A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

Abstract

We have developed a simple protocol for inducing the myocardial differentiation of human induced pluripotent stem (iPS) cells. Human iPS cell-derived embryonic bodies (EBs) were treated with a combination of activin-A, bone morphogenetic protein-4 and wnt-3a for one day in serum-free suspension culture, and were subsequently treated with noggin for three days. Thereafter, the EBs were subjected to adherent culture in media with 5% serum. All EBs were differentiated into spontaneously beating EBs, which were identified by the presence of striated muscles in transmission electron microscopy and the expression of the specific cardiomyocyte markers, NKX2-5 and TNNT2. The beating rate of the beating EBs was decreased by treatment with a rapidly activating delayed rectifier potassium current (Ikr) channel blocker, E-4031, an Ikr trafficking inhibitor, pentamidin, and a slowly activating delayed rectifier potassium current (Iks) channel blocker, chromanol 293B, and was increased by treatment with a beta-receptor agonist, isoproterenol. At a low concentration, verapamil, a calcium channel blocker, increased the beating rate of the beating EBs, while a high concentration decreased this rate. These findings suggest that the spontaneously beating EBs were myocardial cell clusters. This simple protocol for myocardial differentiation would be useful in providing a sufficient number of the beating myocardial cell clusters for studies requiring human myocardium.

The cardiotoxicity of drug candidates is one of the crucial findings that limits drug development. A large number of pharmaceutical companies hope to evaluate the risk of cardiotoxicity of drug candidates using human cardiomyocytes in the early stage of development. We have been trying to examine the embryotoxicity of drug candidates in vitro using human induced pluripotent stem (iPS) cells. A simple and highly efficient protocol for inducing the myocardial differentiation of human iPS cells using biological substances has been strongly desired, especially a system using the embryonic body (EB, spheroids of iPS cells) method.

Primary human myocardium is difficult to obtain commercially because the cells do not proliferate in culture. Embryonic stem (ES) cells and iPS cells from humans are able to differentiate into myocardium, but the mechanisms are different from those required for ES and iPS cells of mice, and the induction has generally been inefficient. The myocardium is differentiated from the mesoderm, one of the three germ layers generated during the differentiation of pluripotent stem cells. Bone morphogenetic protein-4 (BMP-4), activin-A and wnt-3a are mesoderm inducers, and play a crucial role in its development.^1–5) Intracellular signals from all three of these mesoderm inducers are required for mesoderm induction.

Previous studies of methods to induce myocardial development have aimed to provide a simpler protocol and to make the method as efficient as possible. As a simple method, short-term treatment with BMP-4 has been reported to induce mesoderm from human embryonic stem cells.¹⁾ We hypothesized that the complex signals provided by the three mesoderm inducers (BMP-4, activin-A and wnt-3a) would act to synergistically induce mesodermal differentiation at a higher efficiency compared to that induced by the BMP-4 signal alone.

Although the myocardium is induced from the mesoderm, the mesoderm has the potential to differentiate into hematopoietic and muscle (skeletal and cardiac) cells.^6,7) BMP-4 promotes the hematopoietic differentiation of human embryonic stem cell lines.⁸⁾ Because the effects of BMP-4 are abolished by noggin, owing to its blocking BMP-4 binding to the BMP receptors, noggin was previously used to treat cells after the induction of the mesoderm to promote myocardial differentiation and decrease hematopoietic differentiation.⁹⁾ In the present study, to develop a simple protocol (with high efficiency, if possible) for the myocardial differentiation of human iPS cells, the effects of short-term treatment with the complex of mesoderm inducers were examined. The protocol finally established using this approach is illustrated in Fig. 1. The beating EBs generated from EBs were identified to be myocardial cell clusters by transmission electron microscopy, by their expression of the cardiomyocyte marker, NKX2-5, a cardiac homeobox protein and TNNT2, a cardiac muscle troponin T type 2, as well as by the reactions of the EBs to cardiac drugs.

Fig. 1. An Outline of the Method Used for Differentiating Human iPS Cells into Myocardium

Human iPS cells were cultured in an AggreWell™ 800 plate for one to two days until the formation of EBs of uniform size. The EBs were treated with a combination of activin-A, BMP-4 and wnt-3a for 1 d, and were subsequently treated with noggin for 3 d in serum-free suspension culture. Thereafter, the EBs were subjected to adherent culture in media with 5 vol% serum. ^$ AggreWell™ 800 plate (StemCell, Canada), an EZSPHERE® plate (AGC Techno Glass, Shizuoka, Japan) and a low-cell binding 96-well plate with a round-bottom (available from several vendors) could be used to form the EBs of uniform size. AggreWell™: AggreWell™ medium, EBs: Embryonic bodies, Y27632: Rho-associated coiled-coil forming kinase (ROCK) inhibitor, DMEM/F12: Dulbecco’s modified eagle medium F-12 Ham (1 : 1) with GlutaMAX™, BMP-4: recombinant human bone morphogenetic protein-4, Wnt-3a: recombinant human wnt-3a, Activin-A: recombinant human activin-A.

MATERIALS AND METHODS

Human iPS Cell Culture

Human iPS cells¹⁰⁾ were maintained in modified Tenneille serum replacer 1 (mTeSR™1, StemCELL, Vancouver, Canada) which is a serum- and feeder-free medium. The cells were suspended in mTeSR™1 and seeded in 60-mm culture dishes coated with BD Matrigel™ (StemCELL). When the colonies of cells became confluent to the extent that the individual cell colonies did not adhere to the culture dishes, then the colonies were harvested by dispase treatment (1 mg/mL, StemCELL) and subcultured at a 1 : 3 to 4 split ratio.

Myocardial Differentiation

Human iPS cells were suspended in AggreWell™ medium (StemCELL) supplemented with Y-27632 (10 µmol/L), a Rho-associated protein kinase inhibitor. They were then seeded in AggreWell™ 800 plates (StemCELL) at a three to four well per one 60-mm dish ratio, and were incubated at 37°C and 5% CO₂ with saturated humidity. EBs were allowed to form for 1 to 2 d. The EBs were then transferred to low cell-binding plates/dishes (Thermo Fisher Scientific, MA, U.S.A.), suspended in Dulbecco’s modified Eagle medium/F-12 Ham’s medium with GlutaMAX™ (DMEM/F12, StemCELL) supplemented with the B-27® supplement (B27, Life Technologies, Carlsbad, CA, U.S.A.) and non-essential amino acids (NEAA, 2 mmol/L, Life Technologies), and then the EBs were treated with a myocardial-inducing factor.

Combined Effects of Mesoderm Inducers

EBs were treated with either recombinant human BMP-4 alone (BMP-4, 100 ng/mL for the submaximum concentration, HumanZyme, Chicago, IL, U.S.A.) or with a combination of BMP-4 with recombinant human activin-A (activin-A, 100 ng/mL for the submaximum concentration, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and recombinant human wnt-3a (wnt-3a, 100 ng/mL for the submaximum concentration, R&D Systems, Minneapolis, MN, U.S.A.) for 1 d, or with 20% fetal bovine serum (FBS, untreated group) on a low cell-binding culture plate, and were subsequently treated with noggin (300 ng/mL for the submaximum concentration, R&D Systems) or with 20% FBS (untreated group) for 3 d in low-cell binding culture plates. The EBs were then transferred to 96-well culture plates coated with 0.1% gelatin (Wako Pure Chemical Industries, Ltd.) at one EB per one well, and were cultured in DMEM/F12 supplemented with 5% FBS. The EBs were observed under a microscope, and were examined for beating every day for 2 weeks, and the ratio of beating EBs generated from EBs was calculated from the total number of EBs used in each group. The induction of myocardium was judged by the generation of beating cell clusters.

Effects of Noggin on Myocardial Differentiation

EBs were treated with BMP-4, activin-A and wnt-3a for 1 d, and were subsequently treated with noggin for 1–4 d or with 20% FBS (untreated group). The EBs were then transferred to 96-well culture plates coated with 0.1% gelatin at one EB per well, and were cultured in DMEM/F12 supplemented with 5 vol% FBS and 2 mmol/L NEAA. EBs were observed under a microscope, and then were examined for beating every day for 2 weeks.

Identification of the Structure and Function of the EBs as Myocardium

Microstructure and Cardiac Markers

The microstructures of beating EBs were investigated by transmission electron microscopy (H-7600, Tokyo, Hitachi Ltd., Tokyo, Japan). According to the previously described method,¹¹⁾ the beating EBs were rinsed with phosphate buffer solution, fixed with 2.5% buffered glutaraldehyde, dehydrated and embedded in epoxy resin. The semi-thin sections (0.7 µm) of beating EBs were stained with 1% toluidine blue to pick up suitable cells. Ultra-thin sections (0.08 µm) of beating EBs were stained with uranyl acetate and lead citrate, and were qualitatively investigated using a transmission electron microscope.

The key markers of human cardiac lineage, NKX2-5 and TNNT2, were detected using a human cardiomyocyte immunocytochemistry kit (Life Technologies). The staining of beating EBs was performed according to an optimized staining protocol provided by the vendor. The two cardiac markers and the nuclei were stained and observed using a laser scanning microscope (LSM700, Carl Zeiss Meditec AG, Tokyo, Japan).

Effects of Cardiac Drugs

To confirm the cardiac-specific function of the beating EBs, the effects of the following cardiac drugs were examined: a beta-agonist (isoproterenol; MP Biomedicals Japan, Tokyo, Japan), a rapidly activating delayed rectifier potassium current (Ikr) channel blocker (E-4031; self-manufactured), an Ikr channel trafficking inhibitor (pentamidin; Sigma-Aldrich Japan, Tokyo, Japan), a slowly activating delayed rectifier potassium current (Iks) channel blocker (chromanol 293B; R&D Systems) and an L-type calcium channel blocker (verapamil; Sigma-Aldrich Japan). The beating EBs were subcultured in 35 mm dishes (one cluster per dish) coated with 0.1% gelatin and were cultured at 37°C and 5% CO₂ with saturated humidity for a few days. The beating rate of the EB was counted for one minute under a light microscope at approximately 37°C. After the beating rate was steady, drugs were added to the dishes, and fifteen minutes later, the beating rate was determined again. The cardiac drugs were added both cumulatively and singly to assess the different effects. The mean beating rate of the EBs under the drug-free condition was regulated between approximately 40 and 80 beats/min (the normal heart rate in humans) during each test.

Statistical Analysis

The changes in the beating rates of the beating EBs in response to treatment with the cardiac drugs are represented as the means±standard error (S.E.) of the percentage of the beating rate before adding the drug. A statistical analysis was performed using the SAS system (ver. 9.2, SAS Institute Japan, Tokyo, Japan). The changes in the beating rates were analyzed by Dunnett’s test, following a one-way ANOVA. Statistically significant differences were considered to be presented for values of * p<0.05, ** p<0.01, *** p<0.001.

RESULTS

Effects of the Combination of Mesoderm Inducers on Myocardial Differentiation

The rate of induction of myocardium (beating EBs) from EBs treated with BMP-4 alone was 18%, while the rate was 55% for EBs treated with the combination of BMP-4 and wnt-3a, and was 100% for the EBs treated with the combination of BMP-4, wnt-3a and activin-A (Table 1(a)). No beating EBs were induced by treatment with the combination of BMP-4 and activin-A, wnt-3a and activin-A, or by no treatment (20% FBS) (Table 1(a)). The combination of BMP-4, wnt-3a and activin-A was highly efficient for inducing the differentiation of EBs to beating EBs. The beating of the generated EBs was sustained for over a year.

Table 1. The Myocardial Differentiation of the EBs from Human iPS Cells

(a) Effects of BMP-4, activin-A and wnt-3a
Combination	Myocardial differentiation
	Number of EBs	Number of beating EBs	Period between the addition of mesoderm inducers and induction of beating (mean±S.D., d)
BMP-4	40	7 (18%)	7±1
BMP-4+Wnt-3a	40	22 (55%)	9±1
BMP-4+Activin-A	40	0 (0%)	—
Wnt-3a+Activin-A	40	0 (0%)	—
Wnt-3a+Activin-A+BMP-4	40	40 (100%)	5±0
(b) Effects of the noggin treatment period
Period of treatment with noggin (d)	Myocardial differentiation
	Number of EBs		Number of beating EBs
0	48		0 (0%)
1	48		0 (0%)
2	48		40 (83%)
3	48		48 (100%)
4	48		38 (79%)

(a) Human iPS cell-derived EBs were treated with either BMP-4 or with a combination of BMP-4 with activin-A, BMP-4 and wnt-3a for 1 d, and were subsequently treated with noggin for 3 d. (b) Human iPS cell-derived EBs were treated with a combination of activin-A, BMP-4 and wnt-3a for 1 d, and were subsequently treated with noggin for 0 to 4 d. Horizontal line: no differentiation. Parenthesis: percentage of EBs with induced beating, EBs: embryonic bodies, activin-A: recombinant human activin-A, BMP-4: recombinant human bone morphogenetic protein-4, wnt-3a: recombinant human wnt-3a.

Effects of Noggin on the Myocardial Differentiation

Treatment of the EBs with noggin for two, three and four days led to the generation of beating EBs at rates of 83, 100 and 79%, respectively (Table 1(b)). No beating EBs were induced by treatment with noggin for 1 d or by no treatment (20% FBS) (Table 1(b)). The treatment with noggin for 3 d was the most efficient for inducing the differentiation of beating EBs.

Identification of the Structure and Function of the Cells as Myocardium

Microstructure and Cardiac Markers

The beating EBs are shown in Fig. 2(a). Striated muscles and large mitochondria were identified in the beating EBs by electron microscopy (Fig. 2(b)). The expression of the cardiomyocyte marker, TNNT2, and the marker of early cardiac mesoderm and myocardium, NKX2-5, were found in the beating EBs (Fig. 2(c)), as were nuclei. Myofibrils/sarcomeres were observed by staining for TNNT2 (Fig. 2(c)).

Fig. 2. Identification of Beating EBs as Myocardium

(a) Light microscopy of beating EBs: A beating EB was observed at 40× magnification in the EVOS® XL cell imaging system (Life Technologies). (b) Transmission electron microscopy of beating EBs: Preparations were observed at 15000× magnification using a H-7600 instrument (Hitachi). Striated muscle with mitochondria (arrow head) was clearly present. The beating EB was used a few months after the induction of beating. (c) Cardiac-specific marker expression of beating EBs: TNNT2, NKX2-5 and nuclei were stained using the human cardiomyocyte immunocytochemistry kit. (c-1): nucleus (blue), (c-2): TNNT2 (green), (c-3): NKX2-5 (red), (c-4): phase, and (c-5): merged. A beating EB was observed at 20× magnification using a LSM700 laser scanning microscope (Zeiss). The beating EB was used seven days after the induction of beating. EBs: embryonic bodies.

Effects of Cardiac Drugs

The beating rates of EBs were increased by isoproterenol and were decreased by chromanol 293B and E-4031, and also by pentamidin at eighteen hours or later after adding the drug (Figs. 3(a)–(c), (e)). E-4031 evoked an irregular beat. Verapamil caused the beat count to increase at a low concentration and to decrease at a high concentration (Fig. 3(d)). The beat count was not affected by 0.3% dimethylsulfoxide, but increased slightly at the maximum 1% concentration. The mean beating rate (mean±S.E.) in the drug-free controls was as follows: isoproterenol (40±12 beats/min, N=6), chromanol 293B (47±7 beats/min, N=9), E-4031 (38±6 beats/min, N=8), verapamil (49±7 beats/min, N=7), pentamidine (vehicle 51±11, 1 mol/L 66±2, 3 mol/L 55±33, 10 mol/L 84±41, 30 mol/L 69±38 beats/min, N=3).

Fig. 3. The Effects of Cardiac Drugs on the Beating Rate of EBs

Each point with bars represents the mean±S.E. of change from control. (a)–(d): The beating rate was counted for one minute before adding the drugs and at 15 min after adding the drugs, which were added cumulatively. (e): The beating rate was counted at 0.5, 2 18 and 24 h after adding the drugs singly. The beating rate (mean±S.E.) of the drug-free control cultures were as follows: (a) isoproterenol (40±12 beats/min, N=6), (b) chromanol 293B (47±7 beats/min, N=9), (c) E-4031 (38±6 beats/min, N=8), (d) verapamil (49±7 beats/min, N=7), (e) pentamidine (vehicle 51±11 beats/min, 1 mol/L 66±2, 3 mol/L 55±33, 10 mol/L 84±41, 30 mol/L 69±38 beats/min, N=3). ^※ p<0.05, ^※※ p<0.01, ^※※※ p<0.001, statistical significance level vs. control-value (Dunnett’s test).

DISCUSSION

Human myocardial cells would be useful to evaluate the cardiotoxicity of drug candidates, but the isolation and culture of these cells in numbers sufficient for reproducible assays has proven to be difficult. In the present study, we developed a highly efficient and simple protocol to induce beating EBs (myocardial cells) from EBs (human iPS cells). The key point of this protocol is thought to be the components included in the medium, the timing of treatment with these components, and the combination of mesoderm inducers.

The medium used to form the EBs was a commercially available AggreWell® medium. We selected this medium because the myocardial differentiation of EBs was significantly reduced (unpublished observation) when EBs were formed using commercial mTeSR™1 medium containing a sufficient amount of basic fibroblast growth factor to maintain the pluripotency of stem cells. On the other hand, when EBs were formed using the commercial AggreWell® medium, which contains only the minimum amount of basic fibroblast growth factor to ensure cell survival, almost all EBs were differentiated into myocardium. The basic fibroblast growth factor level likely affected the myocardial differentiation.

Mesoderm inducers (BMP-4, activin-A and wnt-3a) were used to treat the EBs in serum-free medium, which was supplemented with B27 as a serum-free supplement. Some components of cell culture media may abolish myocardial differentiation as a result of their prevention of the production of mesoderm inducers. On the other hand, EBs cannot survive under completely serum-free conditions. Therefore, the surrogate serum (B27) promotes EBs survival, and also supports the myocardial differentiation of EBs.

The combination of the three mesoderm inducers was the most efficient for inducing the beating EBs. BMP-4 plays a specific role in the induction of cardiomyocytes in the anterior mesoderm of chick embryos.¹²⁾ In human ES cells, BMP-4 induces mesoderm after short-term treatment, but not after long-term treatment.¹⁾ Wnt-3a enhances the cardiac differentiation of mouse ES cells in the early stage during EB formation, but inhibits cardiac differentiation in the late stage.¹³⁾ BMP-4 and wnt-3a may play crucial roles in stage-specific development, and the treatment with both agents during the early stage, a short-term treatment, may lead to the differentiation of the cardiomyocytes from human iPS cells, while it may inhibit the differentiation in the late stage after a longer-term treatment. Activin-A and further wnt-3a signaling are required for the induction of the primitive streak.⁵⁾ Both activin-A and wnt-3a are required in the early stage of the development of EB formation. It is suggested that all three signaling molecules, activin-A, BMP-4 and wnt-3a, promote mesoderm induction and enhance myocardial induction in the early stage during EB formation.

The absence of noggin led to a failure of the EBs to differentiate in myocardial cells. Noggin plays an important role in our myocardial induction method. Noggin is a BMP-4 antagonist.⁹⁾ Short-term, but not long-term, treatment with BMP-4 initiates the induction of mesoderm, which differentiates into cardiomyocytes and hematopoietic lineage cells.¹⁾ BMP-4 promotes the self-renewal of hematopoietic progenitors.⁸⁾ It has been suggested that the treatment with noggin promotes cardiomyogenesis by inhibiting hematopoiesis. The effect of noggin on myocardial differentiation was most efficient when applied for 3 d. Noggin was used to promote the cardiac progenitor formation from mesoderm by blocking the formation of hematopoietic progenitors. In case of chick embryo, noggin inhibits myocardial differentiation.¹²⁾ It has been suggested that long-term (for 4 d), but not the short-term, treatment with noggin inhibits the differentiation of cardiac progenitors into myocardium. Thus, the actions of noggin on myocardial differentiation appear to be time-dependent.

The only striated muscle cells beating automatically in the human body are the myocardium. The beating EBs generated in the present study were identified as striated muscles (sarcomeres). The beating EBs expressed the cardiomyocyte-specific markers, NKX2-5 and TNNT2. Of note, the beating rate of the beating EBs was increased by isoproterenol, with positive chronotropic action. The inhibition of Ikr or Iks channels is known to delay membrane depolarization and decrease the beat counts. Both E-4031 (an Ikr channel blocker) and chromanol 293B (an Iks channel blocker) decreased the beat counts in our EBs. Verapamil blocks the calcium and Ikr channels,¹⁴⁾ and increases the beat count at low concentrations that shorten the plateau phase (phase-2 action potential) of the action potential by inhibiting calcium channels, and decreases it at high concentrations that may inhibit the Ikr channels. Pentamidin decreases the Ikr channel expression on the cell membrane due to the inhibition of the Ikr channel trafficking.¹⁵⁾ Pentamidin decreased the beat counts after eighteen or more hours of treatment in the present study. These results demonstrated that the beating EBs had the structural and functional properties of myocardium.

A simple myocardial differentiation method was established in the present study. All of the EBs were differentiated into myocardial cell clusters (beating EBs); however, cells with proliferative ability and with different morphology from myocardium appeared in the continuing culture around the myocardial cell clusters. It is thus believed that not all of the cells were differentiated into cardiomyocytes. This simple method would therefore not be 100% from individual cells, but our findings suggest that this method is still highly efficient. This method would shorten the time-consuming myocardial differentiation and can be expected to supply a sufficient number of myocardial cell clusters to perform toxicity studies for an assessment of the cardiotoxicity of a new lead compound. The procedure would still be expensive, because the recombinant biological substances used are expensive. In general, human iPS cells have been pointed out to have differentiation directivity, and moreover, the myocardium induced from human iPS cells is thought to be immature based on the cells’ resting membrane potential, the expression of atrial natriuretic factor and the expression of other factors expressed by human fetal cardiomyocytes.¹⁶⁾ For these reasons, further studies are needed to confirm the efficiency of our protocol using a variety of iPS cell lines, such as commercial human iPS cells and other lines, and also to confirm the quality of the cells, such as electrophysiological confirmation of ion channel expression, the resting membrane potential and other features. Moreover, because the myocardial cells adhere tightly to each other, it is difficult to release the myocardial cells from the beating EBs without causing damage to the proteins/peptides on the cell surface. It will therefore be necessary to establish a myocardial cell isolation method, and the efficiency of myocardial differentiation would thus be evaluated by flow cytometry and other methods.

Conflict of Interest

The authors declare no conflict of interest.

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