© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube
Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
r.h.lekanne{at}amc.uva.nl
* Corresponding author. Department of Anatomy and Embryology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: +31-20-566-5415; fax: +31-20-697-6177.
Received 12 September 2002; accepted 11 February 2003
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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Objective: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and quantitative analyses of cardiogenesis in vivo and in vitro. Methods: Gene expression profiles were made by in situ hybridisation and real-time PCR and electrophysiological profiles by patch clamp analyses of cardiomyocytes derived from time series of differentiating HM1 mouse embryonic stem cells and from embryonic and adult mouse hearts. Results: In embryoid bodies the in situ patterns of expression of
-myosin heavy chain, myosin light chain 2a and sarcoendoplasmic reticulum calcium ATPase 2a were similar to that of the heart muscle-specific marker gene cardiac troponin I. Myosin light chain 2v was expressed in part of the cardiac troponin I-expressing area, indicating heterogeneity within the cardiac cell population. Atrial natriuretic factor expression, indicative of the chamber-type program, could only very occasionally be detected by in situ hybridisation. Quantitative reverse transcriptase PCR showed that all cardiac genes, most notably atrial natriuretic factor, were expressed at relatively low levels, similar to those in embryonic hearts at embryonic day 8.75–9. Analysis of the electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes showed an increase of the upstroke velocity and a shorter duration of the action potential during prolonged differentiation in vitro. When embryonic mouse heart compartments of embryonic day 12.5 were used as a reference, the electrophysiological characteristics of a substantial part of the embryonic stem cell-derived cardiomyocytes were most reminiscent to those observed in the embryonic outflow tract. Conclusion: Together, these data suggest that most cardiomyocytes acquired by differentiation of embryonic stem cells maintain a phenotype reminiscent of that of the cardiomyocytes of the primary heart tube, and hardly any myocytes develop a chamber myocardial phenotype.
KEYWORDS Embryonic stem cells; Developmental biology; Embryology; Gene expression; Membrane currents
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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The vertebrate heart is formed in vivo as a linear tube of so-called primary myocardium. At embryonic day (E)8.25 chamber formation is initiated, accompanied by expression of atrial natriuretic factor (Anf) mRNA in the ventricles and the atria [1]. Differentiation of embryonic stem (ES) cells to the cardiac lineage provides an important model to investigate mechanisms and genes involved in the earliest steps of cardiac development [2]. Moreover, production of cardiomyocytes in an in vitro system offers a potentially unlimited source of donor material for grafting therapies [3]. Both applications of the ES cell differentiation model require proper characterisation of the formed heart cells. A developmental in vivo standard enables discrimination between the initial primary myocardial cells and later developing ventricular and atrial chamber myocytes.
It is the focus of many research groups to analyse how in vitro differentiation of ES cells relates to in vivo cardiogenesis. Besides an inventory of gene expression in ES cell-derived cardiomyocytes, these studies often include electrophysiological characteristics. In these studies the expression of myosin light chain (Mlc)2v [3–10], Anf [5,9] and Mlc2a mRNA [3,10] has been used to ascribe a ventricular or atrial phenotype to ES cell-derived cardiomyocytes. A confusing factor in these studies is that, although these genes are confined to their nominal compartments in the heart after birth [11,12], none of them is restricted to these compartments at the early embryonic stages (before E13). Moreover, quantitative data are scarce. An additional complicating factor is the scarcity of in vivo electrophysiological data on mouse cardiac development that can be used as a standard to characterise ES cell-derived cardiomyocytes. In the present study we assessed the expression of a number of cardiac genes among which those that have been used in ES cell studies to define the phenotypes of ES cell-derived cardiomyocytes to analyse the progress of cardiac differentiation. We performed a quantitative and qualitative comparison between ES cell-derived cardiomyocytes and cardiomyocytes derived from embryonic mice, based on in situ hybridisation (ISH), quantitative reverse transcriptase (RT)-PCR and electrophysiology. The characteristics of the ES cell-derived cardiomyocytes are largely similar to those observed in embryonic cardiomyocytes at E8.75–9. We conclude that the development of ES cell-derived cardiomyocytes includes hardly any formation of chamber myocardium and therefore does not significantly progress beyond that of cardiomyocytes of the embryonic heart tube stage in vivo.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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Mouse locus names according to Mouse Genome Informatics (http://www.informatics.jax.org) are Atp2a1 (sarcoendoplasmic reticulum calcium ATPase), Myh6 (
-myosin heavy chain), Mylc2a (myosin light chain 2a), Mylpc (myosin light chain 2v), Nppa (atrial natriuretic factor), Slc8a1 (sodium calcium exchanger 1), and Tnni3 (cardiac troponin I).
2.1 Cell culturing, animals and tissue preparation
Culturing of undifferentiated mouse ES cells of the line HM1 [13] was performed as described before [14]. Cardiac differentiation was evoked by culturing the cells in aggregates called embryoid bodies (EBs), using the hanging drop assay, essentially as described by Maltsev et al. [15] with some small modifications. After 3 (indicated as day 3+0) days of incubation in hanging drops, EBs were brought in a floating culture, one EB per culture well (24-well plate, COSTAR, diameter ca. 1.5 cm) coated with a layer of 1% sterile agar in differentiation medium to prevent attachment of the EB to the culture well. Differentiation medium contains ISCOVES MDM culture medium (Gibco-BRL) enriched with 20% foetal calf serum (Bodinco, cat. no. 39454), 1x amino acids and 1x penicillin/streptavidin (both from Gibco-BRL). Attachment cultures were established by plating EBs on gelatin-coated culture wells after 4 days in a floating culture [16]. Medium was changed every other day.
FVB mouse embryos were obtained from timed-pregnant animals and used for RNA isolation, electrophysiology and in situ hybridisation. Noon of the day of detection of the vaginal plug was considered E0.5. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). The experiments were approved by The Committee for Experiments on Animals (DEC) of the University of Amsterdam, The Netherlands.
2.1.1 RNA isolation
RNA was isolated from undifferentiated ES cells, from pools of seven to 10 beating EBs cultured for 3+4 until 3+30 days in floating culture or from 3+25 until 3+39 days cultured in attachment culture, and from hearts from E8.5 to 19, neonatal day (N)1, N7 and N14 and adult (3 months) FVB mice. For E8.5–15 two pools of eight to 13 hearts per time point were studied. For E19–N14 two pools of two hearts were used and four adult hearts were used. Total RNA was isolated using an RNA isolation kit (RNeasy, Qiagen) according to the instructions of the manufacturer. To remove contaminating genomic DNA, the RNA preparation (10–100 µg) was subsequently incubated with 10 units RQ1 RNase-free DNase (Promega) for 30 min at 37°C, extracted with phenol and chloroform and finally precipitated and dissolved in 3 mM Tris–HCl, pH 7.5–0.2 mM EDTA. Total RNA concentration was determined spectrophotometrically at 260 nm.
2.1.2 Myocyte isolation for electrophysiology
Isolation of cardiomyocytes from EBs was performed as described by Maltsev et al. [15]. Myocytes from E12.5 mouse hearts were prepared as follows. Pregnant mice were killed by cervical dislocation. The uterus was quickly dissected, rinsed once with Tyrode's solution 1, containing in mM: NaCl 135.1, NaHCO3 4.3, KCl 4.7, CaCl2 2.6, MgCl2 2.0, glucose 11 and Hepes 16.8 (pH was adjusted to 7.3 with NaOH), opened, and the exposed embryos were decapitated. The hearts of the embryos were dissected and separated into three parts: right+left atrium, apical part of the left ventricle, and outflow tract, collected in Eppendorf tubes and rinsed once with ice-cold Tyrode's solution 1. This solution was replaced by dissociation buffer, consisting of Tyrode's solution 1 from which CaCl2 was omitted and 1 mg/ml collagenase B (Roche; Mannheim, Germany) was added, and incubated at 37°C for 30 min. Every 10 min the tissue was gently triturated for 3 s until the tissue was dissociated into single cells and small clusters.
Cells and clusters were allowed overnight to adhere to 3-aminopropyltriethoxysilane-coated coverslips before being used the next day.
2.1.3 Tissue processing for histology
For preparation for ISH and immunohistochemistry, beating EBs were harvested from suspension culture, rinsed twice in PBS and fixed in 4% formaldehyde in PBS for 30 min at RT. Groups of EBs were embedded in 1% sterile agarose (ultraPURETM, BRL, 5510UB) in PBS at 50°C and stored in 70% ethanol at 4°C until further treatment. Agarose blocks were dehydrated in a graded ethanol series and butanol, and embedded in paraplast. For preparation for ISH on sections, embryos were fixed for 4–16 h in freshly prepared 4% formaldehyde in phosphate-buffered saline (PBS, 150 mM NaCl, 10 mM Na-phosphate, pH 7.4) by rocking at 4°C [17]. Embryos were dehydrated in a graded ethanol series and in butanol, and embedded in paraplast.
Serial sections of 15 µm of EBs and embryos were mounted onto microscope slides coated with 3-aminopropyltriethoxysilane.
2.2 Non-radioactive in situ hybridisation
Digoxigenin-labeled probes were made according to the manufacturers specifications, using Dig-UTP (Roche; Mannheim, Germany). Isoform-specific probes were used complementary to the mRNA coding for cardiac troponin (cTnI) [21], Mlc2v [11], Anf [22], Mlc2a [12], sarcoendoplasmic reticulum calcium ATPase (Serca)2 [23] and -myosin heavy chain (Mhc) [24] (see Table 1).
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ISH on sections of EBs and embryos was performed as detailed elsewhere [14,25]. Because of low RNA expression levels in ES cell-derived cardiomyocytes, the signal detection of hybridisation on sections of EBs was enhanced using a tyramide-mediated amplification kit (NEN, Boston). Expression patterns were studied in EBs derived from several independent differentiation experiments at time points between 4 and 35 days of differentiation in floating cultures. Independent experiments showed similar patterns.
2.3 Immunohistochemistry
Mounted sections were deparaffinated and hydrated in xylene and a graded series of ethanol, and washed in PBS. Sections were incubated in 0.5 M NH4Cl with 0.25% Triton X-100 for 30 min to increase permeability of the tissue, washed twice with PBS and blocked for 30 min with freshly prepared bovine serum albumin (BSA) 5% in PBS at RT to reduce non-specific binding. After removal of the BSA, a polyclonal primary antibody against Serca2a (kind gift of F. Wuytack, Department of Physiology, K.U. Leuven, Leuven B-3000, Belgium) was applied 1:3000 in 0.8% BSA in PBS at RT overnight in a moist incubation chamber. The next day, sections were washed 4 times with PBS, blocked with 3% goat serum and 0.8% BSA in PBS for 30 min at RT. To detect bound primary antibody, sections were incubated with goat anti-rabbit antibodies conjugated to alkaline phosphatase (Dako) 1:200 in 0.8% BSA for 2 h at RT in a moist chamber and washed 4 times in PBS. NBT/BCIP (Roche) diluted 1:50 in NTM-T (containing in mM: Tris-9.5 100, NaCl 100, MgCl2 50, Tween-20 0.05%) was used as a chromogenic substrate. The staining reaction was ended by washing with bidistilled water after about 12 min. Sections were dehydrated in a series of ethanol and xylene, and embedded in Entallan.
2.4 Primer design, reverse transcription and real-time polymerase chain reaction
Complementary DNA PCR primers for mouse were designed using Oligo primer analysis (version 4.1, National Biosciences) and Primer Express (version 1.0, PE Applied Biosystems) software from DNA and RNA sequences obtained from GenBank (Table 1). All primer sets had a calculated annealing temperature of 58°C (nearest neighbour method). The sense primer for Na+–Ca2+ exchanger (Ncx)1 was designed in a cardiac-specific part of the transcript [18]. Primers were obtained from Biolegio (The Netherlands).
First-strand complementary DNA was synthesized from 1 µg total RNA as described [19] by priming with a mixture of 2 pmol gene-specific reverse and 125 pmol oligo-dT14VN primer (Biolegio, The Netherlands) in a total volume of 25 µl. For each sample two separate cDNA synthesis reactions were performed.
Quantitative RT-PCR was performed as described [19] using the fluorescent dye SYBR green I, the Light Cycler Instrument (Roche) and software version 3.0 (Roche). Absolute copy numbers were estimated using standard curves made from dilution series of amplicon sequence solutions of known concentration [19]. Approximate average mRNA copy numbers per cell were calculated assuming a 1:1 relationship between RNA and cDNA molecules made during reverse transcription, and that each cell contains the same total amount, 45 pg [20], of RNA. 18S expression levels were used to correct for variations in RNA input. Results are expressed as mean±standard deviation.
Differences in gene expression between time points were tested with a one-way ANOVA. The Student–Newman–Keuls test was used to determine homogeneous subsets of time points per gene. To determine equivalence of in vitro and in vivo expression levels, per pair of in vitro and in vivo time points the squared difference of the values per gene was calculated and summed for all genes. The combination of time points with the least squared difference was considered the combination at which the gene expression patterns in vitro and in vivo correlated best. To avoid undue influence of differences in expression level between genes (a 10 000 times difference between Mlc2v and cardiac troponin I (cTnI) mRNA was observed) the logarithms of the values were used in this calculation. Statistics was performed using SPSS (SPSS Inc. version 11.0.1).
2.5 Electrophysiology
Coverslips containing the attached differentiated ES cells were mounted in a chamber on the stage of an inverted microscope (Nikon Diaphot) and superfused at a rate of approximately 1 ml/min with Tyrode's solution 2 of 33–35°C, containing in mM: NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5 and Hepes 5.0 (pH set at 7.4 with NaOH). Action potentials (APs) and membrane currents were recorded using whole-cell patch clamp, digitised at 2–10 kHz and stored on the hard disk of a computer (Apple Macintosh Quadra 650), equipped with a data-acquisition board (National Instruments). Recording pipettes had resistances of 5–10 M
when filled with a solution containing in mM: K-gluconate 140, KCl 10, Hepes 5.0 (pH set at 7.2 with KOH). Series resistance was compensated for 60–80%.
From spontaneous and stimulated APs the following parameters were measured: AP amplitude (APA), maximum diastolic potential (MDP), upstroke velocity (Vmax), AP duration (APD) at 20, 50 and 80% repolarisation and spontaneous interval. Of each recording the parameters of at least five APs were averaged.
In voltage-clamp, calcium current (ICa,L) was measured in a series of 500-ms clamp steps ranging from –100 to +60 mV, increment 10 mV, interval 5 s. Holding potential (Vhold) was –40 mV to inactivate fast sodium currents. The difference between maximum inward peak and current at the end of the steps was taken as the amplitude of ICa,L. Similarly, the amplitude of the fast sodium current INa was taken as the difference between inward current peak and steady state current recorded during steps from a Vhold of –80 mV. The change in slope during current pulses, applied in current clamp was used to calculate membrane capacitance Cm using Cm=I/dV/dt. Membrane currents were expressed as current densities by dividing Im through Cm. Data are expressed as mean±S.E.M., ANOVA on each electrophysiological parameter was used for testing differences between differentiation stages. Cluster analysis (SPSS, version 11.0.1) was used to divide ES cell data into homogeneous subgroups (Fig. 6D).
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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The aggregation of ES cells into EBs appears to be the trigger for differentiation towards a plethora of phenotypes [5], including beating cardiomyocytes. After at least 2 days in a floating culture, contractions were observed betraying the presence of cardiomyocytes. After 4 days in a floating culture up to 96% of the EBs shows vigorous beating activity throughout the entire EB, owing to an approximately 30% cardiomyocyte content (unpublished data).
3.1 Gene expression patterns
To define whether cTnI mRNA expression can be used as a marker for all cardiomyocytes in EBs, the in vivo pattern of expression in the mouse heart was analysed. In Xenopus [26] and chicken [27] cTnI mRNA expression marks specifically all cardiomyocytes from early stages onward. In rat, cTnI mRNA is expressed in the embryonic heart as well [21]. Ubiquitous expression was observed in the chamber myocardium of the atria and ventricles, and in the primary myocardium of the atrioventricular canal (Fig. 1A) and outflow tract (not shown) of a E9.5 mouse heart. cTnI mRNA expression was found to be restricted to the myocardium. In vitro, cTnI mRNA expression colocalises with Serca2a, another gene expressed in all cardiomyocytes in vivo [28], at the protein (Fig. 2C,A) and the mRNA level (Fig. 2D,F). This corroborates the assumption that cTnI mRNA expression can be used as a general marker for cardiomyocytes in the EB. Moreover, the absence of the skeletal muscle-specific transcription factor MyoD mRNA (data not shown) demonstrates the absence of skeletal muscle differentiation and thereby the cardiac specificity of Serca2a expression in EBs. During the entire differentiation period transcripts of Mlc2a (Fig. 2B) and Mhc (Fig. 2E) were observed in the same areas that expressed cTnI mRNA (Fig. 2C,D), similar to the early embryonic condition in vivo (before E13) [12,29] and as illustrated in Fig. 1D–F for E8.5. Mlc2v mRNA expression was generally observed in a subset of cardiomyocytes (compare Fig. 2G,I). With advancing differentiation time, in an increasing number of EBs Mlc2v mRNA expression became restricted to a part of the cardiomyocyte area (Fig. 2G). Comparison of expression areas of cTnI and Mlc2v mRNAs was substantiated in four independent differentiation experiments in time and significantly fitted a line predicted by a logistic regression model (Fig. 3). In vivo, Mlc2v mRNA is also expressed in a subset of cardiomyocytes. Fig. 1B shows expression of Mlc2v mRNA at E9.5 in the atrioventricular canal and the embryonic ventricle but not in the atria. Anf mRNA expression was hardly detectable in EBs. From 78 EB sections stained with the Anf probe 63 did not show any signal and 15 showed a few very weak positive spots (Fig. 2H) not found in the controls without probe. Fig. 1C shows that in embryonic hearts Anf mRNA is expressed in the developing atrial and ventricular chambers but not in the atrioventricular canal.
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3.2 Gene expression levels
Cardiac gene expression in vivo increased with development for most genes investigated (Fig. 4A). Levels of Mlc2v and Anf mRNA were low until E9, and then increased significantly (P<0.05) till E15 to stay at a variable high level until adulthood (one-way ANOVA, homogeneous subsets E7.5–9 and E15–adult). Early embryonic cTnI mRNA expression was relatively low, showed a perinatal increase and is low again at adulthood. Ncx1 mRNA expression significantly increased until E9 and decreased after birth, whereas Serca2a mRNA levels gradually but significantly rose during embryonic and postnatal life.
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Observations in EBs were based on two independent differentiation experiments. Both time course experiments showed a significant time-dependent effect on gene expression profiles with a similar trend, although peaks in expression varied a few days between the independent experiments (P<0.01; two-way ANOVA on log-transformed data). Developmental profiles of one experiment are given in Fig. 4B. Expression levels of the investigated mRNAs showed an increase within 14 days of culture in which each time point differed significantly from the previous point (P<0.05; one-way ANOVA). Mlc2v and Ncx1 mRNA expression peaked at 10, cTnI and Serca2a at 14 and Anf at 17 days of culture. Levels decreased to a plateau at 17–21 days and were low again at 25 days of differentiation. Expression levels at day 3+4 to 3+25 in vitro correlated best with levels as observed in the embryonic heart in vivo at E8.75–9 (least-squared distance). EBs that were differentiated in attachment cultures showed expression levels similar to E8.5–8.75 (Fig. 4C). Anf mRNA expression in EBs is very low and at day 3+25 to 3+30 fluctuates between 1.1 and 5.1 population-averaged molecules per cell in floating as well as in attached cultures. This argues against a potential stimulating effect of attachment cultures on differentiation to chamber-type cardiomyocytes.
3.3 Electrophysiology
3.3.1 Action potential characteristics from ES cell-derived cardiomyocytes
During the differentiation period a variety of action potential shapes could be recorded, including those reported by Hescheler et al. [30]. Fig. 5A shows that early during differentiation (3+7 days) action potentials can be recorded with a relatively long duration (APD) and a slow upstroke (Vmax), while later during differentiation slow ‘pacemaker-like’ APs were still found, but average action potentials tended to be shorter while having a higher Vmax (Fig. 5A, lower right).
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For comparison with the other results presented in this paper, we determined average action potential characteristics during the differentiation process. We determined the action potential durations APD20, APD50 and APD80, upstroke velocity (Vmax), maximal diastolic potential (MDP) and the spontaneous interval at various stages of differentiation. Fig. 5B shows that during differentiation the average action potential shortened considerably (P<0.0001; ANOVA), while Vmax (Fig. 5C) increased (P<0.001; ANOVA). Due to the variety of action potential configurations, a large scatter was observed especially in Vmax at day 3+10 and later. During differentiation, no significant increase of the negative MDP was observed (P = 0.5; ANOVA) (Fig. 5D). The spontaneous beating interval decreased (P<0.0001; ANOVA), as shown in Fig. 5E. Averaged maxima of ICa,L and INa current densities were plotted against time of differentiation in Fig. 5F. This figure illustrates that INa increased in time of differentiation (P = 0.01; ANOVA). No significant increase in ICa,L was detected (P = 0.4; ANOVA).
3.3.2 Comparison with action potential characteristics from embryonic cardiomyocytes
To asses the electrical maturation of the ES cell-derived cardiomyocytes we compared their electrical properties with those of E12.5 myocytes. Action potentials of cells or small cell clusters from the three compartments showed characteristics that were clearly distinct from each other. Fig. 6A shows representative examples of action potentials recorded from atrial, ventricular and outflow tract myocytes. Fig. 6B,C shows the average properties of APD50 and Vmax, respectively. Vmax and APD50 differed significantly (P<0.03; ANOVA) for all compartments, except for the APD50 from OFT and ventricular cells (P = 0.18; ANOVA). Compared to cells from other regions, atrial cells exhibited a high upstroke velocity (>100 V/s) and a relatively short APD50 that did not show a large dependence on stimulus frequency. Outflow tract cells had an APD50 comparable with that of ventricular cells, but showed a considerably lower Vmax than that found in the other regions. Both outflow tract and ventricular cells showed a negative frequency dependence of the APD50. We compared these three cell types with ES cell-derived cardiomyocytes in a plot of Vmax versus APD, wherein the average data of the embryonic cardiomyocytes are plotted together with those from the individual ES cell-derived cardiomyocytes (Fig. 6D). Cluster analysis indicated that the ES cell data formed three groups: (1) high Vmax, short APD; (2) low Vmax, short APD; and (3) low Vmax, long APD. In this panel the ES cell-data are grouped in ‘early’ and ‘late’, illustrating that a combination of a low Vmax and a long APD is usually found early in differentiation, while at later stages more short APs with high Vmax are found. Comparison with the embryonic heart data showed that ES cell-derived cardiomyocytes from group 2 and 3 did not significantly differ from cells from the outflow tract (i.e., primary myocardium [31]). ES cell-derived cardiomyocytes from group 1 with a high Vmax and a short APD, as found more often late during differentiation, differed not significantly from the embryonic atrial myocyte. The ventricular parameters differed significantly from all three ES groups (P<0.01; bivariate ANOVA).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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Gene expression and electrophysiological data reveal the characteristics of ES cell-derived cardiomyocytes to resemble those of cardiomyocytes from earlier stages of myocardial development than previous authors have concluded from expression of similar genes [3–10,12,30,32]. The difference is possibly due to the fact that in this study compared to others a different combination of genes is used, expression levels were quantified and early embryonic expression patterns were taken into consideration instead of adult/late embryonic patterns to define certain genes as markers of myocardial phenotypes.
4.1 Expression patterns
The expression patterns of several genes in the heart change with development. Many genes have a broader pattern of expression in the embryonic period than in the adult. Mlc2v mRNA expression is shown in the atrioventricular canal, the embryonic ventricle and also in the proximal outflow tract [11], i.e., more broadly than in the ventricle alone as found in the adult. Mlc2v mRNA is selectively expressed in a subset of cardiac myocytes from E7.5 onwards [4] long before ventricular chamber cells are formed. Furthermore, Mlc2v mRNA is also expressed in cells that will not become ventricle but the lower parts of the atrial chambers and the atrioventricular node [31]. Therefore, the expression of Mlc2v mRNA does not indicate the formation of ventricular cells during cardiogenesis in vivo and in vitro per se. At E8.5 Mlc2a and Mhc mRNA are expressed along the entire heart tube in a gradient decreasing towards the downstream part of the heart tube. Because these genes become first confined to the atrial compartment in late embryonic life (from ca. E13 onwards) [12,29] they can only be used as a general myocardial marker in a culture of differentiating cardiomyocytes. Anf is another gene sometimes incorrectly used when applied as a marker to identify putative atrial cells in ES cell cultures [5,9]. Anf mRNA is observed in the developing chamber myocardium of the future atrial appendages and in the ventricular trabeculations that will contribute to the peripheral ventricular conduction system [33] but not in the primary myocardium of the atrioventricular canal, the inflow tract, and the outflow tract (the latter two not visible in Fig. 1C) [1]. Therefore, Anf mRNA expression is used by us as a marker for developing chamber myocardial cells in vitro.
4.2 Expression levels
In general, we found very low expression levels that were most similar to those observed in the early embryonic heart at E8.75–9. For cTnI mRNA, a developmental increase has been described at late embryonic stages [21] but no in vitro data are available. To our knowledge, such quantitative data on Mlc2v mRNA levels are lacking both in vivo and in vitro. In vitro, Mlc2v mRNA is already observed at relatively high levels at day 3+4 as assessed by RT-PCR and ISH, when EBs were beating for 1 or 2 days only. This early appearance is in contrast with other studies [5–7,12,30] in which the delayed expression of Mlc2v mRNA may have led to the idea that Mlc2v gene expression is indicative of development of ventricular working myocardium.
For the embryonic period, quantitative data on Ncx1 and Serca2a mRNAs in mice are lacking. Similar to our observations, Ncx1 mRNA content has been reported to decrease around birth [34]. Serca2a mRNA has been reported to be present from E9 onward in rat [23] (
mouse E7.0), and a functional sarcoendoplasmic reticulum develops with chamber formation [35]. The increase in the levels of Serca2a mRNA during development is in line with earlier studies but these did not encompass the early embryonic period (before E13) [36,37].
Anf mRNA levels increase from E8.25 onwards when formation of working myocardium is initiated [1]. Expression peaks in adulthood, where it is abundantly expressed in the atria [38] and only very weakly in the peripheral conduction system of the ventricles [31]. In vitro, the average Anf mRNA expression was found at levels approximately 100-fold lower then those observed in adult myocardium. This difference is actually much higher because of the preferential expression of Anf mRNA in the adult atria. Anf mRNA expression in differentiating EB cultures has been reported [6,9,12,30], but quantitative data are lacking.
It is not known to what extent the in vivo cardiogenic pathway is recapitulated during ES cell differentiation in vitro. However, undifferentiated ES cells would have to go through a process of differentiation to reach a phenotype reminiscent of that of adult myocytes. In defining this, the use of in vivo gene expression patterns as a developmental marker can be justified.
4.3 Electrophysiology
The electrophysiological experiments showed that on average during differentiation of ES cell-derived cardiomyocytes the APD and the beating interval decreases whereas Vmax increases. Some of these changes are similar to those seen during in vivo development of the murine ventricle. Initially at E9.5, ventricular cells have a relatively low Vmax and an APD50 of 100–200 ms. At E18, Vmax increases and the APD shortens to 74 ms [39,40] and at three neonatal days, APD50 shortens even further to 18 ms [41]. Other changes are different from the in vivo observations. At E9.5 ventricular cells beat spontaneously with a regular interval of about 1 s, whereas from E18 onward the cells become silent at a negative resting potential [40]. We showed that the average beating interval of ES cell-derived cardiomyocytes decreased in time. This suggests that most ES cell-derived cardiomyocytes do not develop into a ventricular phenotype, but towards a nodal phenotype, which mostly resembles primary myocardium [31].
Our observation that INa increases during in vitro differentiation agrees well with the findings of Maltsev et al. [15] and indicates that this increase is involved in the increase in Vmax as observed during differentiation.
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5 Conclusion |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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ES cell-derived cardiomyocytes are not differentiated towards fully mature chamber myocardium as yet. They rather show a phenotype comparable to young embryonic cardiomyocytes in vivo. Several arguments support this interpretation. Firstly, expression levels of genes in differentiating ES cell cultures are similar to levels observed in the primary heart tube stage at E8.75–9. Secondly, markers that are chamber-specific in the late embryonic and adult heart (
Mhc and Mlc2a) [12,29], and the general cardiac markers cTnI and Serca2a [21,28] showed expression in the entire myocardial area of the EB. These observations indicate that at the level of regulation of Mlc2a and Mhc the distinction between a primary or a chamber phenotype has not been made. Thirdly, Anf, the only currently known gene that is exclusively expressed in the forming atrial and ventricular chambers [1], is expressed at very low levels in differentiating EBs, indicating that hardly any chamber myocardium has developed. Fourthly, electrophysiological properties of early ES cell-derived cardiomyocytes were most reminiscent of cells from the outflow tract and further differentiation resulted in cells comparable to those of the embryonic atrium. However, it has to be assessed whether the atrial electrophysiological characteristics are also represented in the embryonic heart tube and as such would represent normal variation of cardiomyocytes in this stage of heart development. Finally, all cardiomyocytes analysed by patch clamp showed automaticity, a characteristic for all embryonic cardiomyocytes and for nodal cells in the mature heart. Taken together, the levels of expression of several key cardiac genes and electrophysiological features strongly suggest that the phenotypes of cardiomyocytes in differentiating EBs are most reminiscent of cardiomyocytes from E8.75–9 mouse hearts where chamber formation has just started. However, the propensity of increasing upstroke velocity of action potentials at later differentiation times could indicate that a first step towards more mature working myocardium has been made. It will be challenging to induce further differentiation of chamber myocardium in vitro and unravel the underlying process using this culture system.
Time for primary review 33 days.
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Acknowledgments |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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We express our gratitude to C.C. Verhoek-Pocock, C. de Gier-de Vries and M.A.W. Markman for expert technical assistance and Dr. P.E.M.H. Habets for providing the embryos. Probes were kindly provided by Drs C.E. Seidman (Anf), K.R. Chien (Mlc2v, MLC2a), S. Schiaffino (cTnI) and K.R. Boheler (
Mhc). This study was supported by Netherlands Heart Foundation, grants 99.170 (ACF), and M96.002 (to AFMM, RHLD and VMC). |
References |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionAcknowledgmentsReferences |
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