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Dual Reporter MESP1mCherry/w-NKX2-5eGFP/w hESCs Enable Studying Early Human Cardiac Differentiation

Dual Reporter MESP1mCherry/w-NKX2-5eGFP/w hESCs Enable Studying Early Human Cardiac Differentiation Abstract Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1mCherry/w-NKX2-5eGFP/w reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-α, CD13, and ROR-2. No enrichment was found for the previously described KDR+PDGFR-α+ progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives. Stem Cells 2015;33:56–67 Embryonic stem cells, Mesoderm, MESP1, NKX2-5, Cardiac differentiation, Cardiac progenitors Introduction Human pluripotent stem cells (hPSCs) offer exceptional opportunities for regenerative medicine, drug discovery and toxicity, disease modeling, and developmental biology because of their ability to differentiate in principle into any cell type of the human body. In particular, their differentiation toward cell types that are challenging to obtain and maintain as primary cultures from tissues with limited or no self-repair capacity, such as the brain and heart, have made them attractive as research models. Previously, we and others have shown that cardiomyogenesis of hPSCs in vitro recapitulates signaling events and sequential transcriptional activation that occurs during heart development in vivo [1, 2]. Moreover, as knowledge of the regulatory mechanisms that govern in vivo cardiac development has advanced, it has been possible to translate this to differentiation protocols based on stepwise temporal modification of signaling pathways. This has led to a remarkable increase in the yield of cardiac cells not only from human embryonic stem cells (hESCs) but also from human induced pluripotent stem cells (reviewed in [3]). However, despite these advances, the cardiac derivatives obtained contain heterogeneous populations of cardiac cells: not only different subtypes of cardiomyocytes but also endothelial cells and smooth muscle cells. This is because the molecular cues underlying specification of early human cardiac progenitors toward specific cardiac subtypes have not been fully elucidated. For future clinical and pharmaceutical applications, better insight into the differentiation of cardiac cell subtypes is necessary. Several studies have shown that different cardiac subtypes are derived from multiple early cardiac progenitors that segregate soon after gastrulation [4-6]. The molecular mechanisms that play a role in the formation of early cardiac progenitors, however, remain largely unknown [4, 7]. In order to study the onset of cardiac differentiation in human, it is necessary to identify early populations from common progenitors at the mesodermal stage of development and identify the molecular pathways involved in the further differentiation of the cardiac lineage. Mesoderm transcription factor MESP1 is important in this context as in vivo lineage tracing in mouse has demonstrated that almost all cells of the heart are derived from a common MESP1-expressing mesodermal progenitor, including myo-, endo, and epicardium, and cells of the conduction system [8-10]. MESP1 is transiently expressed in the anterior mesoderm from the onset of gastrulation prior to cardiac crescent formation [8, 11, 12]. Furthermore, MESP1 overexpression in mouse ESCs showed that MESP1 induces the expression of cardiovascular genes [13-15]. A recent study in mouse illustrated that besides the cardiac lineage, MESP1 derivatives also encompass cells of the hematopoietic- and skeletal myogenic lineages, which emerge depending on the endogenous and exogenous cues present during MESP1 expression [16]. Of note, all these studies have been performed in mice. However, to instigate advances in human heart development, regenerative medicine, disease modeling, and drug testing, it is necessary to understand the earliest steps of cardiac differentiation in a human model. In order to identify, isolate, and characterize MESP1 expressing cells during human cardiomyogenesis, we introduced the mCherry reporter into the MESP1 locus of the NKX2-5eGFP hESC reporter line generated previously [17], leading to a dual reporter system for these early cardiovascular lineage markers in hESCs. We were able to isolate the MESP1-expressing mesoderm progenitors in a cardiac-inducing environment and demonstrate their ability to differentiate further to a highly enriched NKX2-5eGFP expressing cardiac population. We further confirmed the presence of other mesoderm and cardiac progenitor populations in MESP1-mCherry expressing-derivatives, including CD13+ROR-2+ and PDGFR-α+KDR+ populations [18-20]. Although CD13, ROR-2, and PDGFR-α expression were strongly enriched, they were not specifically expressed in the MESP1-mCherry derivatives. In addition, no enrichment was found for the previously described PDGFR-α+KDR+ population. Finally, we demonstrate that this MESP1mCherry-NKX2-5eGFP dual reporter hESC line is a useful tool to study the differentiation of precardiac mesoderm toward early cardiac progenitor cells and their derivatives. It now provides an opportunity to study the molecular mechanisms underlying the onset of progenitor subtypes formation specifically in the cardiac lineage in human. This hESC dual reporter will contribute to providing better insights into cardiomyocyte determination and form the basis for improved differentiation protocols for specifying cardiac subtypes (atrial, ventricular, and pacemaker cells). This will be relevant to future pharmaceutical and clinical applications of specific cardiac cell subtypes. Materials and Methods MESP1 Gene Targeting in hESCs To generate the MESP1 targeting vector, the bacterial artificial chromosome RP11-975L13 (Source Bioscience, Lifesciences, Nottingham, UK) was modified by recombineering (Gene Bridges, Heidelberg, Germany). The mCherry cassette and a selection marker replaced the MESP1 exon 1 sequence (oligonucleotides used for recombineering: MESP1_5arm_PvuI and MESP1_3_arm, Supporting Information Table S1). The modified MESP1 locus and the surrounding 5′ and 3′ homology arms were subsequently subcloned into the MESP1 targeting vector. The final vector comprised a 8.6-kilobase (kb) 5′ homology arm, mCherry, loxP-flanked G418 resistance cassette, and a 2.3-kb 3′ homology arm. The targeting vector was linearized by PvuI digestion and 20 µg was electroporated into 9 × 106 NKX2-5-eGFP reporter hESCs (HES3-NKX2-5eGFP/w) [17] as described previously [21]. Targeted clones were identified by PCR using oligonucleotides specific for mCherry (P1) and a region immediately 3′ of the targeting vector (P2) (Supporting Information Fig. S1, Table S1). Targeted clones were clonally derived and transfected with Cre-recombinase to excise the loxP-flanked neomycin-resistance cassette as described previously [22]. Neomycin excision was confirmed by PCR (Supporting Information Fig. S1, Table S1). Undifferentiated, targeted clones were fixed in 2% paraformaldehyde and were used for immunocytochemistry (Nanog, Tra-181, and Oct-4) to confirm their pluripotent status. For karyotyping, 20 metaphase chromosome spreads were examined for each line. Single-Cell Sorting For single-cell sorting, hESCs were enzymatically passaged using 1× TrypLE Select (Life Technologies, NY, USA). After dissociation, cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% BSA and 2.5 mM EDTA) and filtered through a 40-µm cell strainer (Falcon). Cells were sorted using a BD ARIA III flow cytometer into 96-well flat-bottomed low attachment plates, live cells were gated on the basis of side scatter, forward scatter, and propidium iodide exclusion. Flow cytometric gates were set using control cells labeled with the appropriate isotype control antibody. hESC Culture and Cardiac Differentiation MESP1mCherry/w NKX2-5eGFP/w hESCs were cultured on mouse embryonic fibroblasts on 2.85 cm2 organ dishes (Falcon), in 1 ml hESC medium (Dulbecco's modified Eagle's medium F-12, nonessential amino acids, knockout serum [Gibco by Life Technologies]; basic fibroblast growth factor [Miltenyi Biotec, Bergisch Gladbach, Germany]) and were mechanically passaged [23]. One or two days before differentiation, cells were passaged onto a 12-well tissue culture dish coated with Matrigel (BD Biosciences, New Jersey; growth factor reduced, phenol red-free), in 1 ml of hESC medium per well. On the first day of differentiation, day 0, hESC medium was replaced by the low insulin serum-free medium BPEL (BSA, polyvinyl alcohol, essential lipids, as previously described) [17, 24], containing the cytokines BMP4 (30 ng/ml, R&D Systems, Minneapolis) and Activin A (30 ng/ml, Miltenyi Biotec), and Chir99021 (1.5 µM, Axon Medchem, Groningen, The Netherlands). At day 3 of differentiation, the medium was refreshed with BPEL containing 5 µM Xav939 (R&D). On day 7 of differentiation, the medium was replaced with BPEL without growth factors. Isolation and Reaggregation of MESP1-mCherry Cells On day 3 of differentiation, cells were sorted by flow cytometry as described above, based on mCherry expression (both positive and negative populations). Flow cytometric gates were set using undifferentiated hESCs. Sorted cells were seeded in a 96-well V-shaped nonadherent plate (Greiner Bio One, Alphen a/d Rijn, The Netherlands) at 20,000 cells per well in 50 µl BPEL in the presence of Xav939 (5 µM, R&D) to enhance cardiac differentiation. Aggregation was initiated by a brief centrifugation step for 3 minutes at 480g. Four days after replating, aggregates were refreshed with BPEL without addition of growth factors or inhibitors. Analysis of MESP1-mCherry Derivatives Cardiomyocytes derived from MESP1-mCherry-sorted cells were analyzed by flow cytometry and immunohistochemistry (IHC) on days 3, 5, and 14 of differentiation (0, 2, and 11 days after replating). For IHC, MESP1-mCherry-derived aggregates were fixed in 2% paraformaldehyde (PFA) at day 14. For alpha-actinin and SM-HC IHC, MESP1-mCherry-derived aggregates were dissociated using 1× TrypLE Select, plated on matrigel in BPEL, and fixed after 4 days. For flow cytometric analysis, aggregates were collected, dissociated as described before, and resuspended in FACS washing buffer (PBS with 2% fetal calf serum (FCS) and 2.5 mM EDTA). For analysis of intracellular markers, cells were treated with FIX & PERM Cell Fixation and Permeabilization Kit (Life Technologies). Analyses were carried out using a MACS-Quant VYB flow cytometer (Miltenyi Biotec) and FlowJo Software. Antibodies are listed in Supporting Information Table S4. Specimens were analyzed with confocal laser microscope (Leica TCS SP8). Images were acquired with Leica LAS AF software. MESP1-mCherry expressing cells were visualized in monolayer differentiation at day 3, using a Nuance Multispectral Imaging System. RNA Isolation, Quantitative Real-Time PCR, and Microarray Analysis Total RNA was isolated using either the RNeasy micro (Qiagen) or Nucleospin RNA XS kits (Macherey Nagel, Düren, Germany) according to manufacturer's protocols, and treated with RNase-free DNase. Between 100 ng and 1 µg RNA was reverse transcribed into cDNA with I-script cDNA synthesis kit (Bio-Rad, California, USA), according to manufacturer's protocol. Real-time quantitative PCR was performed on a CFX96 Real-Time System (Bio-Rad). All experiments were carried out in triplicate with iTaq Universal SYBR Green Supermix. Expression levels were normalized to the housekeeping gene hARP and multiplied by 1,000 for the purpose of presentation. Primer sequences are listed in Supporting Information Table S2. Microarray analysis was performed on three biologically independent replicates using Illumina human HT-12v4 arrays (ServiceXS, Leiden, The Netherlands). Illumina microarray data were processed using the lumi package and differentially expressed genes were identified using the limma package, included in R/Bioconductor [25, 26]. ConsensusPathDB-human (http://cpdb.molgen.mpg.de/) web server was used for gene ontology analysis. Additional analysis was performed using Genespring (Agilent Technologies, California, USA). Results Generation of a MESP1mCherry-NKX2-5eGFP Dual Cardiac Reporter hESC line To identify, purify, and characterize human precardiac mesodermal progenitors and study their further differentiation, we generated a dual fluorescent hESC reporter line by inserting the sequence encoding mCherry into the MESP1 locus of an existing NKX2-5eGFP reporter line by homologous recombination (Fig. 1A, 1B). A PCR screening strategy was performed to identify correctly targeted clones (Supporting Information Fig. S1A, S1B). Twenty correctly targeted clones were obtained (11% targeting efficiency) from which four clones showed the loxP-flanked neomycin-resistance cassette was excised following transfection with Cre recombinase (not shown) [22]. We proceeded with clone 2.20B3 (Supporting Information Fig. S1C). hESCs from clone 2.20B3 displayed a high immunoreactivity for NANOG, TRA-181, OCT-4, and EPCAM antibodies, confirming no loss of pluripotency during targeting and selection procedures (Supporting Information Fig. S2A, S2B). Furthermore, chromosome analysis demonstrated no abnormal karyotypes in these targeted lines (Supporting Information Fig. S2C). Open in new tabDownload slide Generation of a MESP1mCherry-NKX2-5eGFP dual reporter hESC line. (A): Schematic overview of wild-type MESP1 allele, MESP1 targeting construct, and targeted MESP1 allele. (B): Schematic overview of the dual cardiac fluorescent reporter line. p: endogenous gene regulatory elements of either MESP1 (MESP1p) or NKX2-5 (NKX2-5p). (C): Cardiac monolayer differentiation protocol in BPEL. (D): MESP1-mCherry expressing cells at day 3 of cardiac differentiation, visualized by a Nuance Multispectral Imaging System. Abbreviations: hESC, human embryonic stem cell; MG, matrigel; Tg, transgenic. Open in new tabDownload slide Generation of a MESP1mCherry-NKX2-5eGFP dual reporter hESC line. (A): Schematic overview of wild-type MESP1 allele, MESP1 targeting construct, and targeted MESP1 allele. (B): Schematic overview of the dual cardiac fluorescent reporter line. p: endogenous gene regulatory elements of either MESP1 (MESP1p) or NKX2-5 (NKX2-5p). (C): Cardiac monolayer differentiation protocol in BPEL. (D): MESP1-mCherry expressing cells at day 3 of cardiac differentiation, visualized by a Nuance Multispectral Imaging System. Abbreviations: hESC, human embryonic stem cell; MG, matrigel; Tg, transgenic. To demonstrate that the targeted MESP1mCherry/w-NKX2-5eGFP/w hESC lines faithfully recapitulated MESP1 activity, hESCs were differentiated as monolayers to cardiac cells by addition of the growth factors BMP4 and Activin-A, as well as GSK-3-β inhibitor Chir99021 from day 0 to day 3, followed by the Wnt-signaling antagonist pathway inhibitor Xav939 from day 3 to day 7 of differentiation (Fig. 1C). Red fluorescence of mCherry was clearly visible in a subset of cells at day 3 of differentiation (Fig. 1D). Cells expressing mCherry appeared to have undergone epithelial-mesenchymal-transition (EMT), based on their mesenchymal-like morphology. MESP1mCherry-NKX2-5eGFP hESCs Differentiate Toward Mesoderm and the Cardiac Lineage Quantitative PCR (qPCR) analysis of the targeted cell line during differentiation toward the cardiac lineage confirmed transient expression of mCherry, peaking at day 3 of differentiation and mirroring endogenous MESP1 expression (Fig. 2A). In agreement with previous in vitro and in vivo studies, expression levels of mesendodermal genes Brachyury (BRY) and Eomesodermin (EOMES) displayed peak values at day 2 of differentiation, before that of MESP1. Expression levels of NKX2-5 were clearly detectable from day 9 of differentiation, just before the appearance of contractile areas. Expression of NKX2-5 further increased at day 12, confirming the ability of the MESP1mCherry-NKX2-5GFP reporter line to differentiate toward the cardiac lineage. Flow cytometric analysis showed that MESP1-mCherry expressing cells first appeared around day 2, peaking at day 4 (8.0% ± 0.5% [mean ± SEM]), and becoming negligible by differentiation day 8 (Fig. 2B, 2C), reflecting the gene expression profile. NKX2-5-eGFP protein was detected later, first appearing on day 8 and continuing to increase to day 14 of differentiation (28.9% ± 1.7%), reflecting our previous findings [17]. These results demonstrated that the MESP1mCherry and the NKX2-5eGFP reporters allowed visualization of early MESP1-expressing progenitors, followed by the formation of NKX2-5-expressing cardiac cells during hESC differentiation. Open in new tabDownload slide Time course of MESP1mCherry-NKX2-5eGFP hESCs differentiating toward the cardiac lineage. (A): mRNA levels of mesendodermal genes BRACHYURY T (T) and EOMESODERMIN (EOMES), precardiac mesoderm gene MESP1, mCherry reporter gene, and early cardiac gene NKX2-5 during monolayer differentiation. Results are normalized for the transcript expression at day 0 (undifferentiated hESCs) (n = 3, error bars indicate SEM). (B): Percentages of mCherry and eGFP protein expression upon monolayer differentiation. (C): Quantification of percentages of mCherry and eGFP expressing cells during monolayer differentiation (n = 4–8; error bars indicate SEM). Abbreviation: hESC, human embryonic stem cell. Open in new tabDownload slide Time course of MESP1mCherry-NKX2-5eGFP hESCs differentiating toward the cardiac lineage. (A): mRNA levels of mesendodermal genes BRACHYURY T (T) and EOMESODERMIN (EOMES), precardiac mesoderm gene MESP1, mCherry reporter gene, and early cardiac gene NKX2-5 during monolayer differentiation. Results are normalized for the transcript expression at day 0 (undifferentiated hESCs) (n = 3, error bars indicate SEM). (B): Percentages of mCherry and eGFP protein expression upon monolayer differentiation. (C): Quantification of percentages of mCherry and eGFP expressing cells during monolayer differentiation (n = 4–8; error bars indicate SEM). Abbreviation: hESC, human embryonic stem cell. Gene Expression Profiling of MESP1-mCherry Progenitors To further characterize the MESP1-mCherry progenitors, we isolated MESP1-mCherry positive cells (MESP1-mCherry pos) at day 3 of differentiation and compared their gene expression profile with MESP1-mCherry negative cells (MESP1-mCherry neg) and undifferentiated hESCs of the same line (Fig. 3A). A correlation heatmap between samples is shown in Supporting Information Figure S3. In three biological replicates, 722 genes showed an increase in expression of ≥1.5-fold when compared with MESP1-mCherry neg cells (Fig. 3B). In agreement with their mesodermal character, MESP1-mCherry pos cell fraction was enriched for EMT- and mesendodermal related genes, including MESP1 itself, SNAI1, SNAI2, MSX1, FOXC1, GSC, MIXL1, EOMES, LHX1, FOXF1, and IRX3. Although the differences were greater in MESP1-mCherry pos cells, mesendodermal and EMT-related gene levels were higher in both MESP1-mCherry pos and neg fractions compared to undifferentiated hESCs, indicating that cells had undergone some form of mesoderm differentiation in the protocol used. Furthermore, the enrichment of N-cadherin (CDH-2) and vimentin (VIM) and decrease of E-cadherin (CDH-1) and EPCAM are also strongly associated with EMT. As expected, transcripts of pluripotency genes, including SOX2, OCT3-4, and NANOG, were significantly decreased. Furthermore, early cardiac genes, such as GATA4, TBX3, MYL4, GATA6, and HAND1, were enriched indicating the precardiac character of MESP1-mCherry progenitors. In addition, genes encoding extracellular matrix proteins were increased, including several collagens, integrins, laminins, and fibronectin, as well as extracellular matrix (ECM)-receptors, such as integrin α-5. These proteins could play an important role in stage-dependent processes such as mesoderm migration or niche formation of the precardiac mesodermal cell population. Open in new tabDownload slide Characterization of MESP1-mCherry expressing cells at day 3 of monolayer differentiation. (A): Transcriptional profiling of three biological replicates of undifferentiated human embryonic stem cells (D0), differentiation day 3 MESP1-mCherry pos cells (D3+), and MESP1-mCherry neg cells (D3−). D3+ samples were enriched for specific transcription factors, cell surface markers, ECM components, EMT-related genes, and signaling pathway ligands and receptors. Clustering of the samples is based on hierarchical clustering of microarray gene expression profiling. Shown are a selection of transcripts expressed in D3+ with a fold change ≥1.5 compared to D3- for three independent paired experiments (n = 3).* indicates differences in only two independent experiments. GEO accession number: GSE56721. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56721. (B): Number of transcripts that was ≥1.5-fold differentially expressed (n = 3) between D3+ and D0, D3− and D0, and D3+ and D3− samples. (C): Signaling pathway analysis of enriched genes in MESP1-mCherry expressing cells. p < .01 for selected pathways. Percentages are based on all enriched genes. (D): Validating microarray results by qRT-PCR analysis of selected transcripts in purified MESP1-mCherry pos and MESP1-mCherry neg cell populations at differentiation day 3 (n = 3, error bars indicate SEM). Values are normalized for hARP levels. Abbreviations: ECM, extracellular matrix; EMT, epithelial-mesenchymal-transition. Open in new tabDownload slide Characterization of MESP1-mCherry expressing cells at day 3 of monolayer differentiation. (A): Transcriptional profiling of three biological replicates of undifferentiated human embryonic stem cells (D0), differentiation day 3 MESP1-mCherry pos cells (D3+), and MESP1-mCherry neg cells (D3−). D3+ samples were enriched for specific transcription factors, cell surface markers, ECM components, EMT-related genes, and signaling pathway ligands and receptors. Clustering of the samples is based on hierarchical clustering of microarray gene expression profiling. Shown are a selection of transcripts expressed in D3+ with a fold change ≥1.5 compared to D3- for three independent paired experiments (n = 3).* indicates differences in only two independent experiments. GEO accession number: GSE56721. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56721. (B): Number of transcripts that was ≥1.5-fold differentially expressed (n = 3) between D3+ and D0, D3− and D0, and D3+ and D3− samples. (C): Signaling pathway analysis of enriched genes in MESP1-mCherry expressing cells. p < .01 for selected pathways. Percentages are based on all enriched genes. (D): Validating microarray results by qRT-PCR analysis of selected transcripts in purified MESP1-mCherry pos and MESP1-mCherry neg cell populations at differentiation day 3 (n = 3, error bars indicate SEM). Values are normalized for hARP levels. Abbreviations: ECM, extracellular matrix; EMT, epithelial-mesenchymal-transition. The microarray data also indicated the expression of genes of various signaling pathways in the MESP1-mCherry pos population, including Notch ligand DLL3, BMP4, FGF17, the BMP-antagonist CER1, Wnt inhibitors DKK1 and DKK4, and other components of (non)canonical Wnt signaling, such as the ligand Wnt5a and (co)-receptors FZD2, ROR-2, and RSPO3. Protein complex analysis showed 100% enrichment of the noncanonical WNT5a/FZD2/ROR-2 complex (data not shown). In agreement with this, pathway analysis showed significant enrichment (p-value ≤ .01) in extracellular matrix organization, Wnt signaling pathway, cardiac progenitor differentiation, and heart development (Fig. 3C). qPCR analysis validated enhanced expression of a selection of the genes upregulated in the microarray data (Fig. 3D). Surface Marker Expression Analysis of MESP1-mCherry Progenitors Further microarray analysis identified several genes encoding mesoderm-associated cell surface proteins, including NCAM1, ROR-2, CD13, CDH-2, CDH-11, and PDGFR-α, which showed increased expression in MESP1-mCherry pos cells (Fig. 3A). FACS analyses showed that large proportions of the MESP1-mCherry pos cells expressed cell surface proteins PDGFR-α (∼70%), CD13 (∼97%), and ROR-2 (∼96%) (Fig. 4A). However, none of these proteins marked the MESP1-mCherry pos population exclusively, since expression was also clearly detected in the MESP1-mCherry neg cell populations (Fig. 4B). Furthermore, only a small percentage in both the MESP1-mCherry pos and neg population was coexpressing PDGFR-α and KDR, in agreement with the microarray analysis, which showed that KDR was not differentially expressed in the MESP1-mCherry pos population. Open in new tabDownload slide Flow cytometry analysis of cell surface markers CD13, ROR-2, KDR, and PDGFR-α on (A) MESP1-mCherry pos and (B) MESP1-mCherry neg-sorted populations at day 3 of differentiation. Open in new tabDownload slide Flow cytometry analysis of cell surface markers CD13, ROR-2, KDR, and PDGFR-α on (A) MESP1-mCherry pos and (B) MESP1-mCherry neg-sorted populations at day 3 of differentiation. Generation of Cardiac Cells from Isolated MESP1-mCherry Progenitors To investigate whether MESP1-mCherry pos progenitors were able to differentiate toward the cardiac lineage, MESP1-mCherry pos cells were isolated on day 3 of differentiation, seeded as aggregates or in monolayer in the presence of Wnt-pathway agonist Xav939 until differentiation day 7, and maintained until differentiation day 14 (Fig. 5A). To evaluate how mesoderm progenitor populations are represented in MESP1-mCherry-sorted derivatives, we initially performed FACS analysis on differentiation day 5 (2 days after replating). ROR-2+CD13+ cells were slightly enriched in the MESP1-mCherry pos population, compared to the MESP1-mCherry neg population; ∼73% ± 9% (mean ± SEM) versus 39% ± 16%, respectively (Fig. 5B). Furthermore, almost all MESP1-mCherry derivatives on day 5 expressed PDGFR-α (∼98% ± 1% [mean ± SEM]), while only 50% ± 4% of the day 5 MESP1-mCherry neg derivatives did. Interestingly, there was no enrichment of a KDR+PDGFR-α+ cardiac progenitor population in the MESP1-mCherry pos population, compared to the MESP1-mCherry neg derivatives (∼22% ± 2% [mean ± SEM] vs. 28% ± 12%, respectively) (Fig. 5B). Similar results were found at other time points during differentiation (days 4–8) (Supporting Information Fig. S4). Open in new tabDownload slide Cardiac differentiation induction from the MESP1-mCherry expressing precardiac mesoderm progenitors. (A): Schematic overview of the cardiac differentiation protocol for MESP1-mCherry cell fractions sorted by flow cytometry at day 3. (B): Flow cytometric analysis of coexpression of cell surface proteins at differentiation day 5 (2 days after replating). (C, D): Enrichment of mRNA transcripts of cardiac progenitor transcription factors at day 8 (n = 3) and day 14 (n = 2) in MESP1-mCherry pos and MESP1-mCherry neg derivatives (5 and 11 days after replating). Open in new tabDownload slide Cardiac differentiation induction from the MESP1-mCherry expressing precardiac mesoderm progenitors. (A): Schematic overview of the cardiac differentiation protocol for MESP1-mCherry cell fractions sorted by flow cytometry at day 3. (B): Flow cytometric analysis of coexpression of cell surface proteins at differentiation day 5 (2 days after replating). (C, D): Enrichment of mRNA transcripts of cardiac progenitor transcription factors at day 8 (n = 3) and day 14 (n = 2) in MESP1-mCherry pos and MESP1-mCherry neg derivatives (5 and 11 days after replating). On day 8 and day 14 of differentiation, the expression of several cardiac-associated genes was enriched in the MESP1-mCherry pos derivatives compared to the MESP1-mCherry neg derivatives, including cardiac genes NKX2-5 and ISL1, smooth muscle cell markers SMA and MYH11, and endothelial marker PECAM-1 (Fig. 5C, 5D). Additionally, NKX2-5 expression, as determined by flow cytometric quantification of eGFP expression, was significantly enriched in the MESP1-mCherry pos population, treated with Xav939 (80% ± 4% [mean ± SEM]) when compared with MESP1-mCherry neg cells under similar differentiation conditions (18% ± 6%) (Fig. 6A–6C). The eGFP+ cells detected in the MESP1-mCherry neg day 14 aggregates may have been derived from a small percentage of MESP1-mCherry pos cells that subsequently appeared in the MESP1-mCherry neg population following sorting. Indeed, low expression levels of mCherry and MESP1 could be detected in negative sorted samples at day 3 and day 4 (1 day after reaggregation) (Supporting Information Fig. S5). Open in new tabDownload slide Day 14 MESP1-mCherry derived aggregates (11 days after sorting). (A): eGFP expression in MESP1-mCherry aggregates at day 14 of differentiation (11 days after replating), with and without treatment of Xav939. Brightfield overlay with eGFP. (B, C): Percentage of cells expressing eGFP in day 14 MESP1-mCherry aggregates, with and without treatment of XAV939 (+sorted: n = 7, −sorted: n = 6, unsorted: n = 2; error bars indicate SEM, ***, p < .001). (D): Immunohistochemical stainings on day 14 MESP1-mCherry aggregates to analyze the presence of cardiac markers, including PECAM-1, SMA, SM-myosin, and α-ACT. (E): Costaining of α-ACT and SM-HC at day 14 to discriminate smooth muscle cells from cardiomyocytes. (F–H): Flow cytometric analysis of cTNT and PECAM-1 expression on day 14 MESP1-mCherry aggregates treated with XAV939 (n = 2, error bars indicate SEM). Abbreviations: α-ACT, α-actinin; SM-HC, SM-heavy chain. Open in new tabDownload slide Day 14 MESP1-mCherry derived aggregates (11 days after sorting). (A): eGFP expression in MESP1-mCherry aggregates at day 14 of differentiation (11 days after replating), with and without treatment of Xav939. Brightfield overlay with eGFP. (B, C): Percentage of cells expressing eGFP in day 14 MESP1-mCherry aggregates, with and without treatment of XAV939 (+sorted: n = 7, −sorted: n = 6, unsorted: n = 2; error bars indicate SEM, ***, p < .001). (D): Immunohistochemical stainings on day 14 MESP1-mCherry aggregates to analyze the presence of cardiac markers, including PECAM-1, SMA, SM-myosin, and α-ACT. (E): Costaining of α-ACT and SM-HC at day 14 to discriminate smooth muscle cells from cardiomyocytes. (F–H): Flow cytometric analysis of cTNT and PECAM-1 expression on day 14 MESP1-mCherry aggregates treated with XAV939 (n = 2, error bars indicate SEM). Abbreviations: α-ACT, α-actinin; SM-HC, SM-heavy chain. The importance of inhibiting Wnt/β-catenin signaling following mesoderm induction to generate NKX2-5-expressing cardiac progenitors was highlighted by the low levels of eGFP in the untreated MESP1-mCherry pos (23% ± 4%) and MESP1-mCherry neg derivatives (0.50% ± 0.4%). Furthermore, in all conditions, the NKX2-5eGFP+ derivatives were spontaneously contracting on differentiation day 14 (data not shown). To determine whether other specific signaling pathways could affect cardiac differentiation from mesoderm, three other major pathways associated with cardiac differentiation were inhibited after MESP1-mCherry sorting and reaggregation (from day 3 to day 7). Inhibitors of these pathways included transforming growth factor beta (TGF-β)/act/Nodal antagonist SB431542, and BMP inhibitor Dorsomorphin. None of these inhibitors led to an increase in GFP expression at day 14 when compared with control treatment. In fact, GFP expression showed a tendency to decrease in the SB431542-treated cells (Supporting Information Fig. S6). Day 14 MESP1-mCherry pos derivatives treated with Xav939, expressed various cardiac lineage markers, including α-Actinin (cardiomyocytes), PECAM-1 (endothelial cells), and SMA and SMC-myosin (expressed in immature cardiomyocytes as well as smooth muscle), indicating the MESP1-mCherry pos cell population can generate all three different cell types of the cardiac lineage (Fig. 6D). In addition, flow cytometric analysis showed enrichment of cTNT (∼51% ± 9% [mean ± SEM]) and PECAM-1 (∼2.2% ± 0.2% [mean ± SEM]) expressing cells in day 14 MESP1-mCherry pos derivatives, compared to the MESP1-mCherry neg derivatives (∼11% ± 5% and 0.62% ± 0.1%, respectively) (Fig. 6E). SIRPA+ VCAM1+ Coexpression Marks NKX2-5-eGFP+ Cardiomyocytes In addition to the characterization and differentiation of MESP1mCherry progenitors and their further differentiation to NKX2-5eGFP cardiac cells, we studied whether the previous described cell surface proteins SIRPA and VCAM1 are specifically marking hESC-derived-cardiomyocytes [27, 28]. We analyzed SIRPA-VCAM1 and cTnT expression on cardiac cells at day 14 of differentiation of NKX2.5-eGFP-HESCs (Supporting Information Fig. S7). We showed that the SIRPA+VCAM1+ double positive population was highly enriched for NKX2-5-eGFP+ cells (Supporting Information Fig. S7A). In addition, the vast majority of NKX2-5-eGFP+ cells were cTnT+ (Supporting Information Fig. S7B). Similar results were described in a recent study by Skelton et al., in which SIRPA+VCAM1+NKX2-5-eGFP+ cardiomyocytes were shown to arise from a SIRPA+NKX2-5-eGFP+ intermediate progenitor population [28]. Discussion Genetically modified reporter lines are proving increasingly valuable for developing precise protocols for the directed differentiation of various lineages from hPSCs [29-32]. Here, we increased the degree of complexity of the processes that can be analyzed by creating a double transgenic reporter hESC line, taking advantage of endogenous activation of the key cardiac transcription factors MESP1 and NKX2-5 [17]. We introduced the fluorescent protein mCherry into the genomic locus of the mesodermal transcription factor MESP1, in NKX2-5eGFP hESCs. As predicted from mouse developmental biology studies, the MESP1mCherry-NKX2-5eGFP line firstly expressed mCherry under conditions of mesoderm induction, and later GFP+ cardiac progenitors and cardiomyocytes emerged in the cultures, demonstrating the value of hESCs as in vitro model of previously inaccessible stages of early human development. MESP1-mCherry pos cells were first detected on day 2 of differentiation with peak levels between days 3 and 4, which is in agreement with our earlier observations during cardiac differentiation of hESCs [1]. We demonstrated that sorted and reaggregated MESP1-mCherry pos cells on differentiation day 3 have a much higher potential to differentiate to cardiovascular derivatives than MESP1-mCherry neg cells, as demonstrated by (a) the appearance of functional beating cardiomyocytes and (b) high numbers of NKX2-5eGFP+ cardiac cells in the population on day 14 of differentiation (11 days after sorting). At the same stage of differentiation, enriched gene and protein expression were found for PECAM-1, SMA, and cTNT, suggesting that MESP1-mCherry cells can differentiate to endothelial cells, smooth muscle cells, and cardiomyocytes, respectively. Microarray analysis of MESP1-mCherry pos and MESP1-mCherry neg cells revealed new insights into the transcriptional profile of human precardiac mesodermal progenitors. In particular, transcription factors and signaling pathways associated with mesoderm formation, EMT, early cardiac development, and components of the ECM were highly enriched in MESP1-mCherry pos cells. This is broadly in accordance with the molecular signature of MESP1-GFP expressing mouse cells [33]. However, disparities possibly related to interspecies differences, differentiation protocols, or technical variations (i.e., random integration of an isolated regulatory sequence of the Mesp1 mouse promoter vs. homologous recombination in the human MESP1 locus) were evident. For example, the expression of cardiac transcription factors such as HAND2, MEIS2, SMARCD3, HEY1, HEY2, and TBX2 was increased in MESP1-GFP positive cells, whereas in our study these genes were not enriched in the MESP1-mCherry pos population. However, expression of these genes was increased at later stages following cardiac differentiation of sorted cells, suggesting that human MESP1-mCherry pos cells may be a more restricted mesodermal population. Transition of undifferentiated epithelial pluripotent stem cells to mesodermal cells requires the activation of EMT-associated genes. Indeed, MESP1-mCherry pos cells exhibit an obvious increase in levels of EMT-related transcription factors SNAI1 and SNAI2. In agreement, increased expression of other EMT-related genes, including Vimentin and CDH-2, and decreased levels of epithelial proteins, such as CDH-1 and EPCAM, suggested that EMT took place in MESP1-mCherry pos cells. In addition, enrichment of many ECM-related transcripts, such as those for collagens, integrins, fibrillin, matrix remodelers, and receptors, like the Apelin Receptor, suggested formation of an appropriate niche for MESP1-expressing cells [34, 35]. Alterations in cell-cell and cell-matrix interactions are crucial for biological processes such as EMT, intracellular interactions, cell-fate decisions, or migration of precardiac MESP1-progenitors from the anterior part of the primitive streak to form the bilaterally situated cardiac crescent in vivo [36-39]. Further study is required to identify the role of specific ECM components and receptors for expansion and further differentiation of MESP1+ cells. Several signaling pathways were also significantly activated in MESP1-mCherry pos cells. In particular, components of the Wnt signaling pathway were affected, including the Wnt-inhibitors DKK1 and DKK4, the (non)canonical pathway ligands WNT4 and WNT5a, and the receptors ROR-2 and FZD2. Previous studies have indicated an important role for activated canonical Wnt-signaling during hESC to mesendoderm differentiation [40-43]. Subsequent inhibition of this pathway by DKK-1 resulted in improved cardiac differentiation, confirming the need to inhibit canonical Wnt-signaling following mesoderm formation for efficient cardiac differentiation [14, 40]. Conversely, at the same stage, activated noncanonical signaling has been associated with driving cardiac differentiation [44]. Noncanonical Wnt signaling has been linked with different molecular mechanisms, including canonical Wnt and/or β-catenin signaling inhibition [42, 45, 46], progenitor proliferation [47], and cellular movements and polarity. Here, we confirmed the importance of Wnt-inhibition in mesoderm to cardiac progression, as we found a major increase in the percentage of NKX2-5eGFP expressing cells upon treatment with the chemical Wnt signaling antagonist, Xav939. Other pathways that have been shown to play crucial roles in the early stages of mesoderm formation, such as the BMP, Notch, and FGF pathways, were also present in the MESP1-mCherry pos population, suggesting crosstalk of components of these pathways during these early precardiac mesoderm stages [41]. We also examined expression of cell surface markers of mesodermal and cardiovascular progenitors during the differentiation of MESP1mCherry-NKX2-5eGFP cells. Several studies have reported that PDGFR-α and Flk1/KDR are expressed on early cardiovascular progenitor cells, giving rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells [5, 19, 20]. In recent studies using hESCs, an early mesoderm population coexpressing CD13 (ANPEP) and ROR-2 has been identified [18, 48]. This population consists of a KDR+PDGFR-α+ subpopulation, which has the potential to give rise to cardiovascular cells. In agreement, we demonstrated a significant increase in CD13, ROR-2, and PDGFR-α on day 3 MESP1-mCherry pos cells and on day 5 derivatives. However, these surface markers were also expressed in a high percentage of the MESP1-mCherry neg populations, indicating that they mark a broader population than only precardiac mesoderm [27]. Interestingly, in MESP1-mCherry pos derivatives we did not find enrichment of KDR+ cells. These findings indicated that, besides the previously described Flk1/KDR+ PDGFR-α+ cardiac progenitor population, other MESP1-derived precardiac mesoderm progenitor populations exist, that (co)express other (cell surface) markers, and have the potential to differentiate toward NKX2-5eGFP expressing cardiac cells. In previous studies, the cardiac potential of other subpopulations besides the Flk1/KDR+ PDGFR-α+ was not tested [18, 20]. One of the major challenges in cardiovascular research is cardiac regeneration and improved cardiac function in heart failure patients. Tissue engineering, cellular therapy, endogenous activation of cardiac progenitor cells, or a combination of these approaches may lead to novel therapeutic strategies [49]. However, a key concern is the selection of the right cell type for transplantation or enhanced in situ proliferation, as this will be important for survival and engraftment, for safety (risk of tumor formation), and for the ability to form the required cell type(s) [50, 51]. A recent study showed the ability of a hESC-derived CD13+ROR-2+KDR+PDGFR-α+ cardiac progenitor population to migrate, couple, and form cardiac cells after being transplanted into ex vivo human fetal ventricular heart tissue, without formation of teratomas [18]. As precardiac MESP1-progenitors are able to form almost all cell types of the heart, it will be of interest to examine both the developmental and regenerative capacity of these cells upon transplantation into both healthy and damaged hearts. Even though many hurdles have to be overcome, transplantation studies using cardiac-lineage-restricted progenitors may shed more light on future heart regeneration therapies. In addition, understanding the molecular cues that are important for expansion of mesodermal cells and their specific differentiation to the cardiac lineage may lead to novel therapies which are based on the progression of endogenous cardiac progenitor cells to functional specialized cardiovascular cells. Conclusions We conclude that the MESP1mCherry/w-NKX2-5eGFP/w dual cardiac fluorescent reporter hESC line in this study is a useful tool for dissecting the molecular and cellular mechanisms related to the earliest steps in cardiac development in live cells. This is novel way to carry out fate analysis of this subpopulation. Purified mesodermal MESP1-mCherry pos cells efficiently differentiated to beating NKX2-5GFP+ cells by inhibition of the Wnt-pathway. Besides a better understanding of underlying mechanisms, this human cardiac dual reporter line will make it feasible to identify and specify culture conditions for expansion of a mesodermal cell population and their further differentiation toward cardiac progenitor cells and specific subtypes of the cardiac lineage in vitro. This is of particular interest for future studies on cardiac toxicity, drug screening, tissue engineering, and regenerative medicine. Acknowledgments We thank Dorien Ward-Van Oostwaard for technical assistance; Kostas Gkatzis for help on statistical analysis using R/Bioconductor, the Department of Molecular Cell Biology, LUMC, for karyotyping analysis. S.C.D.H was supported by the Rembrandt Institute for Cardiovascular Disease. This study was supported by the Rembrandt Institute for Cardiovascular Science (S.H.), NHMRC, Australia (D.A.E.), ZonMw-MKMD-40-42600-98-036 (R.P.), and ERC Advanced STEMCARDIOVASC-323182 (C.L.M.). 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Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC © 2014 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Stem Cells Oxford University Press

Dual Reporter MESP1mCherry/w-NKX2-5eGFP/w hESCs Enable Studying Early Human Cardiac Differentiation

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Oxford University Press
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1066-5099
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10.1002/stem.1842
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Abstract

Abstract Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1mCherry/w-NKX2-5eGFP/w reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-α, CD13, and ROR-2. No enrichment was found for the previously described KDR+PDGFR-α+ progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives. Stem Cells 2015;33:56–67 Embryonic stem cells, Mesoderm, MESP1, NKX2-5, Cardiac differentiation, Cardiac progenitors Introduction Human pluripotent stem cells (hPSCs) offer exceptional opportunities for regenerative medicine, drug discovery and toxicity, disease modeling, and developmental biology because of their ability to differentiate in principle into any cell type of the human body. In particular, their differentiation toward cell types that are challenging to obtain and maintain as primary cultures from tissues with limited or no self-repair capacity, such as the brain and heart, have made them attractive as research models. Previously, we and others have shown that cardiomyogenesis of hPSCs in vitro recapitulates signaling events and sequential transcriptional activation that occurs during heart development in vivo [1, 2]. Moreover, as knowledge of the regulatory mechanisms that govern in vivo cardiac development has advanced, it has been possible to translate this to differentiation protocols based on stepwise temporal modification of signaling pathways. This has led to a remarkable increase in the yield of cardiac cells not only from human embryonic stem cells (hESCs) but also from human induced pluripotent stem cells (reviewed in [3]). However, despite these advances, the cardiac derivatives obtained contain heterogeneous populations of cardiac cells: not only different subtypes of cardiomyocytes but also endothelial cells and smooth muscle cells. This is because the molecular cues underlying specification of early human cardiac progenitors toward specific cardiac subtypes have not been fully elucidated. For future clinical and pharmaceutical applications, better insight into the differentiation of cardiac cell subtypes is necessary. Several studies have shown that different cardiac subtypes are derived from multiple early cardiac progenitors that segregate soon after gastrulation [4-6]. The molecular mechanisms that play a role in the formation of early cardiac progenitors, however, remain largely unknown [4, 7]. In order to study the onset of cardiac differentiation in human, it is necessary to identify early populations from common progenitors at the mesodermal stage of development and identify the molecular pathways involved in the further differentiation of the cardiac lineage. Mesoderm transcription factor MESP1 is important in this context as in vivo lineage tracing in mouse has demonstrated that almost all cells of the heart are derived from a common MESP1-expressing mesodermal progenitor, including myo-, endo, and epicardium, and cells of the conduction system [8-10]. MESP1 is transiently expressed in the anterior mesoderm from the onset of gastrulation prior to cardiac crescent formation [8, 11, 12]. Furthermore, MESP1 overexpression in mouse ESCs showed that MESP1 induces the expression of cardiovascular genes [13-15]. A recent study in mouse illustrated that besides the cardiac lineage, MESP1 derivatives also encompass cells of the hematopoietic- and skeletal myogenic lineages, which emerge depending on the endogenous and exogenous cues present during MESP1 expression [16]. Of note, all these studies have been performed in mice. However, to instigate advances in human heart development, regenerative medicine, disease modeling, and drug testing, it is necessary to understand the earliest steps of cardiac differentiation in a human model. In order to identify, isolate, and characterize MESP1 expressing cells during human cardiomyogenesis, we introduced the mCherry reporter into the MESP1 locus of the NKX2-5eGFP hESC reporter line generated previously [17], leading to a dual reporter system for these early cardiovascular lineage markers in hESCs. We were able to isolate the MESP1-expressing mesoderm progenitors in a cardiac-inducing environment and demonstrate their ability to differentiate further to a highly enriched NKX2-5eGFP expressing cardiac population. We further confirmed the presence of other mesoderm and cardiac progenitor populations in MESP1-mCherry expressing-derivatives, including CD13+ROR-2+ and PDGFR-α+KDR+ populations [18-20]. Although CD13, ROR-2, and PDGFR-α expression were strongly enriched, they were not specifically expressed in the MESP1-mCherry derivatives. In addition, no enrichment was found for the previously described PDGFR-α+KDR+ population. Finally, we demonstrate that this MESP1mCherry-NKX2-5eGFP dual reporter hESC line is a useful tool to study the differentiation of precardiac mesoderm toward early cardiac progenitor cells and their derivatives. It now provides an opportunity to study the molecular mechanisms underlying the onset of progenitor subtypes formation specifically in the cardiac lineage in human. This hESC dual reporter will contribute to providing better insights into cardiomyocyte determination and form the basis for improved differentiation protocols for specifying cardiac subtypes (atrial, ventricular, and pacemaker cells). This will be relevant to future pharmaceutical and clinical applications of specific cardiac cell subtypes. Materials and Methods MESP1 Gene Targeting in hESCs To generate the MESP1 targeting vector, the bacterial artificial chromosome RP11-975L13 (Source Bioscience, Lifesciences, Nottingham, UK) was modified by recombineering (Gene Bridges, Heidelberg, Germany). The mCherry cassette and a selection marker replaced the MESP1 exon 1 sequence (oligonucleotides used for recombineering: MESP1_5arm_PvuI and MESP1_3_arm, Supporting Information Table S1). The modified MESP1 locus and the surrounding 5′ and 3′ homology arms were subsequently subcloned into the MESP1 targeting vector. The final vector comprised a 8.6-kilobase (kb) 5′ homology arm, mCherry, loxP-flanked G418 resistance cassette, and a 2.3-kb 3′ homology arm. The targeting vector was linearized by PvuI digestion and 20 µg was electroporated into 9 × 106 NKX2-5-eGFP reporter hESCs (HES3-NKX2-5eGFP/w) [17] as described previously [21]. Targeted clones were identified by PCR using oligonucleotides specific for mCherry (P1) and a region immediately 3′ of the targeting vector (P2) (Supporting Information Fig. S1, Table S1). Targeted clones were clonally derived and transfected with Cre-recombinase to excise the loxP-flanked neomycin-resistance cassette as described previously [22]. Neomycin excision was confirmed by PCR (Supporting Information Fig. S1, Table S1). Undifferentiated, targeted clones were fixed in 2% paraformaldehyde and were used for immunocytochemistry (Nanog, Tra-181, and Oct-4) to confirm their pluripotent status. For karyotyping, 20 metaphase chromosome spreads were examined for each line. Single-Cell Sorting For single-cell sorting, hESCs were enzymatically passaged using 1× TrypLE Select (Life Technologies, NY, USA). After dissociation, cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% BSA and 2.5 mM EDTA) and filtered through a 40-µm cell strainer (Falcon). Cells were sorted using a BD ARIA III flow cytometer into 96-well flat-bottomed low attachment plates, live cells were gated on the basis of side scatter, forward scatter, and propidium iodide exclusion. Flow cytometric gates were set using control cells labeled with the appropriate isotype control antibody. hESC Culture and Cardiac Differentiation MESP1mCherry/w NKX2-5eGFP/w hESCs were cultured on mouse embryonic fibroblasts on 2.85 cm2 organ dishes (Falcon), in 1 ml hESC medium (Dulbecco's modified Eagle's medium F-12, nonessential amino acids, knockout serum [Gibco by Life Technologies]; basic fibroblast growth factor [Miltenyi Biotec, Bergisch Gladbach, Germany]) and were mechanically passaged [23]. One or two days before differentiation, cells were passaged onto a 12-well tissue culture dish coated with Matrigel (BD Biosciences, New Jersey; growth factor reduced, phenol red-free), in 1 ml of hESC medium per well. On the first day of differentiation, day 0, hESC medium was replaced by the low insulin serum-free medium BPEL (BSA, polyvinyl alcohol, essential lipids, as previously described) [17, 24], containing the cytokines BMP4 (30 ng/ml, R&D Systems, Minneapolis) and Activin A (30 ng/ml, Miltenyi Biotec), and Chir99021 (1.5 µM, Axon Medchem, Groningen, The Netherlands). At day 3 of differentiation, the medium was refreshed with BPEL containing 5 µM Xav939 (R&D). On day 7 of differentiation, the medium was replaced with BPEL without growth factors. Isolation and Reaggregation of MESP1-mCherry Cells On day 3 of differentiation, cells were sorted by flow cytometry as described above, based on mCherry expression (both positive and negative populations). Flow cytometric gates were set using undifferentiated hESCs. Sorted cells were seeded in a 96-well V-shaped nonadherent plate (Greiner Bio One, Alphen a/d Rijn, The Netherlands) at 20,000 cells per well in 50 µl BPEL in the presence of Xav939 (5 µM, R&D) to enhance cardiac differentiation. Aggregation was initiated by a brief centrifugation step for 3 minutes at 480g. Four days after replating, aggregates were refreshed with BPEL without addition of growth factors or inhibitors. Analysis of MESP1-mCherry Derivatives Cardiomyocytes derived from MESP1-mCherry-sorted cells were analyzed by flow cytometry and immunohistochemistry (IHC) on days 3, 5, and 14 of differentiation (0, 2, and 11 days after replating). For IHC, MESP1-mCherry-derived aggregates were fixed in 2% paraformaldehyde (PFA) at day 14. For alpha-actinin and SM-HC IHC, MESP1-mCherry-derived aggregates were dissociated using 1× TrypLE Select, plated on matrigel in BPEL, and fixed after 4 days. For flow cytometric analysis, aggregates were collected, dissociated as described before, and resuspended in FACS washing buffer (PBS with 2% fetal calf serum (FCS) and 2.5 mM EDTA). For analysis of intracellular markers, cells were treated with FIX & PERM Cell Fixation and Permeabilization Kit (Life Technologies). Analyses were carried out using a MACS-Quant VYB flow cytometer (Miltenyi Biotec) and FlowJo Software. Antibodies are listed in Supporting Information Table S4. Specimens were analyzed with confocal laser microscope (Leica TCS SP8). Images were acquired with Leica LAS AF software. MESP1-mCherry expressing cells were visualized in monolayer differentiation at day 3, using a Nuance Multispectral Imaging System. RNA Isolation, Quantitative Real-Time PCR, and Microarray Analysis Total RNA was isolated using either the RNeasy micro (Qiagen) or Nucleospin RNA XS kits (Macherey Nagel, Düren, Germany) according to manufacturer's protocols, and treated with RNase-free DNase. Between 100 ng and 1 µg RNA was reverse transcribed into cDNA with I-script cDNA synthesis kit (Bio-Rad, California, USA), according to manufacturer's protocol. Real-time quantitative PCR was performed on a CFX96 Real-Time System (Bio-Rad). All experiments were carried out in triplicate with iTaq Universal SYBR Green Supermix. Expression levels were normalized to the housekeeping gene hARP and multiplied by 1,000 for the purpose of presentation. Primer sequences are listed in Supporting Information Table S2. Microarray analysis was performed on three biologically independent replicates using Illumina human HT-12v4 arrays (ServiceXS, Leiden, The Netherlands). Illumina microarray data were processed using the lumi package and differentially expressed genes were identified using the limma package, included in R/Bioconductor [25, 26]. ConsensusPathDB-human (http://cpdb.molgen.mpg.de/) web server was used for gene ontology analysis. Additional analysis was performed using Genespring (Agilent Technologies, California, USA). Results Generation of a MESP1mCherry-NKX2-5eGFP Dual Cardiac Reporter hESC line To identify, purify, and characterize human precardiac mesodermal progenitors and study their further differentiation, we generated a dual fluorescent hESC reporter line by inserting the sequence encoding mCherry into the MESP1 locus of an existing NKX2-5eGFP reporter line by homologous recombination (Fig. 1A, 1B). A PCR screening strategy was performed to identify correctly targeted clones (Supporting Information Fig. S1A, S1B). Twenty correctly targeted clones were obtained (11% targeting efficiency) from which four clones showed the loxP-flanked neomycin-resistance cassette was excised following transfection with Cre recombinase (not shown) [22]. We proceeded with clone 2.20B3 (Supporting Information Fig. S1C). hESCs from clone 2.20B3 displayed a high immunoreactivity for NANOG, TRA-181, OCT-4, and EPCAM antibodies, confirming no loss of pluripotency during targeting and selection procedures (Supporting Information Fig. S2A, S2B). Furthermore, chromosome analysis demonstrated no abnormal karyotypes in these targeted lines (Supporting Information Fig. S2C). Open in new tabDownload slide Generation of a MESP1mCherry-NKX2-5eGFP dual reporter hESC line. (A): Schematic overview of wild-type MESP1 allele, MESP1 targeting construct, and targeted MESP1 allele. (B): Schematic overview of the dual cardiac fluorescent reporter line. p: endogenous gene regulatory elements of either MESP1 (MESP1p) or NKX2-5 (NKX2-5p). (C): Cardiac monolayer differentiation protocol in BPEL. (D): MESP1-mCherry expressing cells at day 3 of cardiac differentiation, visualized by a Nuance Multispectral Imaging System. Abbreviations: hESC, human embryonic stem cell; MG, matrigel; Tg, transgenic. Open in new tabDownload slide Generation of a MESP1mCherry-NKX2-5eGFP dual reporter hESC line. (A): Schematic overview of wild-type MESP1 allele, MESP1 targeting construct, and targeted MESP1 allele. (B): Schematic overview of the dual cardiac fluorescent reporter line. p: endogenous gene regulatory elements of either MESP1 (MESP1p) or NKX2-5 (NKX2-5p). (C): Cardiac monolayer differentiation protocol in BPEL. (D): MESP1-mCherry expressing cells at day 3 of cardiac differentiation, visualized by a Nuance Multispectral Imaging System. Abbreviations: hESC, human embryonic stem cell; MG, matrigel; Tg, transgenic. To demonstrate that the targeted MESP1mCherry/w-NKX2-5eGFP/w hESC lines faithfully recapitulated MESP1 activity, hESCs were differentiated as monolayers to cardiac cells by addition of the growth factors BMP4 and Activin-A, as well as GSK-3-β inhibitor Chir99021 from day 0 to day 3, followed by the Wnt-signaling antagonist pathway inhibitor Xav939 from day 3 to day 7 of differentiation (Fig. 1C). Red fluorescence of mCherry was clearly visible in a subset of cells at day 3 of differentiation (Fig. 1D). Cells expressing mCherry appeared to have undergone epithelial-mesenchymal-transition (EMT), based on their mesenchymal-like morphology. MESP1mCherry-NKX2-5eGFP hESCs Differentiate Toward Mesoderm and the Cardiac Lineage Quantitative PCR (qPCR) analysis of the targeted cell line during differentiation toward the cardiac lineage confirmed transient expression of mCherry, peaking at day 3 of differentiation and mirroring endogenous MESP1 expression (Fig. 2A). In agreement with previous in vitro and in vivo studies, expression levels of mesendodermal genes Brachyury (BRY) and Eomesodermin (EOMES) displayed peak values at day 2 of differentiation, before that of MESP1. Expression levels of NKX2-5 were clearly detectable from day 9 of differentiation, just before the appearance of contractile areas. Expression of NKX2-5 further increased at day 12, confirming the ability of the MESP1mCherry-NKX2-5GFP reporter line to differentiate toward the cardiac lineage. Flow cytometric analysis showed that MESP1-mCherry expressing cells first appeared around day 2, peaking at day 4 (8.0% ± 0.5% [mean ± SEM]), and becoming negligible by differentiation day 8 (Fig. 2B, 2C), reflecting the gene expression profile. NKX2-5-eGFP protein was detected later, first appearing on day 8 and continuing to increase to day 14 of differentiation (28.9% ± 1.7%), reflecting our previous findings [17]. These results demonstrated that the MESP1mCherry and the NKX2-5eGFP reporters allowed visualization of early MESP1-expressing progenitors, followed by the formation of NKX2-5-expressing cardiac cells during hESC differentiation. Open in new tabDownload slide Time course of MESP1mCherry-NKX2-5eGFP hESCs differentiating toward the cardiac lineage. (A): mRNA levels of mesendodermal genes BRACHYURY T (T) and EOMESODERMIN (EOMES), precardiac mesoderm gene MESP1, mCherry reporter gene, and early cardiac gene NKX2-5 during monolayer differentiation. Results are normalized for the transcript expression at day 0 (undifferentiated hESCs) (n = 3, error bars indicate SEM). (B): Percentages of mCherry and eGFP protein expression upon monolayer differentiation. (C): Quantification of percentages of mCherry and eGFP expressing cells during monolayer differentiation (n = 4–8; error bars indicate SEM). Abbreviation: hESC, human embryonic stem cell. Open in new tabDownload slide Time course of MESP1mCherry-NKX2-5eGFP hESCs differentiating toward the cardiac lineage. (A): mRNA levels of mesendodermal genes BRACHYURY T (T) and EOMESODERMIN (EOMES), precardiac mesoderm gene MESP1, mCherry reporter gene, and early cardiac gene NKX2-5 during monolayer differentiation. Results are normalized for the transcript expression at day 0 (undifferentiated hESCs) (n = 3, error bars indicate SEM). (B): Percentages of mCherry and eGFP protein expression upon monolayer differentiation. (C): Quantification of percentages of mCherry and eGFP expressing cells during monolayer differentiation (n = 4–8; error bars indicate SEM). Abbreviation: hESC, human embryonic stem cell. Gene Expression Profiling of MESP1-mCherry Progenitors To further characterize the MESP1-mCherry progenitors, we isolated MESP1-mCherry positive cells (MESP1-mCherry pos) at day 3 of differentiation and compared their gene expression profile with MESP1-mCherry negative cells (MESP1-mCherry neg) and undifferentiated hESCs of the same line (Fig. 3A). A correlation heatmap between samples is shown in Supporting Information Figure S3. In three biological replicates, 722 genes showed an increase in expression of ≥1.5-fold when compared with MESP1-mCherry neg cells (Fig. 3B). In agreement with their mesodermal character, MESP1-mCherry pos cell fraction was enriched for EMT- and mesendodermal related genes, including MESP1 itself, SNAI1, SNAI2, MSX1, FOXC1, GSC, MIXL1, EOMES, LHX1, FOXF1, and IRX3. Although the differences were greater in MESP1-mCherry pos cells, mesendodermal and EMT-related gene levels were higher in both MESP1-mCherry pos and neg fractions compared to undifferentiated hESCs, indicating that cells had undergone some form of mesoderm differentiation in the protocol used. Furthermore, the enrichment of N-cadherin (CDH-2) and vimentin (VIM) and decrease of E-cadherin (CDH-1) and EPCAM are also strongly associated with EMT. As expected, transcripts of pluripotency genes, including SOX2, OCT3-4, and NANOG, were significantly decreased. Furthermore, early cardiac genes, such as GATA4, TBX3, MYL4, GATA6, and HAND1, were enriched indicating the precardiac character of MESP1-mCherry progenitors. In addition, genes encoding extracellular matrix proteins were increased, including several collagens, integrins, laminins, and fibronectin, as well as extracellular matrix (ECM)-receptors, such as integrin α-5. These proteins could play an important role in stage-dependent processes such as mesoderm migration or niche formation of the precardiac mesodermal cell population. Open in new tabDownload slide Characterization of MESP1-mCherry expressing cells at day 3 of monolayer differentiation. (A): Transcriptional profiling of three biological replicates of undifferentiated human embryonic stem cells (D0), differentiation day 3 MESP1-mCherry pos cells (D3+), and MESP1-mCherry neg cells (D3−). D3+ samples were enriched for specific transcription factors, cell surface markers, ECM components, EMT-related genes, and signaling pathway ligands and receptors. Clustering of the samples is based on hierarchical clustering of microarray gene expression profiling. Shown are a selection of transcripts expressed in D3+ with a fold change ≥1.5 compared to D3- for three independent paired experiments (n = 3).* indicates differences in only two independent experiments. GEO accession number: GSE56721. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56721. (B): Number of transcripts that was ≥1.5-fold differentially expressed (n = 3) between D3+ and D0, D3− and D0, and D3+ and D3− samples. (C): Signaling pathway analysis of enriched genes in MESP1-mCherry expressing cells. p < .01 for selected pathways. Percentages are based on all enriched genes. (D): Validating microarray results by qRT-PCR analysis of selected transcripts in purified MESP1-mCherry pos and MESP1-mCherry neg cell populations at differentiation day 3 (n = 3, error bars indicate SEM). Values are normalized for hARP levels. Abbreviations: ECM, extracellular matrix; EMT, epithelial-mesenchymal-transition. Open in new tabDownload slide Characterization of MESP1-mCherry expressing cells at day 3 of monolayer differentiation. (A): Transcriptional profiling of three biological replicates of undifferentiated human embryonic stem cells (D0), differentiation day 3 MESP1-mCherry pos cells (D3+), and MESP1-mCherry neg cells (D3−). D3+ samples were enriched for specific transcription factors, cell surface markers, ECM components, EMT-related genes, and signaling pathway ligands and receptors. Clustering of the samples is based on hierarchical clustering of microarray gene expression profiling. Shown are a selection of transcripts expressed in D3+ with a fold change ≥1.5 compared to D3- for three independent paired experiments (n = 3).* indicates differences in only two independent experiments. GEO accession number: GSE56721. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56721. (B): Number of transcripts that was ≥1.5-fold differentially expressed (n = 3) between D3+ and D0, D3− and D0, and D3+ and D3− samples. (C): Signaling pathway analysis of enriched genes in MESP1-mCherry expressing cells. p < .01 for selected pathways. Percentages are based on all enriched genes. (D): Validating microarray results by qRT-PCR analysis of selected transcripts in purified MESP1-mCherry pos and MESP1-mCherry neg cell populations at differentiation day 3 (n = 3, error bars indicate SEM). Values are normalized for hARP levels. Abbreviations: ECM, extracellular matrix; EMT, epithelial-mesenchymal-transition. The microarray data also indicated the expression of genes of various signaling pathways in the MESP1-mCherry pos population, including Notch ligand DLL3, BMP4, FGF17, the BMP-antagonist CER1, Wnt inhibitors DKK1 and DKK4, and other components of (non)canonical Wnt signaling, such as the ligand Wnt5a and (co)-receptors FZD2, ROR-2, and RSPO3. Protein complex analysis showed 100% enrichment of the noncanonical WNT5a/FZD2/ROR-2 complex (data not shown). In agreement with this, pathway analysis showed significant enrichment (p-value ≤ .01) in extracellular matrix organization, Wnt signaling pathway, cardiac progenitor differentiation, and heart development (Fig. 3C). qPCR analysis validated enhanced expression of a selection of the genes upregulated in the microarray data (Fig. 3D). Surface Marker Expression Analysis of MESP1-mCherry Progenitors Further microarray analysis identified several genes encoding mesoderm-associated cell surface proteins, including NCAM1, ROR-2, CD13, CDH-2, CDH-11, and PDGFR-α, which showed increased expression in MESP1-mCherry pos cells (Fig. 3A). FACS analyses showed that large proportions of the MESP1-mCherry pos cells expressed cell surface proteins PDGFR-α (∼70%), CD13 (∼97%), and ROR-2 (∼96%) (Fig. 4A). However, none of these proteins marked the MESP1-mCherry pos population exclusively, since expression was also clearly detected in the MESP1-mCherry neg cell populations (Fig. 4B). Furthermore, only a small percentage in both the MESP1-mCherry pos and neg population was coexpressing PDGFR-α and KDR, in agreement with the microarray analysis, which showed that KDR was not differentially expressed in the MESP1-mCherry pos population. Open in new tabDownload slide Flow cytometry analysis of cell surface markers CD13, ROR-2, KDR, and PDGFR-α on (A) MESP1-mCherry pos and (B) MESP1-mCherry neg-sorted populations at day 3 of differentiation. Open in new tabDownload slide Flow cytometry analysis of cell surface markers CD13, ROR-2, KDR, and PDGFR-α on (A) MESP1-mCherry pos and (B) MESP1-mCherry neg-sorted populations at day 3 of differentiation. Generation of Cardiac Cells from Isolated MESP1-mCherry Progenitors To investigate whether MESP1-mCherry pos progenitors were able to differentiate toward the cardiac lineage, MESP1-mCherry pos cells were isolated on day 3 of differentiation, seeded as aggregates or in monolayer in the presence of Wnt-pathway agonist Xav939 until differentiation day 7, and maintained until differentiation day 14 (Fig. 5A). To evaluate how mesoderm progenitor populations are represented in MESP1-mCherry-sorted derivatives, we initially performed FACS analysis on differentiation day 5 (2 days after replating). ROR-2+CD13+ cells were slightly enriched in the MESP1-mCherry pos population, compared to the MESP1-mCherry neg population; ∼73% ± 9% (mean ± SEM) versus 39% ± 16%, respectively (Fig. 5B). Furthermore, almost all MESP1-mCherry derivatives on day 5 expressed PDGFR-α (∼98% ± 1% [mean ± SEM]), while only 50% ± 4% of the day 5 MESP1-mCherry neg derivatives did. Interestingly, there was no enrichment of a KDR+PDGFR-α+ cardiac progenitor population in the MESP1-mCherry pos population, compared to the MESP1-mCherry neg derivatives (∼22% ± 2% [mean ± SEM] vs. 28% ± 12%, respectively) (Fig. 5B). Similar results were found at other time points during differentiation (days 4–8) (Supporting Information Fig. S4). Open in new tabDownload slide Cardiac differentiation induction from the MESP1-mCherry expressing precardiac mesoderm progenitors. (A): Schematic overview of the cardiac differentiation protocol for MESP1-mCherry cell fractions sorted by flow cytometry at day 3. (B): Flow cytometric analysis of coexpression of cell surface proteins at differentiation day 5 (2 days after replating). (C, D): Enrichment of mRNA transcripts of cardiac progenitor transcription factors at day 8 (n = 3) and day 14 (n = 2) in MESP1-mCherry pos and MESP1-mCherry neg derivatives (5 and 11 days after replating). Open in new tabDownload slide Cardiac differentiation induction from the MESP1-mCherry expressing precardiac mesoderm progenitors. (A): Schematic overview of the cardiac differentiation protocol for MESP1-mCherry cell fractions sorted by flow cytometry at day 3. (B): Flow cytometric analysis of coexpression of cell surface proteins at differentiation day 5 (2 days after replating). (C, D): Enrichment of mRNA transcripts of cardiac progenitor transcription factors at day 8 (n = 3) and day 14 (n = 2) in MESP1-mCherry pos and MESP1-mCherry neg derivatives (5 and 11 days after replating). On day 8 and day 14 of differentiation, the expression of several cardiac-associated genes was enriched in the MESP1-mCherry pos derivatives compared to the MESP1-mCherry neg derivatives, including cardiac genes NKX2-5 and ISL1, smooth muscle cell markers SMA and MYH11, and endothelial marker PECAM-1 (Fig. 5C, 5D). Additionally, NKX2-5 expression, as determined by flow cytometric quantification of eGFP expression, was significantly enriched in the MESP1-mCherry pos population, treated with Xav939 (80% ± 4% [mean ± SEM]) when compared with MESP1-mCherry neg cells under similar differentiation conditions (18% ± 6%) (Fig. 6A–6C). The eGFP+ cells detected in the MESP1-mCherry neg day 14 aggregates may have been derived from a small percentage of MESP1-mCherry pos cells that subsequently appeared in the MESP1-mCherry neg population following sorting. Indeed, low expression levels of mCherry and MESP1 could be detected in negative sorted samples at day 3 and day 4 (1 day after reaggregation) (Supporting Information Fig. S5). Open in new tabDownload slide Day 14 MESP1-mCherry derived aggregates (11 days after sorting). (A): eGFP expression in MESP1-mCherry aggregates at day 14 of differentiation (11 days after replating), with and without treatment of Xav939. Brightfield overlay with eGFP. (B, C): Percentage of cells expressing eGFP in day 14 MESP1-mCherry aggregates, with and without treatment of XAV939 (+sorted: n = 7, −sorted: n = 6, unsorted: n = 2; error bars indicate SEM, ***, p < .001). (D): Immunohistochemical stainings on day 14 MESP1-mCherry aggregates to analyze the presence of cardiac markers, including PECAM-1, SMA, SM-myosin, and α-ACT. (E): Costaining of α-ACT and SM-HC at day 14 to discriminate smooth muscle cells from cardiomyocytes. (F–H): Flow cytometric analysis of cTNT and PECAM-1 expression on day 14 MESP1-mCherry aggregates treated with XAV939 (n = 2, error bars indicate SEM). Abbreviations: α-ACT, α-actinin; SM-HC, SM-heavy chain. Open in new tabDownload slide Day 14 MESP1-mCherry derived aggregates (11 days after sorting). (A): eGFP expression in MESP1-mCherry aggregates at day 14 of differentiation (11 days after replating), with and without treatment of Xav939. Brightfield overlay with eGFP. (B, C): Percentage of cells expressing eGFP in day 14 MESP1-mCherry aggregates, with and without treatment of XAV939 (+sorted: n = 7, −sorted: n = 6, unsorted: n = 2; error bars indicate SEM, ***, p < .001). (D): Immunohistochemical stainings on day 14 MESP1-mCherry aggregates to analyze the presence of cardiac markers, including PECAM-1, SMA, SM-myosin, and α-ACT. (E): Costaining of α-ACT and SM-HC at day 14 to discriminate smooth muscle cells from cardiomyocytes. (F–H): Flow cytometric analysis of cTNT and PECAM-1 expression on day 14 MESP1-mCherry aggregates treated with XAV939 (n = 2, error bars indicate SEM). Abbreviations: α-ACT, α-actinin; SM-HC, SM-heavy chain. The importance of inhibiting Wnt/β-catenin signaling following mesoderm induction to generate NKX2-5-expressing cardiac progenitors was highlighted by the low levels of eGFP in the untreated MESP1-mCherry pos (23% ± 4%) and MESP1-mCherry neg derivatives (0.50% ± 0.4%). Furthermore, in all conditions, the NKX2-5eGFP+ derivatives were spontaneously contracting on differentiation day 14 (data not shown). To determine whether other specific signaling pathways could affect cardiac differentiation from mesoderm, three other major pathways associated with cardiac differentiation were inhibited after MESP1-mCherry sorting and reaggregation (from day 3 to day 7). Inhibitors of these pathways included transforming growth factor beta (TGF-β)/act/Nodal antagonist SB431542, and BMP inhibitor Dorsomorphin. None of these inhibitors led to an increase in GFP expression at day 14 when compared with control treatment. In fact, GFP expression showed a tendency to decrease in the SB431542-treated cells (Supporting Information Fig. S6). Day 14 MESP1-mCherry pos derivatives treated with Xav939, expressed various cardiac lineage markers, including α-Actinin (cardiomyocytes), PECAM-1 (endothelial cells), and SMA and SMC-myosin (expressed in immature cardiomyocytes as well as smooth muscle), indicating the MESP1-mCherry pos cell population can generate all three different cell types of the cardiac lineage (Fig. 6D). In addition, flow cytometric analysis showed enrichment of cTNT (∼51% ± 9% [mean ± SEM]) and PECAM-1 (∼2.2% ± 0.2% [mean ± SEM]) expressing cells in day 14 MESP1-mCherry pos derivatives, compared to the MESP1-mCherry neg derivatives (∼11% ± 5% and 0.62% ± 0.1%, respectively) (Fig. 6E). SIRPA+ VCAM1+ Coexpression Marks NKX2-5-eGFP+ Cardiomyocytes In addition to the characterization and differentiation of MESP1mCherry progenitors and their further differentiation to NKX2-5eGFP cardiac cells, we studied whether the previous described cell surface proteins SIRPA and VCAM1 are specifically marking hESC-derived-cardiomyocytes [27, 28]. We analyzed SIRPA-VCAM1 and cTnT expression on cardiac cells at day 14 of differentiation of NKX2.5-eGFP-HESCs (Supporting Information Fig. S7). We showed that the SIRPA+VCAM1+ double positive population was highly enriched for NKX2-5-eGFP+ cells (Supporting Information Fig. S7A). In addition, the vast majority of NKX2-5-eGFP+ cells were cTnT+ (Supporting Information Fig. S7B). Similar results were described in a recent study by Skelton et al., in which SIRPA+VCAM1+NKX2-5-eGFP+ cardiomyocytes were shown to arise from a SIRPA+NKX2-5-eGFP+ intermediate progenitor population [28]. Discussion Genetically modified reporter lines are proving increasingly valuable for developing precise protocols for the directed differentiation of various lineages from hPSCs [29-32]. Here, we increased the degree of complexity of the processes that can be analyzed by creating a double transgenic reporter hESC line, taking advantage of endogenous activation of the key cardiac transcription factors MESP1 and NKX2-5 [17]. We introduced the fluorescent protein mCherry into the genomic locus of the mesodermal transcription factor MESP1, in NKX2-5eGFP hESCs. As predicted from mouse developmental biology studies, the MESP1mCherry-NKX2-5eGFP line firstly expressed mCherry under conditions of mesoderm induction, and later GFP+ cardiac progenitors and cardiomyocytes emerged in the cultures, demonstrating the value of hESCs as in vitro model of previously inaccessible stages of early human development. MESP1-mCherry pos cells were first detected on day 2 of differentiation with peak levels between days 3 and 4, which is in agreement with our earlier observations during cardiac differentiation of hESCs [1]. We demonstrated that sorted and reaggregated MESP1-mCherry pos cells on differentiation day 3 have a much higher potential to differentiate to cardiovascular derivatives than MESP1-mCherry neg cells, as demonstrated by (a) the appearance of functional beating cardiomyocytes and (b) high numbers of NKX2-5eGFP+ cardiac cells in the population on day 14 of differentiation (11 days after sorting). At the same stage of differentiation, enriched gene and protein expression were found for PECAM-1, SMA, and cTNT, suggesting that MESP1-mCherry cells can differentiate to endothelial cells, smooth muscle cells, and cardiomyocytes, respectively. Microarray analysis of MESP1-mCherry pos and MESP1-mCherry neg cells revealed new insights into the transcriptional profile of human precardiac mesodermal progenitors. In particular, transcription factors and signaling pathways associated with mesoderm formation, EMT, early cardiac development, and components of the ECM were highly enriched in MESP1-mCherry pos cells. This is broadly in accordance with the molecular signature of MESP1-GFP expressing mouse cells [33]. However, disparities possibly related to interspecies differences, differentiation protocols, or technical variations (i.e., random integration of an isolated regulatory sequence of the Mesp1 mouse promoter vs. homologous recombination in the human MESP1 locus) were evident. For example, the expression of cardiac transcription factors such as HAND2, MEIS2, SMARCD3, HEY1, HEY2, and TBX2 was increased in MESP1-GFP positive cells, whereas in our study these genes were not enriched in the MESP1-mCherry pos population. However, expression of these genes was increased at later stages following cardiac differentiation of sorted cells, suggesting that human MESP1-mCherry pos cells may be a more restricted mesodermal population. Transition of undifferentiated epithelial pluripotent stem cells to mesodermal cells requires the activation of EMT-associated genes. Indeed, MESP1-mCherry pos cells exhibit an obvious increase in levels of EMT-related transcription factors SNAI1 and SNAI2. In agreement, increased expression of other EMT-related genes, including Vimentin and CDH-2, and decreased levels of epithelial proteins, such as CDH-1 and EPCAM, suggested that EMT took place in MESP1-mCherry pos cells. In addition, enrichment of many ECM-related transcripts, such as those for collagens, integrins, fibrillin, matrix remodelers, and receptors, like the Apelin Receptor, suggested formation of an appropriate niche for MESP1-expressing cells [34, 35]. Alterations in cell-cell and cell-matrix interactions are crucial for biological processes such as EMT, intracellular interactions, cell-fate decisions, or migration of precardiac MESP1-progenitors from the anterior part of the primitive streak to form the bilaterally situated cardiac crescent in vivo [36-39]. Further study is required to identify the role of specific ECM components and receptors for expansion and further differentiation of MESP1+ cells. Several signaling pathways were also significantly activated in MESP1-mCherry pos cells. In particular, components of the Wnt signaling pathway were affected, including the Wnt-inhibitors DKK1 and DKK4, the (non)canonical pathway ligands WNT4 and WNT5a, and the receptors ROR-2 and FZD2. Previous studies have indicated an important role for activated canonical Wnt-signaling during hESC to mesendoderm differentiation [40-43]. Subsequent inhibition of this pathway by DKK-1 resulted in improved cardiac differentiation, confirming the need to inhibit canonical Wnt-signaling following mesoderm formation for efficient cardiac differentiation [14, 40]. Conversely, at the same stage, activated noncanonical signaling has been associated with driving cardiac differentiation [44]. Noncanonical Wnt signaling has been linked with different molecular mechanisms, including canonical Wnt and/or β-catenin signaling inhibition [42, 45, 46], progenitor proliferation [47], and cellular movements and polarity. Here, we confirmed the importance of Wnt-inhibition in mesoderm to cardiac progression, as we found a major increase in the percentage of NKX2-5eGFP expressing cells upon treatment with the chemical Wnt signaling antagonist, Xav939. Other pathways that have been shown to play crucial roles in the early stages of mesoderm formation, such as the BMP, Notch, and FGF pathways, were also present in the MESP1-mCherry pos population, suggesting crosstalk of components of these pathways during these early precardiac mesoderm stages [41]. We also examined expression of cell surface markers of mesodermal and cardiovascular progenitors during the differentiation of MESP1mCherry-NKX2-5eGFP cells. Several studies have reported that PDGFR-α and Flk1/KDR are expressed on early cardiovascular progenitor cells, giving rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells [5, 19, 20]. In recent studies using hESCs, an early mesoderm population coexpressing CD13 (ANPEP) and ROR-2 has been identified [18, 48]. This population consists of a KDR+PDGFR-α+ subpopulation, which has the potential to give rise to cardiovascular cells. In agreement, we demonstrated a significant increase in CD13, ROR-2, and PDGFR-α on day 3 MESP1-mCherry pos cells and on day 5 derivatives. However, these surface markers were also expressed in a high percentage of the MESP1-mCherry neg populations, indicating that they mark a broader population than only precardiac mesoderm [27]. Interestingly, in MESP1-mCherry pos derivatives we did not find enrichment of KDR+ cells. These findings indicated that, besides the previously described Flk1/KDR+ PDGFR-α+ cardiac progenitor population, other MESP1-derived precardiac mesoderm progenitor populations exist, that (co)express other (cell surface) markers, and have the potential to differentiate toward NKX2-5eGFP expressing cardiac cells. In previous studies, the cardiac potential of other subpopulations besides the Flk1/KDR+ PDGFR-α+ was not tested [18, 20]. One of the major challenges in cardiovascular research is cardiac regeneration and improved cardiac function in heart failure patients. Tissue engineering, cellular therapy, endogenous activation of cardiac progenitor cells, or a combination of these approaches may lead to novel therapeutic strategies [49]. However, a key concern is the selection of the right cell type for transplantation or enhanced in situ proliferation, as this will be important for survival and engraftment, for safety (risk of tumor formation), and for the ability to form the required cell type(s) [50, 51]. A recent study showed the ability of a hESC-derived CD13+ROR-2+KDR+PDGFR-α+ cardiac progenitor population to migrate, couple, and form cardiac cells after being transplanted into ex vivo human fetal ventricular heart tissue, without formation of teratomas [18]. As precardiac MESP1-progenitors are able to form almost all cell types of the heart, it will be of interest to examine both the developmental and regenerative capacity of these cells upon transplantation into both healthy and damaged hearts. Even though many hurdles have to be overcome, transplantation studies using cardiac-lineage-restricted progenitors may shed more light on future heart regeneration therapies. In addition, understanding the molecular cues that are important for expansion of mesodermal cells and their specific differentiation to the cardiac lineage may lead to novel therapies which are based on the progression of endogenous cardiac progenitor cells to functional specialized cardiovascular cells. Conclusions We conclude that the MESP1mCherry/w-NKX2-5eGFP/w dual cardiac fluorescent reporter hESC line in this study is a useful tool for dissecting the molecular and cellular mechanisms related to the earliest steps in cardiac development in live cells. This is novel way to carry out fate analysis of this subpopulation. Purified mesodermal MESP1-mCherry pos cells efficiently differentiated to beating NKX2-5GFP+ cells by inhibition of the Wnt-pathway. Besides a better understanding of underlying mechanisms, this human cardiac dual reporter line will make it feasible to identify and specify culture conditions for expansion of a mesodermal cell population and their further differentiation toward cardiac progenitor cells and specific subtypes of the cardiac lineage in vitro. This is of particular interest for future studies on cardiac toxicity, drug screening, tissue engineering, and regenerative medicine. Acknowledgments We thank Dorien Ward-Van Oostwaard for technical assistance; Kostas Gkatzis for help on statistical analysis using R/Bioconductor, the Department of Molecular Cell Biology, LUMC, for karyotyping analysis. S.C.D.H was supported by the Rembrandt Institute for Cardiovascular Disease. This study was supported by the Rembrandt Institute for Cardiovascular Science (S.H.), NHMRC, Australia (D.A.E.), ZonMw-MKMD-40-42600-98-036 (R.P.), and ERC Advanced STEMCARDIOVASC-323182 (C.L.M.). 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Stem Cells – Oxford University Press

Published: Jan 1, 2015

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