieved confluence and then seeded at 100,000–250,000 cells per cm2 in mTeSR1 or E8 supplemented with 5 μM ROCK inhibitor Y-27632 (Selleckchem) (day −3) for 24 h14,21. Cells were then cultured in mTeSR1 or E8, which was changed daily. At day 0, cells were treated with 6 μM CHIR99021 (Selleckchem) for 24 h in RPMI medium, followed by a change with RPMI medium at day 1. 2.5–5 μM IWP2 (Tocris) was added at day 3 and removed during the medium change at day 5. Cardiac progenitor cells can also be efficiently generated in RPMI/B27− medium using our previous GiWi protocol11.
Epicardial cell generation via activation of canonical Wnt signalling
At day 6, cardiac progenitor cells were singularized with Accutase at 37 °C for 5 min and then seeded onto a gelatin- or SyntheMax-coated cell culture plate at 20,000–80,000 cells per cm2 (or a 1:3–1:12 split) in LaSR basal medium (advanced DMEM/F12 with 100 μg ml−1 ascorbic acid) or RPMI/Vc/Ins medium (100 μg ml−1 ascorbic acid and 1 μg ml−1 human recombinant or bovine insulin (Sigma)) with 5 μM ROCK inhibitor Y-27632 for 24 h. The addition of 1% human recombinant albumin (Sigma) or fetal bovine serum (FBS) can improve cell attachment and survival, but they are not required. At day 7, cells were treated with 1–9 μM CHIR for 2 days in LaSR basal medium or RPMI/insulin/Vc medium. CHIR-containing medium was aspirated on day 9 and cells were cultured in LaSR basal medium or RPMI/insulin/Vc medium without CHIR99021 for 3–5 additional days, as explained previously14.
Long-term maintenance of hPSC-derived epicardial cells
To expand the epicardial cells, confluent cells on day 12 of differentiation or day 4 after expansion were split 1:3 to 1:9 at a density of 0.03 to 0.09 million cells per cm2 using Versene (Life Technologies) or Accutase and routinely passaged onto gelatin-coated plates in LaSR basal medium or RPMI/Vc/Ins medium and 0.5 μM A83-01 (Stemgent) or 2 μM SB431542 (Stemgent) with medium changed daily until the cells reached confluence. Overnight treatment of 5 μM Y27632 and 1% human recombinant albumin on single cells during passage was used to improve cell attachment and survival, but they were not required once cells attached. H13 hESC-derived day 50 epicardial cells were subjected for karotype analysis at WiCell Research Institute.
Single-cell passage and EMT induction
Confluent WT1+ cells were singularized with Accutase at 37 °C for 5 min and then seeded onto a gelatin-coated cell culture plate at a density of 10,000 cells cm−2 in LaSR basal medium supplemented with 5 μM Y-27632 for 24 h. After 24 h, the medium was changed to LaSR basal medium and cells were treated with TGF-β1 or bFGF (R&D Systems) as indicated. Medium was changed every 3 days until analysis.
Cardiac-fibroblast-derived extracellular matrix (CF-ECM) to transfer epicardial cells in vivo to the infarcted heart
Immunodeficient mice were purchased from Harlan Laboratories and all procedures were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee. As described previously50, myocardial infarction was induced 48 h before the transplantation. CF-ECM scaffolds were generated according to a previous report50. Briefly, cardiac fibroblasts isolated from porcine hearts were plated at a density of 0.1–0.25 million cells per cm2 in high-glucose DMEM supplemented with 10% FBS and cultured at 37 °C and 5% CO2 for 10–14 days. The combined cardiac fibroblasts and extracellular matrix were removed from the culture dish by incubation with 2 mM EDTA solution at 37 °C after a 10- to 14-day culture. The resulting cell sheet was treated with molecular grade water followed by 0.15% peracetic acid (PAA buffer) for 24–48 h at 4 °C with constant agitation to denude the cells. The resulting matrix was then rinsed with sterile water followed by PBS several times. CF-ECM scaffold was then seeded with 1–5 million ES03 eGFP hPSC-derived epicardial cells and incubated for 3 h before transfer to the epicardial surface of the MI area. After transplantation, the chest was closed. After 2, 6 and 12 days, the mouse hearts were harvested and excised for histology.
As explained in a previous study13, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then stained with primary and secondary antibodies (Supplementary Table 6) in PBS plus 0.4% Triton X-100 and 5% non-fat dry milk (Bio-Rad). Nuclei were stained with Gold Anti-fade Reagent with DAPI (Invitrogen). An epifluorescence microscope (Leica DM IRB) with a QImaging Retiga 4000R camera was used for imaging analysis.
As described in a previous report14, cells were dissociated into single cells with Accutase for 10 min and then fixed with 1% paraformaldehyde for 20 min at room temperature and stained with primary and secondary antibodies (Supplementary Table 6) in PBS plus 0.1% Triton X-100 and 0.5% BSA. Data were collected on a FACS Caliber flow cytometer (Beckton Dickinson) and analysed using FlowJo. FACS gating was based on the corresponding isotype antibody control.
Genomic DNA extraction and genomic PCR
Quick Extract DNA Extraction Solution (Epicentre Cat. no. QE09050) was used to rapidly extract genomic DNA from hESCs according to the manufacturer’s instructions. Genomic PCR was carried out using GoTaq Green Master Mix (Promega Cat. no. M7123). PCR primer sequences are provided in Supplementary Table 7.
RT-PCR and quantitative RT-PCR
As explained previously14, total RNA was prepared with the RNeasy mini kit (QIAGEN) and treated with DNase (QIAGEN). 1 μg RNA was reverse transcribed into cDNA via Oligo (dT) with Superscript III Reverse Transcriptase (Invitrogen). Real-time quantitative PCR was done in triplicate with iQSYBR Green SuperMix (Bio-Rad). GAPDH was used as an endogenous housekeeping control. PCR primer sequences are provided in Supplementary Table 7.
RNA sequencing and data analysis
Total RNA of day 12, 19-9-7 iPSC-derived epicardial cells and day 48, 19-9-11, ES03, H9 hPSC-derived epicardial cells were prepared with the Direct-zol RNA MiniPrep Plus kit (Zymo Research) according to the manufacturer’s instructions. Human primary epicardial RNAs from four different donors were provided by our collaborator (AstraZeneca, Sweden). Samples were performed in IIIumina HiSeq 2500 by the Biotechnology Center at the University of Wisconsin-Madison. The resulting sequence reads were mapped to the human genome (hg19) using HISAT51, and the RefSeq transcript levels (RPKMs) were quantified using the python script rpkmforgenes.py52. Hierarchical clustering of whole transcripts were then plotted using GENE-E. Fastq files of hPSCs38,
As described in a previous report43, cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce) in the presence of Halt Protease and Phosphatase Inhibitor Cocktail (Pierce). Proteins were then separated by 10% Tris-Glycine SDS/PAGE (Invitrogen) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk in TBST, the membrane was incubated with primary antibody (Supplementary Table 6) overnight at 4 °C. The membrane was then washed, incubated with an anti-mouse/rabbit peroxidase-conjugated secondary antibody for 1 h at room temperature or overnight at 4 °C, and developed by SuperSignal chemiluminescence (Pierce).
Data are presented as mean ± standard error of the mean (s.e.m.). Statistical significance was determined by Student’s t-test (two-tail) between two groups, and three or more groups were analysed by one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
The final processed data and raw fastq files were submitted to Gene Expression Omnibus (GEO) with the accession number GSE84085. Source data for some of the figures in this study are available in figshare with the identifier http://dx.doi.org/10.6084/m9.figshare.3971748 (ref. 54). The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information.
How to cite this article: Bao, X. et al. Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions. Nat. Biomed. Eng. 1, 0003 (2016).
We thank D.A. Roenneburg and X. Wang for their technical support. We also thank members of the Palecek group for critical discussion of the manuscript. This work was supported by NIH grant EB007534 (S.P.P.), NSF grant 1547225 (S.P.P.), and a fellowship from the University of Wisconsin Stem Cell and Regenerative Medicine Center (X.B.).
Non-contracting hESC-derived pro-epicardial cells at day 12.
Spontaneously contracting hESC-derived cardiomyocytes at day 12.
Spontaneously contracting hESC-derived cardiomyocytes at day 12.
Non-contracting iPSC-derived pro-epicardial cells at day 12.