Effect of Cell Labeling on the Function of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3'-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca2＋ influx by L-type Ca2＋ channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.

Introduction

Cell labeling knowledge has significantly increased, resulting in the development of numerous labeling agents, including fluorescent probes, superparamagnetic n class="Chemical">iron oxide, rapan> class="Chemical">diotracers, and genetic modifications (1). Indeed, a wide variety of labeling agents have been used to distinguish target cells from neighboring cells and monitor the functional effects of target cells. Several studies have reported that the labeling mechanism typically involves the adherence and diffusion of a lipophilic cyanine-based dye across the phospholipid cell membrane bilayer. Other modes include transporters, endocytosis, and phagocytosis (2, 3).

The transportation of labeling agents into the cells could change the biological responses of cells, such as metabolism, proliferationpan>, anpan>d migpan> class="Species">ration (4-6). Labeling agent-rich regions cause changes in the physical properties of the cell surface and thereby determine cellular characteristics, i.e., the cell surface, repulsion between cells, subsequent migration, and the uptake of ion (7-9). Ideal labeling agents should have high labeling efficiency and not have harmful effects on cellular function. In previous studies, various labeling agents have been used to efficiently label cardiomyocytes (CMs) for in vivo tracking (10-13). However, these studies did not focus on the functional effects on CMs. In particular, the influence of labeling on the physiological functions of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) should be verified. hiPSC-CMs are considered a promising option for future applications in various fields (14-16). The electrophysiological dysfunction of hiPSC-CMs can lead to negative application consequences, such as abnormal drug reactions when assessing cardiac toxicity or lethal arrhythmia in heart failure treatment with injected CMs (17-19). In addition, low cell retention and engraftment rates are another safety risk for stem cell-based therapies, so the labeling of CMs is required for long-term monitoring (20-22). Despite the importance of hiPSC-CM function and labeling, no studies have evaluated the effect of labeling agents on hiPSC-CMs.

Therefore, the purpose of this study was to investigate the effects of three different labeling agents on the cellular functions of CMs. Thus, CMs were labeled with 3’-n class="Chemical">dioctadecyloxapan> class="Chemical">carbocyanine perchlorate (DiO), quantum dots (QDs), and plasmid DNA expressing EGFP using Lipo2K. The labeled cells were subsequently characterized at the cellular functional and molecular levels. The results of this study may provide useful tools to expand the potential applications of CM labeling.

Materials and Methods

Human iPSC-derived cardiomyocyte culture

hn class="Disease">iPSC-derived CMs (iCellⓇ) anpan>d cell culture medium (iCell Cardiomyocyte Maintenance Medium) were purchased from Cellular Dynamics International (CDI, Madison, WI, USA). Cells were thawed and handled according to the manufacturer’s guidelines. The cells were plated at 2×104 cells/well and maintained at 37℃ with 5% CO2 and 95% humidity. Cells began to beat spontaneously within one or two days of plating. Cell labeling was performed at 7 days after plating.

Cell labeling and the measurement of labeling efficacy

The CMs were labeled with n class="Chemical">DiO (Vybranpan>t cell labelinpan>g solutionpan>, Molecular Probes, Eugene, OR), QD 525 (Thermo Fisher Scientific, Rockford, IL, USA), anpan>d pcDNA3-EGFP usinpan>g pan> class="Chemical">Lipofectamine 2000 (Lipo2K; Invitrogen, Carlsbad, CA, USA). For labeling, cells were treated with DiO, 5 μM dye working solution for 10 min. After treatment, the dye working solution was removed, and the cells were washed three times with growth medium. For EGFP labeling using Lipo2K, CMs were transfected with 100 ng of pcDNA3-EGFP using Lipo2K according to the manufacturer’s instructions. Branched polyethyleneimine (BPEI: 25 kDa) was prepared at an N/P ratio of 20/1. The cells were seeded on well plates, the culture medium was replaced with transfection medium containing Lipo2K complexes, and the plate was incubated for 6 h at 37℃. Then, the transfection medium was replaced with fresh growth medium, and the cells were maintained for 2 days. After incubation, the cells were washed twice with DPBS (pH 7.4). For QD labeling, cells were incubated in medium containing 1 μM QD 525 for 6 h at 37℃, and cells were washed three times with DPBS. Then, the samples were analyzed using laser-scanning confocal microscopy (LSM 710, Carl Zeiss, Thornwood, NY, USA).

Cell viability assay

To measure cell viability after labeling, CMs were seeded on 96-well plates (5×103 cells/well) and incubated for 1 day. After labeling, cell counting kit-8 solution (10 μl; Dojindo Molecular Technologies Inc., Kumamoto, Jn class="Gene">apan) was added to each well. After further inpan>cubationpan> for 2 h at 37℃, the absorbanpan>ce of each well was measured at 450 nm usinpan>g a microplate reader (iMmark, Microplate Absorbanpan>ce Reader, Bio-pan> class="Gene">Rad, CA, USA).

Real-time quantitative PCR

Total RNA was extracted from heart tissue with n class="Chemical">TRIzol (Invitrogen, Carlsbad, CA, USA) anpan>d reverse-tranpan>scribed usinpan>g a complementary DNA reverse tranpan>scriptionpan> kit (Invitrogen, Carlsbad, CA, USA). Reactionpan>s were performed inpan> a real-time PCR thermocycler (Applied Biosystem, Foster City, CA, USA) usinpan>g pan> class="Chemical">SYBR green as the fluorescence dye. The mRNA expression of the target genes was normalized to that of the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the comparative threshold cycle method. Quantitative PCR was performed using SYBR Green PCR Master Mix (2×; Promega, Madison, WI, USA) and 0.2 μM of the following gene-specific primers: forward primer, 5’-TTCCTATTACCTCGGGGCAC-3’ and reverse primer, 5’-TGCCATAGAGATCTGGCAGC-3’ for SCN5A; forward primer, 5’-ATACCAGCCGCTCTC CAGTT-3’ and reverse primer, 5’-ACTGCCCATTAACTT GGCCT-3’ for CACNA1c; forward primer, 5’-ACTACAT CATCCGGGAAGGC-3’ and reverse primer, 5’-CAGCAG GCAGATCTCTCCAA-3’ for HCNA4; forward primer, 5’-TCCTCTGTGTGGGTTCCAAA-3’ and reverse primer, 5’-CCGGACTCATTCACTGCATC-3’ for KCNQ1; forward primer, 5’-CATCTACTGCAACGACGGCT-3’ and reverse primer, 5’-GACAGCCCCATCCTCATTCT-3’ for KCNH2; forward primer, 5’-CCCAATTGCTGTTTTCATGG-3’ and reverse primer, 5’-GTCTCTCATGGCAATCACGG-3’ for KCNJ2; and forward primer, 5’-CCCATGTTCGTCATGG GTGT-3’ and reverse primer, 5’-TGGTCATGAGTCCTTC CACGATA-3’ for GAPDH. The cycle conditions were as follows: primary denaturation at 95℃ for 3 min, 40 cycles of 6 s at 95℃, 60 s at 60℃, and 60 s at 72℃, followed by fluorescence measurement. The expression of target genes was normalized using GAPDH as a reference gene.

Electrophysiological recording

Conventional whole-cell patch-clamp was performed to record the action potential (AP) and currents. The signals were amplified and digitized with an Axopatch 200B amplifier (Axon Instruments, Foster, CA, USA) and a Digidata 1440B AD-DA converter (Axon Instruments). pClamp 10.1 (Axon Instruments) and Origin 8.0 (Microcal, Northampton, MA, USA) were used for the analysis of AP parameters and current amplitudes. Microglass pipettes (World Precision Instruments, Sarasota, FL, USA) were pulled to the resistance of 2∼3 MΩ with a PP-830 puller (Narishige, Tokyo, Jn class="Gene">apan). The extracellular buffer solutionpan> for recordinpan>g AP activity conpan>tainpan>ed (inpan> mM) 145 pan> class="Chemical">NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 1.8 CaCl2, and 5 glucose adjusted to pH 7.4 with NaOH. The intracellular solution contained (in mM) 120 K-aspartate, 20 KCl, 5 NaCl, 2 CaCl2, 5 EGTA, 10 HEPES, and 5 MgATP adjusted to pH 7.25 with KOH. The extracellular buffer solution for ICa,L contained (in mM) 145 CsCl, 10 HEPES, 1 MgCl2, 1.8 CaCl2 and 5 glucose adjusted to pH 7.4 with CsOH. The intracellular solution for ICa,L contained (in mM) 106 CsCl, 5 NaCl, 20 TEA-Cl, 10 HEPES, 5 MgATP, and 1 EGTA adjusted to pH 7.25 with CsOH. ICa,L was recorded using a stepwise protocol with a holding potential of −50 mV and depolarization from −40 to 40 mV in 10-mV increments. The extracellular buffer solution for INav contained (in mM) 135 CsCl, 20 NaCl, 10 HEPES, 1 MgCl2, 1.8 CaCl2, 0.1 CdCl2, and 5 glucose adjusted to pH 7.4 with CsOH. The intracellular solution for INav contained (in mM) 135 CsCl, 5 NaCl, 10 HEPES, 5 MgATP, and 10 EGTA adjusted to pH 7.25 with CsOH. To assess the peak amplitude of the INav density, cells were held at −100 mV and depolarized to various test step voltages (from −80 to 40 mV in 10 mV steps).

Statistical analysis

Quantitative data were expressed as the mean±standard deviation. Statistical comparisons were carried out using ANOVA tests (SPSS Inc., Chicago, IL, USA). A probability level of less than 0.05 was considered statistically significant.

Results

The n class="Disease">hiPSC-CMs were thawed, plated inpan> platinpan>g medium, mainpan>tainpan>ed inpan> mainpan>tenanpan>ce medium, anpan>d beginpan> to beat sponpan>tanpan>eously withinpan> 2 days of platinpan>g. After the cells were labeled with three different agents (pan> class="Chemical">DiO, QDs, and EGFP using Lipo2K), the fluorescence labeling efficiency was analyzed on days 1, 7, and 14. One day after culture, 97.5 and 99.4% of the inoculated hiPSC-CMs were labeled with DiO and QDs, whereas only 33.7% expressed GFP (Fig. 1). The number of hiPSC-CMs labeled with DiO and QDs was maintained over the culture time. The labeling efficacy of both DiO and the QDs remained similar over 14 days. In contrast, the number of GFP-labeled cells and viability decreased significantly over the culture time. Cytotoxicity of Lipo2K has been reported for many cell lines, but EGFP-labeling using Lipo2K showed much lower viability in hiPSC-CMs compared to COS (CV-1 in Origin, and carrying SV40 genetic material) and other immortal cell lines (23, 24). This result suggests that Lipo2K, a liposomal transfection reagent with exogenous DNA is not a suitable labeling method for hiPSC-CMs.

Fig. 1

Analysis of the labeling efficiency and cell viability of three labeling agents: DiO, QD, and GFP using Lipo2K. (A, B) The efficiency of the three agents was analyzed by green fluorescence at 1 day, 7 days, and 14 days after labeling. (C) Cell viability after labeling was analyzed on the same days.

Next, we examined whether n class="Chemical">DiO anpan>d QD labelinpan>g inpan>fluences the mRNA expressionpan> of the essential cardiac ionpan> chanpan>nels (K+ chanpan>nel; pan> class="Gene">KCNA4, KCNQ1, KCNH2, and KCNJ2, L-type Ca2+ channel; CACNA1c, voltage-dependent Na+ channel; SCN5A, and hyperpolarization-activated cyclic nucleotide-gated channel; HCN4) of hiPSC-CMs (Fig. 2). There was no significant difference in the mRNA expression between the control cells and the labeled cells over the 14 days after labeling. When the mRNA expression of SCN5A, CACNA1c, and KCNJ2 in the control cells increased, that of the labeled cells also increased. At 7 days after labeling, the mRNA expression of CACNA1c, KCNH2, KCNA4, and KCNQ1 in the labeled group was significantly different from that in the control group. Over time, the difference in KCNH2, KCNA4, and KCNQ1 mRNA expression was reduced. Overall, there was no difference in the mRNA expression between the DiO and QD labeled groups.

Fig. 2

The effects of DiO and QD labeling on mRNA expression of cardiac ion channels. (A∼G) Changes in the expression of SCN5A, CACNA1c, KCNA4, HCN4, KCNQ1, KCNH2, and KCNJ2 in hiPSC-CMs were observed at 7 and 14 days after DiO and QD labeling. All the data were analyzed using paired t-tests, where p<0.05 (*) and p<0.01 (**) were considered statistically significant.

Finally, we validated whether the action of n class="Chemical">DiO anpan>d QDs affect the CM electrophysiological profile. The effects of pan> class="Chemical">DiO and QDs on the APs and ion channel currents were evaluated in the hiPSC-CMs at day 7 after labeling because the change in ion channel gene expression was the greatest at day 7 after labeling. APs from nontreated control or DiO- and QD-labeled hiPSC-CMs were recorded and analyzed (Fig. 3). DiO and QD labeling agents did not affect the AP amplitude (APA), maximum diastolic potential (MDP), maximum upstroke velocity (Vmax), and action potential duration 90% (APD90). The parameters are presented in Table 1.

Fig. 3

The AP activity of hiPSC-CMs was recorded after 7 days of DiO and QD labeling, and its characteristics were analyzed. (A∼C) Spontaneous AP activity of control, DiO, and QD-labeled hiPSC-CMs. The action potentials were analyzed as the APA, MDP, Vmax, and APD90 (Table 1). All the data were analyzed using paired t-tests, where a p<0.05 was considered statistically significant.

Table 1

Analysis of action potential properties in control, DiO, QD-labeled hiPSC-CMs

Labeling probe	APA (mV)	MDP (mV)	Vmax (V/s)	APD90 (mV)
Control (n=5)	86.3±4.8	−59.2±4.1	8.1±2.3	447.3±37.8
DiO (n=5)	91.4±5.5	−52.0±5.1	13.1±6.9	473.5±49.0
QDs (n=5)	84.8±1.9	−51.0±1.9	7.76±1.4	457.9±67.3

The voltage-dependent Na+ current (INav) and L-type n class="Chemical">Ca2+ current (ICa,L) inpan> pan> class="Disease">hiPSC-CMs were recorded using the voltage-clamp patch-clamp method. The subtracted currents were obtained by substitution of extracellular Na+ with NMDG+ and by applying nifedipine, a specific inhibitor of ICa,L. The peak amplitude of INav at −50 mV was analyzed between control and DiO- or QD-labeled CMs. The amplitude of INav in DiO- and QD-labeled cells was not different from that in control cells (Fig. 4A and 4B). The peak amplitude of ICa,L at 0 mV was analyzed. Compared to the control CMs, QD-labeled CMs had an increased amplitude of ICa,L, but the DiO-labeled CM amplitude showed no conversion (Fig. 4C and 4D).

Fig. 4

Effect of DiO and QD labeling on the voltage-dependent Na+ current (INav) and L-type Ca2+ current (ICa,L) in hiPSC-CMs. (A, B) Peak amplitudes of INav were analyzed after replacing Na+ in the extracellular solution with NMDG+. The peak inward current densities recorded in DiO- and QD-labeled CMs compared to unlabeled control CMs at −50 mV. (C, D) Peak amplitudes of ICa,L were analyzed after the application of a selective inhibitor, nifedipine (1 μM). A significant increase in ICa,L was observed in QD-labeled CMs compared to control CMs. All the data were analyzed using paired t-tests, where *p<0.05 was considered statistically significant.

Discussion

Many hurdles remain to the widespread acceptance of labeling for cell tracking and labeling for stem cell-derived cardiomyocytes because the funpan>ctionpan>al chanpan>ges of labelinpan>g agents inpan> cells are not fully unpan>derstood. Although these materials have biocompatibility inpan> the cells, manpan>y previous studies have shownpan> that the pan> class="Chemical">carbocyanine dyes with highly lipophilic nature and nano-materials such as QD specifically led to changes in bio-physiological function, cellular signaling pathways and interference with cell-cell communication (4-6,25, 26). Indeed, carbocyanine dyes showed at high concentrations in cancer cell lines (6). The QD negatively affected cell growth and migration functions (25, 26). Thus, if these labeling agents are to be used for stem cell-derived cardiomyocytes, its effects on physiological properties, biochemical properties of modified cells, and cellular function should be clearly defined.

This study is the first to evaluate the functional effect of labeling agents on n class="Disease">hiPSC-CMs. pan> class="Disease">hiPSC-CMs were labeled with DiO and QDs with high efficiency and without cytotoxic effects. However, labeling with EGFP using Lipo2K, a transfection reagent, significantly reduced cell viability. Labeling with DiO and QDs did not have negative electrophysiological influences, such as arrhythmogenic effects, including the early and delayed afterdepolarizations of AP or prolongation of AP in hiPSC-CMs. Rather, QD labeling increased Ca2+ influx through the enhancement of ICa,L.

n class="Gene">Direct labelinpan>g is more conpan>sistent anpan>d effective thanpan> inpan>pan> class="Gene">direct labeling in hiPSC-CMs. We selected DiO and QDs for the direct labeling method because DiO and QD labeling have proven to be sufficient for various cell labeling applications in vitro. Cells labeled with both types of labels showed higher fluorescence intensities than untreated cells (Fig. 1). Generally, one of the disadvantages of direct labeling methods is that the labeling agent becomes diluted when cells proliferate, resulting in decreased fluorescence intensity of the labeling agent per cell (27-30). This factor usually limits the time available for the observation of directly labeled cells. However, hiPSC-CMs exhibit very low levels of proliferation (31, 32). Thus, the dilution of the labeling agent in the cell by cell division does not occur. Indeed, the intensity of hiPSC-CMs labeled with DiO and QDs was similar on the first and the 14th day after labeling (Fig. 1). These results indicate that direct labeling methods are suitable for hiPSC-CMs with low proliferative activity. The labeling efficacy of the indirect system was also tested with genetic modification using EGFP gene transfection by Lipo2K. It was difficult to transfect the hiPSC-CMs using a nonviral gene transfer delivery system because of the low transfection rates and cell toxicity.

n class="Disease">hiPSC-CMs possess similar conpan>tractibility, synpan>chronpan>icity, anpan>d electrochemical properties as cardiac tissue (33, 34). Therefore, pan> class="Disease">hiPSC-CMs are used for cellular cardiomyoplasty to regenerate the myocardium (35, 36). Indeed, recent studies reported that CMs from pluripotent stem cells were successfully transplanted and repaired the myocardium when injected into the damaged heart of immunosuppressed animals (17,37-40). In addition, hiPSC-CMs have been developed to replace animal testing for cardiac safety assays (41, 42). For these developments, the functions of hiPSC-CMs, including the repolarization or depolarization of cardiac ion channels and calcium flux, should be maintained. Therefore, the ideal labeling method of hiPSC-CMs should be noncytotoxic and not have harmful effects on other cellular functions. Our results demonstrate the electrophysiological safety of DiO and QD labeling in hiPSC-CMs. Both labeling agents did not significantly change the AP.

Functional immaturity is a well-known property of n class="Disease">hiPSC-CMs. Accordinpan>g to previous reports, pan> class="Disease">hiPSC-CMs have an immature Ca2+ handling ability and fewer contractile proteins than mature CMs (43, 44). In mature CMs, the excitation-contraction (EC) coupling is mediated mainly by calcium-induced calcium release, whereas it is predominantly due to the trans-sarcolemmal influx of Ca2+ in hiPSC-CMs (43). Our results demonstrate that QD labeling enhances Ca2+ influx (Fig. 4C) and mRNA expression of CACNA1c (Fig. 2B). DiO and QD labelings promoted mRNA expression of Ca2+ and K+ ion channels. In both control and labeled groups, mRNA expression of Ca2+ and K+ ion channels increased over 14 days. Interestingly, mRNA expression of Ca2+ and K+ ion channels, which was expressed in 14 days in control group, was expressed in 7 days in DiO and QD labeled groups (Fig. 2B, 2C, 2E, and 2F). Increased Ca2+ influx and enhanced ion channel expression may improve the Ca2+ handling and electrophysiological properties of immature EC coupling in hiPSC-CMs, although we do not present direct evidence of Ca2+ and CM contraction.

In conclusion, we demonstn class="Species">rated that pan> class="Chemical">DiO and QD labeling of hiPSC-CMs is stable, long-lasting, non-toxic, and may allow safe and long-term cell tracking to monitor CM-based therapies. Since this study was conducted in vitro, it is necessary to confirm the safety and efficacy of the labeled CMs in vivo in further studies.