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MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Human embryonic stem cells (hESCs) are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease.
To better understand the transition between the human embryonic and cardiac “miRNA-omes”, we report here the first miRNA profiling study of cardiomyocytes derived from hESCs (hESC-CMs). Analyzing 711 unique miRNAs, we then identify several interesting miRNAs, including miR-1, miR-133, and miR-208, that have been previously reported to be involved in cardiac development and disease and that show surprising patterns of expression across our samples. We also identify novel miRNAs such as miR-499 that are strongly associated with cardiac differentiation, and which shares many predicted targets with miR-208. Over-expression of miR-499 and miR-1 resulted in upregulation of important cardiac myosin heavy chain genes in embryoid bodies; miR-499 over-expression also caused upregulation of the cardiac transcription factor MEF2C.
Taken together, our data give significant insight into the regulatory networks that govern hESC differentiation, and highlights the ability of miRNAs to perturb, and even control, the genes that are involved in cardiac specification of hESCs.
Human embryonic stem cell–derived cardiomyocytes (hESC-CMs) hold great promise for myocardial regeneration after infarction. A number of groups have reported successful transplantation of hESC-CMs into rodent models of myocardial infarction1–3. However, a significant obstacle still exists with consistent derivation of purified hESC-CM populations. In order to precisely drive differentiation of hESCs into cardiac cells, a more comprehensive understanding of the regulatory networks that are involved in this process is needed. hESC-CM transcriptional profiling has already been reported2, however no group has yet focused on the rapidly evolving field of microRNAs (miRNAs) and their expression profiles in hESC-CMs.
MiRNAs play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression4. Hundreds of human miRNAs have been discovered, and each is predicted to target tens or hundreds of different mRNAs. hESCs are known to express miRNAs that are often undetectable in adult organs5–7, and some of these miRNAs are believed to regulate ESC self-renewal and differentiation8. A growing body of evidence has also implicated miRNAs in heart disease and development9. As examples, miRNAs −21, −195, and −208 have demonstrated roles in heart failure, hypertrophy, and response to stress10–12, while miRNAs −1 and −133 are known to regulate cardiac development, differentiation, and some aspects of disease13–16. Although previous studies have highlighted miRNA profiles of hESCs and embryoid bodies5, 6, to date the miRNA signature of hESC-CMs remains unknown.
To better understand the transition between the human embryonic and cardiac “miRNA-omes”, we report here the miRNA profiles of cardiomyocytes derived from human embryonic stem cells. Our results confirm the presence of a signature group of miRNAs that is upregulated in hESCs, and whose expression is significantly altered during differentiation to a cardiac lineage. We also identify novel patterns of miRNA expression, as well as individual miRNAs such as miR-499, that suggest a central role for miRNAs in pluripotency, differentiation, and maintenance of the cardiac phenotype.
All studies used cells derived from the female H7 hESC line (Wicell, Madison, WI) and were maintained in the undifferentiated state using mTeSR™ 1 medium (StemCell Technologies, Vancouver, Canada).
Prior to differentiation, hESCs were switched to mouse embryonic fibroblast conditioned medium. For these experiments, hESC-CMs were generated using a method adapted from a previously reported protocol for efficient cardiogenesis3.
Undifferentiated hESCs and hESC-CMs were plated onto 8 chamber slides and fixed with acetone on ice for 20 minutes, then stained for immunofluorescence with the appropriate antibodies. Microscopy was performed using a ZEISS Axiovert microscropy (Sutter Instrument).
Cultures were dissociated with 0.05% trypsin, permeabilized with methanol (and incubated with alpha- sarcomeric actinin (Sigma A7811) or isotype control) in 20% heat inactivated goat serum with rat anti-mouse Fc block. Samples were washed, incubated with the appropriate secondary antibody (0.5 ug/106 cells), and incubated at room temperature for 15 min in the dark. Samples were washed with 4 ml PBS and resuspended in 200 ul of 0.5% PFA prior to analysis on a FACSCalibur machine.
Functional experiments were performed with Lenti-miR™ MicroRNA Precursors and a miRZip anti-miR-499 microRNA construct (System Biosciences, Mountain View, CA). All experiments were performed a minimum of three times. SIN lentivirus was prepared by transient transfection of 293T cells. hESCs were stably transduced with Lenti-miR™ MicroRNA Precursors or miRZip anti-miR-499 microRNA construct at a multiplicity of infection (MOI) of 10. For the miRNA over-expression studies, the infectivity was determined by copGFP expression as analyzed on FACScan (BD Bioscience). The copGFP positive cell populations were isolated by fluorescence activated cell sorting (FACS) Vantage SE cell sorter (Becton Dickinson) followed by plating on feeder cell layer for long-term culturing.
hESC colonies were dispersed into cell aggregates containing approximately 500 to 1,000 cells using 1 mg/mL collagenase IV. The cell aggregates were then suspension-cultured in ultra-low attachment cell culture dishes with hESC differentiation medium (without basic fibroblast growth factor): 80% Dulbecco’s modified Eagle’s medium/F12, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 0.1 mN non-essential amino acids, 20% Knockout Serum Replacement. Culture media was changed every two days, for a total of 6 days.
Using the miRNeasy Mini Kit (Qiagen Inc., Valencia, CA), total RNA containing miRNAs was isolated separately from biological duplicates of H7 hESCs and H7 hESC-CMs for microarray profiling. As positive control, total RNA was isolated from left ventricular tissue derived from two 20-week old human fetus hearts. As negative control, total RNA was isolated from IMR90 fetal fibroblasts (ATCC, Manassas, VA). We prepared a total of eight distinct RNA samples for miRNA profiling. For embryoid body experiments, total RNA was isolated from biological triplicates (each experiment repeated minimum three times) of H7 hESCs and H7 embryoid bodies (day 6). Total RNA concentration and purity were analyzed by spectrophotometry.
The expression of human embryonic cell markers (OCT4, NANOG, REX1) and cardiac-specific markers (ANF, MEF2C, GATA4) were compared before and after hESC differentiation. 18S was used as housekeeping gene control. The primer sets used in the amplification reaction are as follows:
Microarray assay was performed using a service provider (LC Sciences, Houston, TX). The assay started with 4 to 8 μg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore, Billerica, MA) and the small RNAs (< 300 nt) isolated were 3′-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a μParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies, Houston, TX)17. On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other control RNA, and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent chemistry. Hybridization used 100 μL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 or Cy5 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Devices, Sunnyvale, CA) and digitized using Array-Pro image analysis software (Media Cybernetics, Bethesda, MD).
See online Supplemental Information.
For mRNA qPCR, 2 μg of total RNA from each sample was reversed transcribed with Superscript III (Invitrogen). For each sample, qRT-PCR was performed in triplicate on an ABI 7900HT instrument (Applied Biosystems) using Taqman primer probe sets (Applied Biosystems) for each gene of interest and 18S endogenous control primer probe set for normalization. Representative results are shown as fold expression relative to undifferentiated hESCs unless otherwise stated. Error bars reflect one standard deviation from the mean of three biological replicates unless otherwise stated.
MiRNA qPCR was performed using a service provider (LC Sciences). Briefly, single-stranded cDNA was synthesized from total RNA samples using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. cDNA was then PCR amplified using Human TaqMan® MicroRNA Assays (Applied Biosystems) for the following human miRNA: miR-1, miR-302c, miR-302d, miR-133a, and miR-16. We included miR-16 because it has been reported to exhibit relatively stable expression across multiple tissue types18. Data were normalized to the endogenous control genes RNU48 and U4719. Results are shown as fold expression relative to undifferentiated hESCs unless otherwise noted. Error bars indicate one standard deviation from the mean of three technical replicates unless otherwise stated.
Cells were collected in RIPA lysis buffer and briefly sonicated to shear DNA and reduce sample viscosity. The protein concentration of the supernatant was determined by the Bio-Rad Protein Assay. Each sample supernatant in equal volume loading buffer was run on 12% Tris-glycine SDS-PAGE gels and transferred to Hybond ECL membrane (Amersham). Protein blots were analyzed with GATA4 (Abcam) and MEF2C (Abcam) antibodies and developed by ECL assay (Amersham).
Spontaneously beating clusters were microsurgically dissected from hESC-derived embryoid bodies 14–21 days after initiating differentiation. After collagenase dissociation and plate attachment, electrophysiological experiments of isolated single hESC-CMs were performed using the whole-cell patch-clamp technique with an Axopatch 200B amplifier and the pClamp9.2 software (Axon Instruments Inc., Foster City, CA). A xenon arc-lamp was used to view GFP fluorescence at 488/530 nm (excitation/emission). Patch pipettes were prepared from 1.5 mm thin-walled borosilicate glass tubes using a Sutter micropipette puller P-97 and had typical resistances of 4–6 MΏ when filled with an internal solution containing (mM): 110 K+ aspartate, 20 KCl, 1 MgCl2, 0.1 Na-GTP, 5 Mg-ATP, 5 Na2-phospocreatine, 1 EGTA, 10 HEPES, pH adjusted to 7.3 with KOH. The external Tyrode’s bath solution consisted of (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH adjusted to 7.4 with NaOH. Current-clamp recordings were performed at 37 °C within 4 to 7 days after Lenti-miR™ transduction.
Non-microarray data are presented as mean±S.D. Data were compared using standard or repeated measures, using ANOVA where appropriate. Pairwise comparisons were performed using a two-tailed Student’s t–test. Differences were considered significant for P-values<0.05.
To determine the miRNA-omes of hESC-CMs, cardiomyocytes were differentiated from H7 hESCs based on a published protocol3. Flow cytometry revealed greater than 90% of cells were positive for the cardiac marker α-sarcomeric actinin (Figure 1a). hESC-CMs spontaneously beat in culture (Videos S1–S4) and exhibited a muscle-like morphology that was similar to isolated left ventricular human fetal heart cells (Figure 1b). While hESCs stained positive for OCT4 (Figure 1c), hESC-CMs stained positive for the cardiac specific markers Troponin-T, MEF2C, and Connexin 43 (Figure 1d). RT-PCR further revealed that hESC-CMs express cardiac genes (NKX2.5, ANF, GATA4, αMHC (MYH6), βMHC (MYH7), and βMHC7b (MYH7B)), with a corresponding decrease in embryonic genes (OCT4, NANOG, REX1) (Figures 1e,f). By quantitative PCR (qPCR), we noted similar, though not identical, gene expression levels in hESC-CMs as compared to fetal heart, suggesting that these cells are different from fetal heart, as has been previously described2.
Next, we analyzed 711 unique miRNAs in hESC-CMs and human fetal left ventricles using the Sanger miRBase Version 10.0 miRNA expression microarrays (LC Sciences, Houston, TX). MiRNA profiles for H7 hESCs and human fetal fibroblasts from our previous publication20 were also included in the analysis. The expression levels of a select group of miRNAs were confirmed with qPCR (Figure S1; see Table S1 for full normalized data set, and Materials and Methods).
We first performed principal component analysis (PCA), which is a reductionist method that provides an overview of the degree of variation between samples (Figure S2). PCA indicated that the miRNA expression profile is distinct for each of the four samples groups, with fetal heart and hESC-CMs exhibiting the closest distance – and thus the most similar miRNA profiles, and hESCs the furthest from the other three groups. Using clustered heatmaps of subsets of miRNAs, we organized miRNAs into three broad categories: miRNAs significantly expressed in hESCs (Figure 2a) and were similar to previous studies5, 7, miRNAs significantly expressed in the cardiac phenotype (heart and hESC-CMs) (Figure 2b), and miRNAs significantly expressed in fetal samples (heart and fibroblasts) (Figure S3) (see Figure S4 for full heatmaps). These miRNA subsets are further refined in Figures 2c–f. Some miRNAs increased during differentiation (Figure 2d) but were not cardiac-specific since they were also expressed in fibroblasts, while other miRNAs decreased during differentiation (Figure 2e). Of interest in this former group is miR-145, which has been previously reported to be upregulated during differentiation and is an inhibitor of OCT4, SOX2, and KLF421.
We were of course particularly interested in miRNAs that appeared to be cardiac-specific since these miRNAs may be critical for driving cardiac differentiation. Known cardiovascular-related miRNAs include miRNAs −1, −133, and −208, which have roles in heart failure, hypertrophy, stress response, cardiac and skeletal muscle development, and differentiation10, 11, 14–16. In our profiles, these miRNAs show robust expression in hESC-CMs and (with the exception of miR-208a) fetal heart, but are nearly undetectable in hESCs and fibroblasts (Figure 2f). Interestingly, we observed co-expression of miRNAs and their host genes22. For example, miR-208a is transcribed from an intron of the myosin heavy chain αMHC (MYH6, see Figure S5), and its expression therefore follows that of αMHC: higher expression in hESC-CMs relative to fetal heart (see Figure 1f). In contrast, miR-208b is transcribed from intronic regions of βMHC (MYH7), and its expression thus follows that of βMHC: mildly higher expression in fetal heart compared to hESC-CMs (see Table S1 for quantitative miRNA expression data).
Of the cardiac phenotype-associated miRNAs identified in Figure 2b, we observed one miRNA, miR-499-5p, that showed statistically significant expression in the cardiac phenotypes but was undetectable in hESCs and fibroblasts (Figure 2f), a pattern similar to miR −1, −133 and −208b. This miRNA has been previously reported to be important in human fetal cardiomyocyte differentiation23 and regulation of murine muscle myofibers24. Like the miR-208 family, miR-499 is located in an intronic region of a myosin heavy chain gene, βMHC7B (Figure S5), and its trend in expression follows that of βMHC7B: higher expression in fetal heart compared to hESC-CMs. Using TargetScan (www.targetscan.org), an online database of predicted biological targets of miRNAs based on the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA, we found that miR-499 shared many targets with miR-208 and had some overlap with miR-1 targets (Figure 3a). Canonical pathway analysis of the predicted targets for miR-499 using Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) revealed that many targeted genes are involved in early cardiogenesis (e.g., the transcription factor GATA425) and embryogenesis. Targeted canonical pathways included Wnt/β-catenin signaling26, TGF-β signaling, and cell cycle regulation (see Table 1 for selected pathways, and Table S2 for full data set). These findings suggest that miR-499 may be involved in maturation of the cardiomyocyte via inhibition of embryogenesis and cardiogenesis pathways.
Next, we wanted to know whether the predicted targets of miR-499, along with miR-1 and miR-208, were significantly downregulated during cardiac differentiation as these miRNAs become more highly expressed. For example, are the target genes of miR-499 downregulated in fetal heart relative to hESCs due to the higher expression of miR-499? Using microarray data from our previous study2 (GEO accession number GSE13834), we used Gene Set Enrichment Analysis (GSEA)27 to analyze the enrichment of predicted miRNA targets in three successive stages of cardiac differentiation relative to hESCs: beating embryoid body (EB, an intermediate stage of cardiac differentiation), hESC-CM, and fetal heart. In Figure 3b, the y-axis represents the normalized enrichment score (NES), which is the primary statistic for examining GSEA results. NES>1 means the targets are more highly expressed in the cardiac cell type, and NES<1 means the targets are more highly expressed in hESCs (see Table S3 for quantitative data). As expected, a custom set of 26 genes that are each associated with pluripotency was more highly enriched in undifferentiated hESCs compared to the three cardiac differentiation stages. In contrast, the predicted targets of the miR-302 cluster, which was significantly upregulated in hESCs and has been previously associated with pluripotent cell types20, had the opposite pattern of expression: higher enrichment in the three stages of cardiac differentiation relative to hESCs, as expected for an “embryonic” miRNA. Importantly, the predicted target genes for miR-1, −208, and −499 each exhibited similar enrichment patterns wherein the target genes were gradually reduced in expression during cardiac differentiation, thus underscoring the similarity of miR-499 with the better characterized miR-1 and miR-208 miRNAs. We also performed GSEA in a mouse system using previously published microarray data for murine ESCs differentiating to cardiomyocytes28, 29 (GEO accession numbers GSE5671 and GSE10970). Similar to our human results, we found that the predicted targets for miR-499, miR-1, and miR-208 became successively less enriched during cardiac differentiation (Figure S6).
We next wanted to determine how over-expression of miR-1 and miR-499 might affect cardiac gene expression in differentiating hESCs. Using Lenti-miR MicroRNA Precursor lentiviral constructs (System Biosciences, Mountain View, CA, Figure S7a), we stably transduced H7 hESCs with miR-1 or miR-499 (Figure 4a). qPCR confirmed upregulation of the two miRNAs (Figure S7b). Cells were differentiated into EBs and qPCR then used to determine embryonic, mesodermal, and cardiac gene changes on day 6 (Figure 4b). Interestingly, we noted significant upregulation of MEF2C in miR-499EBs relative to miR-1EBs and WTEBs. Knockdown of miR-499 using a miRZip anti-miR-499 microRNA construct (System Biosciences) that specifically inhibits the −5p and not the −3p isoform did not upregulate MEF2C. MEF2C is required for activation of cardiac contractile genes, and for cardiac structural development30, suggesting that miR-499 may be an important factor in differentiating hESCs. This is further evidenced by upregulation of βMHC in miR-499EBs relative to WTEBs. miR-1EBs exhibited upregulation of all three myosin heavy chain isoforms. Furthermore, GATA4 was significantly upregulated in miR-1EBs but not in miR-499EBs, suggesting that miR-1 and miR-499 each have distinct yet positive roles in cardiomyocyte biology. Western blots confirmed higher protein expression of MEF2C in miR-499EBs and GATA4 in miR-1EBs relative to the other cell lines (Figure S7c). Clearly these results highlight the ability of specific miRNAs such as miR-499 and miR-1 to alter the expression of genes involved in the cardiac specification of hESCs.
Two studies have reported cardiac conduction abnormalities in murine hearts either after transfection with miR-131 or in mutants that lack miR-1 expression14. To further explore the functional effects of miR-1 and miR-499, we also transduced post-differentiated hESC-CMs with each miRNA. miR-1 transduction statistically decreased the beating rates of spontaneously-contracting EB clusters, although miR-499 had no effect (6 independent batches, P<0.05; Figure 5a). Consistently, the pacemaker current (If), which is known to play a major role in phase 4 depolarization, decreased its amplitude with miR-1 over-expression (Figure 5b). This finding confirms a previous report in which miR-1 was shown to decrease If by targeting the pacemaker genes HCN2 and HCN4 in hypertrophic cardiomyocytes32.
This study seeks to generate a systems-level overview of the miRNA profiles in hESCs, hESC-CMs, and human fetal heart. Our results indicate that these profiles are highly dynamic and specific during cardiac differentiation of hESCs. Several interesting miRNAs, including miR-1, miR-208 and miR-133, that have been previously reported to be important in cardiac development and disease, exhibited significant changes during cardiac differentiation. Other miRNAs such as miR-499 that have poorly defined roles in development and differentiation also showed cardiac-specific expression. In silico analysis of the predicted targets for selected miRNAs revealed significant overlap between miR-208 and miR-499, both of which are transcribed from the introns of myosin heavy chains.
Admittedly, the field of computational predictions of miRNA targets is still in its infancy, and so we cannot assume complete confidence in their accuracy until further validation through functional assays is performed. However, the analysis of target gene sets by GSEA, rather than by individual genes, lends increased statistical power to the in silico analysis of target predictions. Using this algorithm, we have shown that miR-499 shares many similarities with previously identified cardiac-specific miRNAs in terms of gene targets and expression pattern. In general, the targets of miR-1, −208, and −499 exhibited very similar pattern of expression: high enrichment in EBs, moderate enrichment in hESC-CMs (though miR-1 targets were still highly expressed in hESC-CMs), and significantly reduced enrichment in fetal heart. We were surprised by some of the continued target expression in hESC-CMs, which may be due to the heterogeneity of the hESC-CMs used in Cao et al. in which only 43% were positive for the cardiac marker cardiac Troponin T, potential biological differences between hESC lines (H7 vs. H9), and also the poorly understood roles these miRNAs play during cellular differentiation. This latter point is based on the observation that some miRNAs are co-expressed with their target mRNAs33, and thus may represent complex, incoherent feedback loops34 that help fine-tune the expression levels of important transcription factors.
Lastly, with the ultimate goal of discovering factors that might enhance cardiac differentiation, we selected miR-499 and miR-1 for functional studies. Each miRNA had significant, though different, positive effects on cardiac differentiation, suggesting a powerful role for miRNAs in influencing hESC fate decisions. What is likely necessary for efficient differentiation of hESCs to cardiomyocytes is the simultaneous expression of miR-1, miR-499, and others such as miR-208, that together regulate that molecular programs of hESC-CMs. In the future, we believe miRNAs will play a key role in achieving higher yields of hESC-CMs that can then be used for transplantation studies and, ultimately, clinical therapies.
We are grateful to Dr. Joseph Gold at Geron Corporation for his assistance with hESC differentiation; Dr. Christoph Eicken at LC Sciences for his help with miRNA microarrays; and Dr. Travis Antes at System Biosciences for lentiviral constructs.
Funding Sources: This work is supported by Stanford Bio-X Fellowship (KDW) and NIH DP2 OD004437, HL099776, HL089027, and BWF (JCW).
Conflict of Interest Disclosures None.