Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.


Introduction
Severe heart failure is a progressive and treatment-resistant disease for which the only complete cure is heart transplantation [1]. In addition, many of the etiological problems leading to heart failure remain unsolved. Direct reprogramming of somatic cells to produce induced pluripotent stem cells (iPSCs) through the forced expression of several embryonic stem (ESC)-specific transcription factors is a prominent recent advance in stem cell biology [2], and human iPSCs hold great promise as new tools for investigating mechanism of disease and as cell sources for transplantation therapy [3]. Human iPSC-derived cardiomyocytes (iPSC-CMs) are thus potential future cell sources for novel cardiomyocyte transplantation therapies for severe heart failure [4,5].
Successful differentiation of human iPSCs into cardiomyocytes was first reported in 2009 [6]. Subsequently, human iPSC-CMs have been developed as in vitro models for cardiac electrophysiological studies and drug screening [7]. Disease modeling of cardiac disease using patient-derived iPSC-CMs was also reported in the field of inherited arrhythmia diseases [8][9][10][11][12] and inherited cardiomyopathies [13][14][15]. Furthermore, iPSC-CMs have been engrafted successfully into the hearts of experimental animals [16] and used to improve cardiac function after ischemic cardiomyopathy in a porcine model [17]. Thus, research using iPSC-CM will be important in clarifying disease mechanisms and establishing novel therapies [18][19][20].
A wide range of donor cell types, gene-introducing vehicles, and combinations of reprogramming factors have been used for generating iPSCs [21]; however, minimally invasive methods have the significant practical advantage for generating iPSCs from patients [22]. We previously reported the generation of T cellderived iPSCs (TiPSCs), which involves a non-invasive method and requires only small volumes of peripheral blood, which means an increased number of eligible patients [23]. Further to this, the present study sought to clarify whether TiPSCs with genomic T cell receptor (TCR) gene rearrangements could differentiate into functional cardiomyocyte in vitro, by examining the morphology, gene expression pattern, and electrophysiological properties of TiPSC-CMs.

Cell culturing
The human TiPSC lines, T05 and T07 were generated from human peripheral T cells by introducing OCT3/4, SOX2, KLF4, and c-MYC with Sendai virus vectors [23]. The human iPSC line, F16 was generated from human fibroblasts by introducing OCT3/ 4, SOX2, KLF4, and c-MYC with retrovirus vectors [24]. The human ESC line (KhES-2) [25] was obtained from the Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University and used in conformity with the Guidelines for Derivation and Utilization of Human Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science, and Technology, Japan. These cell lines were cultured in human iPSC medium consisting of DMEM/F12 medium (Invitrogen) supplemented with 20% Knock-out Serum Replacement (KSR; Invitrogen), 1 mM L-glutamine, 1 mM non-essential amino acids, 0.1 mM b-mercaptoethanol, 50 U penicillin and 50 mg/ml streptomycin (Invitrogen), and 4 ng/ml basic fibroblast growth factor (bFGF; WAKO). The human iPSC medium was changed every other day until the colonies were picked. The iPSCs and ESCs were maintained on irradiated mouse embryonic fibroblast (MEF) feeder cells [26] from wild-type ICR mice in human iPSC medium which was changed every 2-3 days, and the cells were passaged using 1 mg/ml collagenase IV (Invitrogen) every 5-6 days.

In vitro differentiation of iPSCs and ESCs
The iPSCs and ESCs were harvested using 1 mg/ml collagenase IV, and transferred to ultra-low attachment plates (Corning) in differentiation medium. The differentiation medium consisting of Minimum Essential Medium Alpha Medium (Gibco) supplemented with 2 mM L-glutamine (Invitrogen), 0.1 mM nonessential amino acids (Sigma), 0.1 mM 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin (Invitrogen), and 20% fetal bovine serum (Gibco). The medium was replaced every second or third day. To promote cardiac differentiation, recombinant human Wnt3a was added to the differentiation medium for the first 4 days of culture at 100 ng/ml [27]. The time window of differentiation for analyzing the beating embryoid bodies (EBs) and cardiomyocytes was 30-60 days from starting the differentiating conditions.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from cells using TRIZOL reagent (Invitrogen) and RNasefree DNase I (Qiagen), according to the manufacturer's instructions. The concentration and purity of the RNA were determined using an ND-1000 spectrophotometer (Nanodrop). The cDNA was synthesized using the Superscript First-Strand Synthesis System (Invitrogen). PCR was performed with rTaq (TAKARA). The primers used for PCR are listed in Table S1.

Quantitative RT-PCR analysis
Total RNA was isolated and used for reverse transcription with random primers as described by the manufacturer. Real-time quantitative RT-PCR was performed using MX3000P (Stratagene), with SYBR Premix ExTaq (TAKARA). The amount of mRNA was normalized to GAPDH mRNA. Primer sequences and cycling conditions are listed in Table S1.

Electron microscopy
Contracting EBs were fixed with 2.5% glutaraldehyde in 60 mM HEPES, pH 7.4 for 120 min at room temperature, and washed three times with 0.2 M phosphate buffer. Cells were postfixed in 1% OsO 4 in 60 mM HEPES, pH 7.4 for 120 min at room temperature, and stained en bloc in 2% uranyl acetate for 30 min at room temperature. Tissues were dehydrated with gradually increasing concentrations of ethanol for resin embedding, before thin sectioning and staining.
Field potential recordings using the on-chip multi-electrode array system Multi-electrode array (MEA) chips from Multi Channel Systems were coated with fibronectin (Sigma-Aldrich). Beating EBs were plated and incubated at 37uC, and MEA measurements were performed at 37uC, as previously described [7]. The signals were initially processed, and the obtained data were subsequently analyzed with MC Rack (Multi Channel Systems). Data for analysis were extracted from 2-5 min of the obtained data. The recorded extracellular electrograms were used to determine local field potential duration (FPD), defined as the time interval between the initial deflection of the field potential(FP) and the maximum local T wave. FPD measurements were normalized (corrected FPD: cFPD) to the activation rate using Bazett's correction formulae: cFPD = FPD/(RR interval) 1/2 , where RR represents the time interval (in seconds) between two consecutive beats. Isoproterenol hydrochloride (Sigma-Aldrich), E4031 (Sigma-Aldrich), and verapamil hydrochloride (Sigma-Aldrich) were prepared as 1 or 10 mM stock solutions. The FPs were recorded for 5 min, and then drug was added to the medium. After 2-3 min of incubation, the FPs were again measured for 5 min.

Characterization of in vitro cardiac differentiation ability of TiPSCs
First, we examined the in vitro cardiac differentiation ability of TiPSCs. We used suspension culture to induce the TiPSCs to form beating EBs, as shown previously for ESCs and iPSCs [28]. The TiPSCs made beating EBs in the same way as ESCs and fibroblast-derived iPSCs ( Figure 1A, 1B, Movie S1), confirming that TiPSCs could differentiate into beating cardiomyocytes in suspension culture in vitro.
We also examined the temporal gene expression pattern of derivatives of TiPSCs and ESCs during the in vitro differentiation process. While the expression of undifferentiated pluripotent markers OCT3/4, NANOG, and SOX2 decreased similarly, the cardiac-associated transcription factors, NKX2.5 and MEF2C, and the cardiac-specific structural genes, aMHC and MLC2V, showed high expression in the late stages of floating culture ( Figure 1C). On the other hand, primitive streak, mesoderm, and cardiomesoderm markers, MESP1 and BRACHYURY, showed transient expression at the early stage of differentiation ( Figure 1C). These results indicated that the expression patterns of undifferentiated and cardiac markers in the derivatives of TiPSCs and ESCs were similar to that observed during embryonic development.

Molecular and cellular characterization of TiPSC-CMs
We next examined expression of the cardiac-specific markers of TiPSC-CMs by further analyzing the derived beating EBs. RT-PCR revealed that our TiPSC-CMs properly expressed cardiomyocyte markers and showed decreased pluripotent marker expression (Figure 2A). Immunostaining confirmed the proper expression of cardiomyocyte markers, a-Actinin, Troponin-I, Titin, ANP, GATA4, and Nkx2.5 in the TiPSC-CMs ( Figure 2B).
We then checked the expression of proteins associated with the functionality of cardiomyocytes other than sarcomeric components in TiPSC-CMs. Connexin43 is the most abundant connexin in the ventricular myocardium; it constitutes gap junctions, which are localized to the cardiomyocyte plasma membrane and electrically couple cardiomyocytes to orchestrate action potential propagation [29]. Immunostaining revealed that Connexin43 to the cardiomyocyte plasma membrane and to the intercellular junctional regions in TiPSC-CMs, suggesting the proper development of gap junctions and intact electric coupling ( Figure 3A). The activity of the cardiac sarcoplasmic reticulum Ca 2+ transport ATPase (SERCA2a) and Na + -Ca 2+ exchanger control cardiomyocyte relaxation and contraction by regulating intracellular Ca 2+ [30]. In our TiPSC-CMs, SERCA2a was localized to the cytoplasm and Na + -Ca 2+ exchanger was localized to the plasma membrane ( Figure 3A). Electron microscopy of the TiPSC-CMs revealed subcellular morphology identical to that observed in native cardiomyocytes such as the sarcomeric organization and gap junctions ( Figure 3B). Ca 2+ oscillations are part of the fully that cardiomyocyte differentiation was characterized by a continuous decrease in the expression of pluripotent markers (OCT4, NANOG, and SOX2) coupled with an initial increase in mesoderm and cardiomesoderm markers (MESP1 and Brachyury). This was followed by the expression of cardiacrelated transcription factors (NKX2.5 and MEF2C), and finally by cardiac-specific structural genes (aMHC and MLC2V). The graph shows an average of three independent examinations. Error bars show means 6 s.d. doi:10.1371/journal.pone.0085645.g001 differentiated cardiac phenotype and key to controlled cardiomyocyte relaxation and contraction. To evaluate intracellular Ca 2+ behavior, Ca 2+ transients were measured from spontaneously contracting TiPSC-CMs using Fluo-4AM. Line-scan images of Ca 2+ transient and its average fluorescence intensity in TiPSC-CMs showed Ca 2+ oscillations ( Figure 3C) that indicate electrophysiological function typical of native cardiomyocytes. These findings indicated that the generated TiPSC-CMs were functional in regulating intracellular Ca 2+ .

Pharmacolobical characterization of TiPSC-CMs
To further evaluate the functionality of TiPSC-CMs, we examined the pharmacological effects of various cardioactive drugs by measuring FPs of the spontaneously beating EBs (Movie S2). To check the response to adrenergic stimulation, we added isoproterenol to the beating EBs derived from TiPSCs, and found a dose-dependent increase in beating frequency, indicating an intact b-adrenergic signaling cascade ( Figure 4A). Next, we investigated the effects of ion channel-specific inhibitors on the FP waveform, to determine whether TiPSC-CMs show a native cardiomyocyte-like response to ion channel-specific inhibitors. The IKr blocker E4031 dose-dependently prolonged FPDs in the TiPSCs-CMs (Figure 4B), and the L-type calcium channel blocker, verapamil dose-dependently shortened the length of FPD ( Figure 4C). These results indicated that the TiPSC-CMs have identical electrophysiological responses to native cardiomyocytes.

Discussion
The cardiac differentiation ability of iPSCs and functionality of human iPSCs-derived cardiomyocytes have been characterized in previous reports [6,7,31,32]. However, the only evaluation of functionality in derivatives of human pluripotent stem cells that contain genomic rearrangements in the TCR gene locus was in hepatic cells derived from human TiPSCs [33]. TiPSC differentiation into the three germ-cell layers was shown for in vivo teratoma formation [23,[34][35][36][37], and mouse mature T cells that contained genomic rearrangements in the TCR gene locus successfully generated monoclonal mice by the nuclear transfer method [38]. However, the functionality of cardiomyocytes differentiated from human TiPSCs in vitro has not been evaluated. In the present study, we demonstrated several characteristics in TiPSC-CMs that are seen in native cardiomyocytes, and also were previously described as cardiac-specific and essential characters in ESC-CMs and iPSC-CMs without genomic rearrangement [6,39]. Our results thus confirm the potential application of TiPSCs to clinical regenerative therapy for cardiovascular disease and to cardiac disease modeling.
For cardiac cell-based drug screens and cardiac disease research, the minimally invasive methods for tissue sampling involved in generating TiPSCs would be useful for increasing the number of patients who could be subjected to iPSC generation. Our results confirmed the potential of TiPSC-CMs as a cell source of cardiomyocytes with electrophysiological features resembling those of native cardiomyocytes, Therefore, TiPSC-CMs are possible and appealing cell sources for cardiac cell-based drug screens and cardiac disease research.
On another front, TiPSC-CMs have two advantages for transplantation therapy. First, the descendents of TiPSCs that transform into malignant tumors are easily identifiable by their rearrangement patterns. When iPSC-CMs obtained from dermal fibroblasts or other somatic cells are transplanted into diseased patients, there is no good procedure for following the progenies. In animal models, several marker genes can be used to chart the progression and consequences of iPSC-CM transplantation, e.g., GFP and luciferase genes are easily detected and frequently used in experiments with animal models. However, it is not desirable to insert exogenous marker genes into the genomes of human iPSCs for clinical use. In contrast, TiPSCs already have a signature in their specific TCR gene rearrangement, and this could be tracked in TiPSCs descendents [23].
Second, the minimally invasive methods for tissue sampling involved in generating TiPSCs would be especially appealing to allogenic iPSC derivative transplantation because increasing the number of patients subjected to iPSC generation is important for providing HLA-matched cells. Indeed, an HLA-haplotype bank of pluripotent stem cell lines would also be beneficial for reducing the high cost and time spent in generating iPSCs from individual patients [40]. Because TiPSCs can be generated using less invasive tissue sampling compared to dermal fibroblasts by skin biopsy sampling, they are well suited for providing such HLA-matched cell lines for the target population.
In conclusion, TiPSC-CMs have the potential to provide a unique and valuable cell source for clinical regenerative therapy in cardiovascular disease and for cardiac disease modeling.

Supporting Information
Movie S1 The beating EB derived from TiPSCs in suspension culture.