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Heart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation.
We treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)–polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation.
We observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium.
These studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy.
Human cardiomyocytes (hCM) differentiated from various human embryonic stem cell (hESC) lines have been shown to exhibit properties similar to endogenous hCM, including cardiac-specific gene expression (1), sarcomere ultrastructure (2,3) and characteristic action potentials (4–6). Successful engraftment of isolated beating clusters into various models of myocardial disease has generated enthusiasm for hESC as a source of new hCM for myocardial cell therapy (1,5,7,8).
The differentiation of hESC into hCM, however, is a low-yield process, as most groups have relied on spontaneous differentiation in the presence of serum (9–11). For an effective therapy, the cardiac cells need to be generated in large numbers and with sufficient purity to avoid the formation of other tissues (11–13). Xu et al. (10) reported increased cardiogenesis with the addition of the de-methylation agent azacytidine, followed by Percoll gradient separation. Mummery et al. (14,15) subsequently showed an increased cardiomyocyte yield by co-culturing hESC with a mouse endoderm-like cell line with or without serum. Other groups have since reported increased cardiogenesis by the addition of various culture additives, such as activin A and bone morphogenetic proteins 2 and 4 (16,17).
Pathways regulating the activity of p38 mitogen-activated protein kinase (p38MAPK) have been implicated in regulating mammalian hCM proliferation (18) and neural differentiation (19). Recently, Graichen et al. (20) detected the expression of p38MAPK in hESC as well as differentiating hEB and suggested that its inhibition could induce cardiogenesis. They reported enhanced cardiogenesis from hESC grown in co-culture with visceral endoderm cells in the presence of a p38MAPK inhibitor (20).
In this study, we used SB203580, a small-molecule inhibitor of p38MAPK, to study the dynamics of cardiogenesis with p38MAPK inhibition. We report that inhibition of p38MAPK during the stage of hESC differentiation that coincides with ectoderm/mesoendoderm divergence results in directed, accelerated differentiation of hCM, and that the resulting hCM maintain properties, such as genomic stability and survival in vivo, that are essential for cell transplant therapy.
All hESC procedures were approved by the Stem Cell Research Oversight Committee at the University of California (San Francisco, CA, USA). The H9 hESC line (WA09) expressing enhanced green fluorescent protein (GFP) under the control of the ubiquitin C promoter (21) was maintained on irradiated CF1 mouse embryonic fibroblasts (MEF), as described previously (22). All reagents were purchased from Invitrogen, Carlsbad, CA, USA, except where indicated. hESC were cultured between passages 35–90 in KSR medium [knock-out Dulbecco’s modified Eagle medium (DMEM) containing 20% knock-out serum replacer, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 12 ng/mL recombinant human basic fibroblast growth factor (bFGF) (R&D Systems, Minneapolis, MN, USA)]. MEF were cultured in DMEM containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA, SH 30071.03), 2 mM L-glutamine and 1× penicillin and streptomycin. hESC colonies were passaged every 3–4 days on fresh MEF using 1 mg/mL collagenase IV in KSR medium without bFGF and manual dissociation of hESC colonies.
hESC were differentiated by hEB formation as described previously (22). Proliferating hESC colonies were washed with calcium/magnesium-free phosphate-buffered saline (PBS) (PBS-cmf) and incubated with 1 mg/mL collagenase IV for 5 min at 37°C, washed with PBS-cmf, then resuspended in small clumps in differentiation medium [knock-out DMEM medium containing 20% FBS (Hyclone SH 30070.03), 0.1 mM non-essential amino acids, 2 mM L-glutamine and 0.1 mM 2-mercaptoethanol] and placed in low-attachment 6-well plates. The medium was replaced every other day. On day 7 following re-suspension, approximately 25–30 hEB/well were plated on 0.1% gelatin-coated, 12-well plates in the same medium.
SB203580 (Calbiochem, Gibbstown, NJ, USA) was prepared in dimethylsulfoxide (DMSO) at 10 mM, and diluted in medium to the indicated final concentrations. SB203580 was removed by rinsing cultures with PBS-cmf, then resuspending hEB in differentiation medium.
Flow cytometric analysis was performed as described previously (22). hESC differentiated for 30 days in culture were harvested using 0.05% trypsin–ethylene diamine tetra acetic acid (EDTA) and fixed in 2% paraformaldehyde. Cells were permeabilized using perm/wash buffer (554723; BD Biosciences, San Jose, CA, USA), blocked with cold blocking buffer (PBS with 20% horse serum and 0.5 mM EDTA) and incubated on ice in blocking buffer with Alexa 647-conjugated anti-α myosin heavy chain (MHC) (1:50 dilution) or isotype control antibody (1:50 dilution). Anti-αMHC (αMHC ab15; Abcam, Cambridge, MA, USA) and isotype control antibodies were conjugated to Alexa Fluor 647 using an Alexa labeling kit (A-20186; Molecular Probes/Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Following incubation with antibody, cells were washed with blocking buffer and analyzed on a FACSCalibur flow cytometer (BD Biosciences) using standard filter sets.
The lineage fate of differentiated hESC was determined by staining 30-day-old adherent differentiating hEB in culture plates. Cells were fixed with 2% paraformaldehyde for 20 min at 21°C, then permeabilized and blocked in 10% horse serum, 1% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS for 1 h at 21°C. hEB were incubated with primary antibody (1–5 μg/mL) in blocking buffer for 1 h at 21°C or overnight at 4°C. Primary antibodies used were mouse anti-human α-fetoprotein (Sigma-Aldrich, St. Louis, MO, USA, A8452), mouse anti-human nestin (R&D Systems MAB1259), mouse anti-human βIII tubulin (R&DSystemsMAB1195), mouse anti-humans mooth muscle actin (R&D Systems MAB1420) and mouse anti-human cardiac troponin T (cTnT; Lab Vision Corporation, Fremont, CA, USA, MS-295). Cells were washed with blocking buffer, incubated with a 1:200 dilution of phycoerythrin (PE)-conjugated donkey anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) for 30 min at 21°C, washed with 1% Triton X-100 in PBS-cmf, and then analyzed by fluorescence microscopy using a Nikon Eclipse TE300 microscope.
Genomic DNA (300–500 ng) from treated and untreated 30-day-old differentiated hEB was isolated using a QiaEasy DNA kit for tissue (Qiagen, Valencia, CA, USA). DNA was labeled with Cy3 and Cy5 using the BioPrime DNA labeling system (Invitrogen), hybridized to custom HumArray 3.2 human chromosome arrays, and analyzed for chromosomal composition in the Array Core at the UCSF Helen Diller Family Comprehensive Cancer Center according to published methods (http://cancer.ucsf.edu/array/protocols/index.php, 3 June, 2010) (23). Differentially labeled human male reference genomic DNA was run as a control. The custom HumArray 3.2 prepared in the Array Core contains 2464 bacterial artificial chromosome clones spotted in triplicate and distributed uniformly across the genome. Each clone contains at least one Sequence Tagged Site (STS) and is mapped to the human genome sequence. Clones containing unique sequences near the telomeres and genes known to be significant in cancer and medical genetics are included on these arrays.
For analysis of hESC-derived hCM, beating areas from approximately 10 hEB were visualized with a Leica, Wetzlar, Germany, MZ6 microscope and manually excised using an 18-g needle. The collected tissue samples were treated with 0.05% trypsin–EDTA to generate a single-cell suspension prior to RNA isolation. RNA was isolated and cDNA synthesized from c. 50 000 hEB-derived cells or proliferating hESC using the TaqMan gene expression cells-to-CT kit (Ambion, Austin, TX, USA). cDNA was quantitated using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies/Thermo Scientific, Wilmington, DE, USA, ND software version 3.3.0). Linear pre-amplification of target sequences was accomplished using the Applied Biosystems PreAmp system. Relative expression was determined using a TaqMan assay (Applied Biosystems (ABI), Foster City, CA, USA) on an ABI 7300 real-time (RT)–polymerase chain reaction (PCR) system with the following commercially inventoried primer pairs (Applied Biosystems): α-fetoprotein (Hs00173490_m1), smooth muscle actin (Hs00242273_m1), βIII tubulin (Hs00964965_m1), GATA4 (Hs00171403_m1), Nkx2–5 (Hs00231763_m1), αMHC (Hs00411908_ m1), MLC4 (Hs00267321_m1), cTnT (Hs001 65960_m1) and GAPDH (4326317E). Cycle times to detection were normalized against a reference gene, GAPDH, and relative changes were calculated using ABI version 1.4 sequence detection software.
Beating areas (c. 20–30) were excised from p38i-treated and untreated cultures at day 45 of differentiation and fixed in 2% glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4. Tissue was post-fixed in 2% osmium tetroxide, block-stained in 2% aqueous uranyl acetate, dehydrated in acetone and embedded in LX-112 resin (Ladd Research Industries Inc, Williston, VT, USA). Ultrathin sections were contrast stained with 0.8% lead citrate. Samples were analyzed on a JEOL JEM-1230 electron microscope (JEOL USA, Inc. Portland, OR, USA) and photographed with a Gatan ultrascan USC1000 digital camera (Gatan, Inc., Pleasanton, CA, USA). At least 30 sections were analyzed for each condition.
All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California. Teratomas were generated as previously described (22,24). Treated and untreated hEB were differentiated for 7 days in suspension, then 5 × 105 hEB-derived cells were mixed with an equal volume of 1 mg/mL Phaseolus vulgaris lectin (PHA-P L1668; Sigma), pelleted and incubated in growth medium overnight at 37°C, 5% CO2 in a 0.4-μm MILLICELL (Millipore, Billerica, MA, USA). At least two cell pellets were grafted under each kidney capsule of 8-week-old male CB17 SCID-Beige mice (three mice per condition) using standard techniques (24,25). The resulting teratomas were analyzed 10 weeks after grafting. Teratomas were fixed in 10% buffered formalin, embedded in paraffin, and 5-μm sections stained with hematoxylin and eosin (H&E) to identify tissue structures.
hESC treated at differentiation days 4–6 with a 10-μm final concentration of SB203580 were differentiated for 30 days and hCM enriched by Percoll gradient using established methods (10). hCM were dissociated into single cells using Accutase (Sigma A6964) and 106 cells were delivered into the hearts of 12-week-old CB17 SCID-Beige mice (Taconic, Hudson, NY, USA) using closed-chest ultrasound-guided injection, as published previously (26). Hearts were harvested at 60 days post-injection, fixed in 10% buffered formalin and embedded in paraffin. Sections (5 μm) were stained with H&E for tissue analysis, and with anti-GFP antibody (Invitrogen A11122) to determine retention of injected hESC-derived hCM. Antigen retrieval was performed by incubating the sections with trypsin for 5 min at 37°C followed by incubations with anti-GFP (1:100), rabbit-on-rodent horseradish peroxidase (HRP) (1:200; Biocare Medical RMR622) and 3,3′-diaminodbenzidine (DAB) chromogen substrate (Biocare Medical, Concord, CA, USA, BDB2004).
Percentages of beating hEB were determined as a ratio of hEB exhibiting spontaneous contractile activity to total number of hEB plated. At least 120 hEB were plated for each group in each experiment, with five replicates performed for each experiment. Multiple synchronously beating areas within one hEB were counted as one beating hEB. For comparison among groups [i.e. dose and interval of inhibitor treatment, cytometric analysis of αMHC expression and quantitative RT–PCR (qPCR) analysis of relative expression], analysis of variance (ANOVA) with Fisher’s post-hoc test was used. Where ANOVA indicated significant differences among groups, multiple comparisons were made using Student’s t-test with Bonferroni correction. A P-value less than 0.05 was considered significant.
To determine whether inhibition of p38MAPK alters the dynamics of hESC differentiation, we exposed hEB to the p38MAPK inhibitor, SB203580, for sequential time periods during in vitro differentiation (Figure 1A) and counted the number of beating hEB on day 21, based on earlier reports that contractile activity peaks by this time-point (9). We observed a significant increase in hCM formation, as evidenced by an increase in the percentage of hEB with spontaneous contractile activity, with exposure to the inhibitor during days 2–6 of differentiation; a maximal effect was observed with exposure specifically from days 4 to 6 (26 ± 4 versus 12 ± 1.5; P < 0.0001). Addition of the compound at later time-points had no significant effect. To establish the optimal concentration of SB203580 for directed differentiation of hEB into hCM, we generated a dose–response curve for exposure from days 4 to 6, which established an optimal concentration of 5–10 μM (Figure 1B). As hEB may contain non-uniform cell numbers, we quantitated the percentage of hEB-derived cells expressing αMHC by flow cytometry (Figure 2). This demonstrated that inhibition of p38MAPK between days 4 and 6 resulted in c. 2.1-fold increase in the number of αMHC-expressing hCM (26.3 ± 4.6 versus 12.3 ± 3.9; P < 0.01).
To correlate the increase in numbers of hESC-derived hCM with p38MAPK inhibition to gene expression, we compared cardiac-specific gene expression by qPCR between total hEB at day 4 prior to treatment and at 14 days of differentiation relative to undifferentiated hESC (Figure 3A). This analysis showed that levels of αMHC and cTnT were increased in treated hEB compared with spontaneously differentiated hEB (αMHC, 3.6-fold increase, P < 0.01; cTnT, 1.3-fold increase, P < 0.03). These data suggested an increase in cardiac-specific gene expression in total hEB cultures exposed to the p38MAPK inhibitor. To establish whether cardiac-specific gene expression was specifically up-regulated in hEB with spontaneous contractile activity, we manually dissected beating areas from the surrounding hEB tissue at 30 days of differentiation, and analyzed for expression of early cardiac transcription factors, GATA4 and Nkx2-5, and sarcomeric proteins, cTnT, αMHC and myosin light chain 4 (MLC4). Expression of these proteins was very low in non-beating areas, with no detectable difference between treated and untreated samples (Figure 3B). As expected, the expression of GATA4, Nkx2-5, cTnT, αMHC and MLC4 in beating areas was substantially increased compared with non-beating areas (Figure 3B), with no detectable expression of the early germ layer markers Pax6 (ectoderm), Pdx1 (endoderm) and Brachyury (mesoderm) (data not shown). Remarkably, beating areas from cultures that had been treated with p38MAPK inhibitor demonstrated substantially increased expression of these transcripts, compared with beating areas isolated from untreated cultures (GATA4, 3.1-fold increase, P < 0.001; Nkx2-5, 7.7-fold increase, P < 0.001; cTnT, 6.7-fold increase, P < 0.001; αMHC, 5.2-fold increase, P < 0.001; MLC4, 1.9-fold increase, P < 0.03).
While the increase in expression of GATA4, Nkx2-5, cTnT, αMHC and MLC4 in beating areas of cultures treated with p38MAPK inhibitor may in part be the result of an increase in the number of hESC-derived hCM, we also hypothesized that this could be the result of accelerated differentiation. This was supported by our observation that, at day 14 of differentiation, treated hEB already showed an increase in cTnT and αMHC expression compared with spontaneously differentiating control hEB (Figure 3A). To examine the rate of hCM development at the ultrastructural level, we examined hEB at day 45 of differentiation by transmission electron microscopy (TEM) for evidence of sarcomere formation (Figure 4). Compared with the scattered areas of myofibril bundles seen among hEB from untreated cultures, hEB that had been treated with the p38MAPK inhibitor showed extensive regions of myofibril formation, with nascent Z-bodies and numerous mitochondria, consistent with developing sarcomeres.
The genomic stability of hESC has been shown to be sensitive to culture conditions (27,28). To determine whether p38MAPK inhibition affected genomic stability, we analyzed treated hEB by array comparative genomic hybridization (aCGH). This is a sensitive method for detecting chromosomal abnormalities at the level of individual regions of the genome, allowing for the detection of single copy gains and losses compared with diploid even in the presence of normal cell contamination. This analysis demonstrated that treatment with SB203580 did not affect the chromosomal content of hESC-derived hEB at day 30 of differentiation (Figure 5).
As p38MAPK inhibition directed the accelerated differentiation of hEB toward hCM, we wanted to know whether this inhibition affected the pluripotency of treated hESC. To accomplish this, we evaluated treated hEB differentiated for 30 days by immunostaining with antibodies against markers of the three embryonic germ layers: nestin and βIII tubulin (ectoderm), smooth muscle actin and cTnT (mesoderm) and α-fetoprotein (endoderm) (Figure 6A). We observed evidence of differentiation into all three embryonic germ layers in hEB that had been treated with SB203580, suggesting that while p38MAPK inhibition may direct differentiation toward hCM, it does not completely disrupt differentiation of other tissues derived from mesoderm, or from ectoderm or endoderm.
To determine whether p38MAPK inhibition influenced the expression of early genes associated with embryonic germ layer differentiation, we performed qPCR analysis of α-fetoprotein, smooth muscle actin and βIII tubulin in day-14 cultures. There were no significant differences between treated and untreated hEB, and each showed an appropriate increase in expression of all three proteins with differentiation (Figure 6B).
While qPCR results suggested that treated hESC maintained their ability to differentiate into all three germ layers, we wanted to determine whether this was true in vivo using a teratoma formation assay. Analysis of teratoma tissues showed no qualitative difference in the distribution of ectoderm, mesoderm or endoderm from the p38MAPK inhibitor-treated versus untreated differentiated hESC (Figure 7), suggesting that although p38MAPK inhibition directs hESC differentiation toward hCM, differentiation toward the three embryonic germ layers is not disrupted.
To determine the viability of p38MAPK inhibitor-exposed hCM in vivo, we differentiated GFP-expressing, p38MAPK inhibitor-treated hESC for 30 days and purified hCM by Percoll gradient separation (10). We injected 106 hCM into the hearts of CB17 SCID-Beige mice and evaluated cell survival 60 days after injection. The injected hESC-derived hCM were retained in the mouse hearts, as demonstrated by the presence of GFP-expressing cells 60 days post-injection (Figure 8A–D). In addition, there was no evidence of local teratoma formation (Figure 8E, F).
In the current study, we have shown that treatment of hESC with an inhibitor of p38MAPK (a) accelerated the directed differentiation of hESC into hCM in a developmental stage-specific manner, (b) did not affect hESC pluripotency, (c) did not induce genomic instability and (d) maintained viability of transplanted hCM in mouse myocardium.
During the course of our experiments, another group reported increased cardiogenesis after p38MAPK inhibition in endoderm co-cultures using hESC lines (20). Despite seemingly similar results, our study differs from their work in several important ways. Foremost, they differentiated hESC using serum-free medium conditioned by visceral endoderm-like mouse cells, whereas we used serum induction, a frequently employed method for spontaneous hESC differentiation (2,9,17). Second, they studied HES2, HES3, HES3-GFP hESC lines, while we carried out our studies using another extensively characterized (H9) hESC line. The increase in cardiogenesis observed irrespective of differentiation protocol across three hESC lines [our work and (20)] suggests that the effects of p38MAPK inhibition are not hESC line-specific. Third, the previously reported study examined cardiogenesis in a dose-dependent manner only, while we evaluated the effect of SB203580 in a developmental stage-specific manner. We show that the combination of timing and dose has an additive effect on cardiogenic enrichment. Our results emphasize the need for identifying the appropriate time-points during differentiation in culture to achieve a maximal effect. Each study adds significant details to the primary observation of p38MAPK inhibition as promoting hESC cardiogenesis, and the similar results using different hESC lines and different culture conditions (feeder-free serum replacement versus visceral endoderm co-culture) suggest that p38MAPK activity is important during hESC differentiation, and its manipulation may be useful in directing the differentiation of other mesoderm-derived tissues as well.
Others have observed that p38MAPK controls an early switch between commitment to mesoderm versus ectoderm during mouse embryonic stem cell (mESC) differentiation (19,29). There appear to be two waves of p38MAPK activity, one peaking at an early time-point between days 2 and 5 of mESC differentiation, and one at a later time-point between days 12 and 16. The early activity appears to control the decision between mesoderm and ectoderm (29), which is consistent with our finding that inhibition of p38MAPK at an early time-point (days 4–6) shifts differentiation toward cardiogenesis.
The early up-regulation of cardiac-specific genes in p38MAPK-inhibited hEB prompted our investigation into the ultrastructural maturity of treated versus untreated hCM. Surprisingly, we observed more mature sarcomere organization in the treated hEB. Previous studies have shown a progressive increase in the amount and organization of sarcomere structures coinciding with the developmental stage of hEB (3) or differentiation conditions (30). While we observed more extensive myofibrillar arrangements and evidence of nascent sarcomere formation earlier in treated cells than in untreated cells, we did not see characteristics of fully mature hCM in any of our samples. This variability in ultrastructural maturity between cultures, and even compared with fetal hearts, has been noted by others and thought to be because of differences between individual culture systems (3). This variability emphasizes the need to specify the differences between hESC differentiation protocols, the use of various culture additives, and the role of different culture systems, such as MEF and visceral endoderm-like cells (30).
In summary, we show that inhibition of p38MAPK during differentiation of hESC in co-culture with MEF results in accelerated differentiation of hCM and a 2.1-fold increase in hCM yield. We demonstrate that this enrichment is dependent on inhibiting p38MAPK at the time of ectoderm–mesoendoderm discrimination. We show that p38MAPK inhibition does not abolish pluripotency, as treated hESC remain competent at forming tissues from all three embryonic germ layers, and that the resulting hCM are genetically stable, can be purified by established methods (Percoll gradient) and are viable upon in vivo transplantation into mouse myocardium. These studies highlight further the utility of this method for producing hCM for therapeutic purposes.
This work was supported by a Comprehensive Research Grant from the California Institute for Regenerative Medicine (RC1-00104), a Public Health Service Grant (HL085377) from NHLBI, and a gift from the Pollin Foundation to HSB; and funds from the UCSF Cardiac Stem Cell Foundation to YY. We thank Frank King for technical advice and Angela Feraco for assistance with statistical analysis.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.