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The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following Wnt signaling modulation cardiac differentiation

The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following... Owing to the beneficial properties of amniotic fluid-derived stem cells (AFSCs), including pluripotency and the lack of ethical issues associated with embryonic stem cells (ESCs), they should be a promising cell source for regenerative medicine. However, how to differentiate AFSCs into contracting cardiomyocytes has not been established. In this study, a well-established, direct cardiac differentiation protocol involving the modulation of Wnt signaling was used to differentiate Oct 3/4 AFSCs into cardiomyocytes. By day 14 of cardiomyocyte differentiation, these AFSCs expressed cardiac-specific genes (i.e., cardiac troponin T and myosin light chain 2v) and proteins but could not spontaneously contract. Using the patch-clamp technique, we further characterized the electrophysiological properties of human ESC-derived cardiomyocytes (hESC-CMs) and differentiated AFSCs. We used different configurations to investigate membrane potentials and ion currents in differentiated AFSCs and hESC-CMs. Under cell-attached voltage- or whole- cell current-clamp modes, we recorded spontaneous action currents (ACs) or action potentials (APs) in hESC-CMs but not in differentiated AFSCs. Compared to hESC-CMs, differentiated AFSCs showed significantly diminished activity of both BK and IK channels, which might lead to a lack of spontaneous ACs and APs in differentiated AFSCs. These Ca Ca results indicated that this well-established Wnt signaling modulating cardiac differentiation protocol was insufficient to induce the differentiation of functional cardiomyocytes from Oct 3/4 AFSCs. Therefore, AFSC may not be an ideal candidate for cardiomyocyte differentiation. Introduction tissues with functional stem cell-derived cardiomyocytes. After severe myocardial injury, such as myocardial It was reported that exogenous bone-marrow-derived c- + 3 + infarction, the regenerative ability of mammalian hearts is kit hematopoietic stem cells and endogenous c-kit 1 4 very limited, which may lead to impaired cardiac systolic cardiac progenitor cells restored the infarcted myo- function, heart failure or even death. Ideally, post-infarct cardium, supporting the concept that stem cells may be cardiac contractility could be restored by replacing scar effective for cardiac regeneration. However, several stu- dies have shown that c-kit stem cells, including hema- topoietic stem cells and cardiac progenitor cells, do not 5–7 Correspondence: Y-W. Liu (wen036030@gmail.com) or S-N. Wu (snwu@mail. efficiently differentiate into cardiomyocytes. Addition- ncku.edu.tw) ally, over the last decade, hundreds of patients have Division of Cardiology, Department of Internal Medicine, National Cheng received c-kit stem cell therapy, with conflicting results Kung University Hospital, College of Medicine, National Cheng Kung University, 8–13 138 Sheng-Li Rd. North District, Tainan 70403, Taiwan regarding the improvement in cardiac function. Institute of Clinical Medicine, National Cheng Kung University Hospital, Human embryonic stem cells (hESCs) are pluripotent. College of Medicine, National Cheng Kung University, Tainan, Taiwan There is no doubt that using a well-established cardiac Full list of author information is available at the end of the article. Edited by M. V. Nikilson Chirou © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Liu et al. Cell Death Discovery (2019) 5:59 Page 2 of 11 differentiation protocol, hESCs can differentiate into undifferentiated cells (undifferentiated AFS cells: 1 ± 0.25; 14–16 contracting cardiomyocytes. hESC-derived cardio- day 0: 0.13 ± 0.01; day 5: 8.24 ± 1.67; day 10: 11.38 ± 2.7; myocytes (hESC-CMs) can sufficiently repair damaged day 14: 18.67 ± 2.52; undifferentiation vs. day 5: p = 14–19 cardiac tissues and result in favorable cardiac repair. 0.0016; undifferentiation vs. day 10: p = 0.0012; undiffer- Although cardiac regeneration using hESC-CMs is pro- entiation vs. day 14: p < 0.001; Fig. 2c). mising, significant obstacles limit their clinical applica- At differentiation day 14, we performed flow cytometry using both differentiated AFSCs and hESC-CMs to tion. For example, after hESC-CM transplantation, the recipients will need the life-long use of strong immuno- evaluate expression changes in cardiac-specific sarco- suppressive drugs to prevent rejection of these trans- meric proteins (i.e., cTnT, MLC2a, and MLC2v) (Fig. 2d). planted cells ; nevertheless, these drugs may cause These differentiated cells were not positive for MLC2a, several major adverse events, such as kidney injury, ser- indicating that they were not atrial cardiomyocytes. ious infection, and malignancy. Additionally, the use of Compared to undifferentiated AFSCs, these differentiated hESCs for research or therapy has complex social and AFSCs had significantly higher expression levels of ven- ethical issues. tricular cardiomyocyte markers, i.e., cTnT and MLC2v Amniotic fluid-derived stem cells (AFSCs) express the (Table 1;Fig. 2d). The percentages of differentiated transcription factor Oct-4, indicating that they should be AFSCs expressing cTnT and MLC2v were 60.2 and 21,22 pluripotent. Importantly, owing to low major histo- 27.5%, respectively. Although the percentage of differ- compatibility complex (MHC) class I antigen expression entiated AFSCs expressing cTnT was 60.2%, a typical and the absence of MHC class II antigens, AFSCs may bimodal distribution of cardiomyocytes was not detected 21–23 have immune privilege. Moreover, unlike hESCs, (Fig. 2d), indicating that these differentiated AFSCs were using AFSCs for research does not have any major ethical not true ventricular cardiomyocytes or were immature issues. Owing to these beneficial properties, AFSCs should ventricular cardiomyocytes. Because the expression pat- be a good candidate for regenerative medicine research. tern of MLC2v may provide information regarding the 24,25 Accordingly, we aimed to investigate whether AFSCs maturity of differentiated cells, we used flow cyto- could be differentiated into contracting cardiomyocytes metry to evaluate MLC2v expression and found a low in vitro. percentage of MLC2v expression in differentiated AFSCs. Moreover, we performed and quantified immuno- Results fluorescence staining on day 14 of cardiac differentiation (Table 2; Fig. 3). Obvious cTnT expression was observed AFSC characteristics Undifferentiated AFSCs predominantly exhibited a in differentiated AFSCs and hESC-CMs (Fig. 3a), but low fibroblast-like morphology (Fig. 1a). Flow cytometry expression of MLC2v was noted in differentiated AFSCs indicated that undifferentiated AFSCs and hESCs (Fig. 3b). Furthermore, differentiated AFSCs did not expressed the pluripotent stem cell markers, i.e., Nanog, express MLC2a (Fig. 3c). These immunofluorescence Oct3/4, and SSEA4 (Table 1; Fig. 1b). At cardiac differ- staining results were compatible with the flow cytometry entiation day 14, the expression of these 3 pluripotent results. Our results indicated that the differentiated stem cell markers significantly reduced in both differ- AFSCs were not atrial cardiomyocytes and were likely entiated AFSCs and hESC-CMs (Table 1; Fig. 1b). This immature ventricular cardiomyocytes or cardiomyocyte- finding indicated that ASFCs possessed pluripotent like cells. characteristics, similar to those of hESCs and induced pluripotent stem cells. Characterization of the electrophysiological properties of differentiated AFSCs and hESC-CMs Cardiac differentiation of AFSCs Although differentiated AFSCs expressed cardiac- Using the direct cardiac differentiation protocol based specific markers (i.e., cTnT and MLC2v), they did not on the Wnt signaling pathway (Fig. 2a), differentiated contract spontaneously. Therefore, it is necessary to AFSCs were elongated and larger in size than undiffer- identify the difference in electrophysiological character- entiated cells (Fig. 1a). During the differentiation period, istics between differentiated AFSCs and hESC-CMs. By significant changes in cardiac gene expression, i.e., posi- using whole-cell current-clamp recordings, we evaluated tive expression of both cardiac troponin T (cTnT) and membrane potential in differentiated AFSCs and hESC- myosin light chain (MLC) 2v, were observed since dif- CMs. A periodic change in membrane potential was ferentiation day 10 (Fig. 2b). A quantitative reverse tran- clearly detected in hESC-CMs (Supplementary figure 1) scription polymerase chain reaction (qRT-PCR) analysis but not in differentiated AFSCs. The hESC-CM ampli- of cTnT expression was performed on days 0, 5, 10, and tude, firing frequency, and initial rate of increase in 14. Relative cTnT gene expression was significantly higher spontaneous APs were 64 ± 4 mV, 0.29 ± 0.03 Hz, and on days 5, 10, and 14 in differentiated ASFCs than in 0.72 mV/ms (n = 9), respectively. To study spontaneous Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 3 of 11 Fig. 1 Characterization of undifferentiated and differentiated amniotic fluid-derived stem cells (AFSCs). a Representative images showed the appearance of undifferentiated and differentiated AFSCs, human embryonic stem cell (hESC) and hESC-derived cardiomyocytes (hESC-CMs). Undifferentiated AFSCs exhibited a heterogeneous morphology with a preponderance of fibroblastoid, mesenchymal-like cell shapes. After 14 days of differentiation, the morphology of AFSCs exhibited a rod-like appearance, different from that of human embryonic stem cell-derived cardiomyocytes. Scale bar, 200 µm. b Undifferentiated AFSCs and human embryonic stem cells (hESCs) expressed the pluripotent stem cell markers Nanog, Oct3/4, and SSEA4. At cardiac differentiation day 14, the expression of these 3 pluripotent stem cell markers significantly reduced in both differentiated AFSCs and hESC–derived cardiomyocytes (hESC-CMs) 2+ + ACs in hESC-CMs, we utilized cell-attached current Characterization of large-conductance Ca -activated K 26,27 recordings. The resting membrane potential of these (BK ) channel activity in AFSCs Ca hESC-CMs was approximately −65 mV. Biphasic current Because we were unable to detect the APs and ACs of waveforms (i.e., ACs) induced across the membrane patch differentiated AFSCs, we were interested in ion channel by intracellular APs were observed in hESC-CMs (Sup- expression. Thus, we characterized membrane ion cur- plementary figure 2) but not in differentiated AFSCs. rents in undifferentiated and differentiated AFSCs. These These currents with irregular amplitudes are thought to AFSCs were immersed in a high K solution (145 mM) 2+ be electrical manifestations of APs as cell-attached vol- containing 0.1 μMCa . When inside-out current tage-clamp recordings are achieved. The spontaneous recordings were established, BK channel activity was Ca ACs of hESC-CMs with a firing frequency of 0.36 ± 0.01 observed at different levels of holding potential (Fig. 4a). Hz (n = 8) were measured. Notably, the emergence of Unexpectedly, in undifferentiated AFSCs, but not in dif- ACs appears when a spike in the downward deflection ferentiated AFSCs, we detected high activity of BK Ca occurs. channels, as previously reported in human cardiac Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 4 of 11 Table 1 Median fluorescence intensity (MFI) for surface markers of amniotic fluid derived stem cells and human embryonic stem cells MFI Undifferentiated Differentiated hESCs hESC- AFSCs AFSCs CMs Nanog 648 168 632 108 Oct 3/4 551 127 5048 170 SSEA4 1512 146 5622 136 cTnT 89 1319 72 806 MLC2a 182 464 101 320 MLC2v 167 1230 111 475 AFSC indicated amniotic fluid derived stem cell cTnT cardiac troponin T, hESC-CMs human embryonic stem cell derived cardiomyocytes, MFI median fluorescence intensity, MLC myosin light chain, Oct 3/4 octamer-binding transcription factor 3/4, SSEA4 stage-specific embryonic antigen-4 fibroblasts. Based on the I–V relationship of these channels (Fig. 4b), the single-channel conductance in these undifferentiated AFSCs was 185 ± 4 pS (n = 11). Moreover, the addition of 10 μM 2-guanidine-4-methyl- quinazoline (GMQ) significantly increased the probability of channel openings (control vs. GMQ: 0.019 ± 0.004 vs. 0.039 ± 0.007, n = 9/group, p = 0.008), whereas 1 μM verruculogen effectively decreased open BK channels Ca (control vs. verruculogen: 0.019 ± 0.004 vs. 0.002 ± 0.001, Fig. 2 Gene and protein expression in amniotic fluid-derived n = 9/group, p = 0.004) (Fig. 4c). However, neither GMQ stem cells (AFSCs) following cardiac differentiation. a Schematic nor verruculogen was capable of modifying the single- representation of the cardiac differentiation protocol involving the modulation of Wnt signaling. b Gene expression of cardiac troponin T channel conductance of these channels. (cTnT) and myosin light chain 2 v (MLC2v) in those differentiating Each large current downward deflection, with an AFSCs since day 10 of cardiac differentiation was confirmed by reverse amplitude of 3.1 ± 0.1 pA (n = 12), indicated the opening transcription-polymerase chain reaction (RT-PCR). P, positive control of a single BK channel. Notably, the current deflection Ca (human embryonic stem cell-derived cardiomyocytes, hESC-CMs); Un, coincided with the emergence of a depolarizing waveform, undifferentiated AFSCs; 0, differentiation day 0; 5, differentiation day 5; 10, differentiation day 10; 14, differentiation day 14. c Relative gene observed as an upward deflection. A change in the expression of cTnT was measured by quantitative RT-PCR (qRT-PCR). depolarizing waveform may result from the opening tra- Compared to undifferentiated AFSCs, cTnT gene expression was jectory of the channel, which was distorted with a current significantly increased since day 5 of cardiac differentiation. d Flow relaxation of approximately 14 ms. When the open state cytometry analysis of cell surface markers staining determined the probability of a single BK channel was derived and percentage of cTnT-positive, MLC2a-positive, and MLC2v-positive cells Ca after 14-day cardiac differentiation. Isotype controls are in blue and plotted over time (Fig. 4d, e), the probability of channel the surface markers are in red. Expression of each surface marker or openings tended to be positively correlated with changes isotype control was analyzed on 100,000 cells. The percentages of in the amplitude of depolarizing waveforms (Fig. 4f). The hESC-CMs expressing cTnT, MLC2a, and MLC2v were 93.2, 41, and time lag was 4.9 ± 0.2 ms (n = 12) (Fig. 4g). 74.2%, respectively. The percentages of differentiated AFSCs expressing cTnT, MLC2a, and MLC2v were 60.2, 0.1, and 27.5%, respectively. Compared to hESC-CMs, the differentiated AFSCs did not Characterization of intermediate-conductance 2+ + show a typical bimodal distribution of cardiac differentiation Ca -activated K (IK ) channel activity in AFSCs Ca When the membrane was maintained at 0 mV relative to the bath solution, we could detect the IK channel Ca activity of undifferentiated AFSCs (Fig. 5a). Again, the channels (i.e., single-channel amplitude versus Δvoltage) IK activity of differentiated AFSCs was not detected. As was established (Fig. 5b). The single-channel conductance Ca different levels of membrane potential were applied to the was 28.1 ± 1.1 pS (n = 9). Moreover, the addition of either undifferentiated AFSCs, the I–V relationship for IK 1-[(2-chlorophenyl)diphenylmethyl]- H-pyrazole Ca Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 5 of 11 (TRAM-34) (1 μM) or 2-chloro-α,α-diphenyl benzenea- probability of IK -channel openings (control vs. TRAM- Ca cetonitrile (TRAM-39) (1 μM) significantly decreased the 39: 0.011 ± 0.001 vs. 0.0015 ± 0.0005, n = 9/group, p = 0.001), while verruculogen (1 μM) did not have any effect (control vs. verruculogen: 0.011 ± 0.001 vs. 0.0011 ± Table 2 Quantification of the fluorescence intensity for 0.0014, n = 9/group, p = 0.06) (Fig. 5c). When the pipette immunofluorescent staining in differentiated amniotic solution included 1 μM chlorotoxin (a Cl channel fluid derived stem cells and human embryonic stem cell- blocker, a gift from Dr. Woei-Jer Chuang, Department of derived cardiomyocytes Biochemistry, College of Medicine, National Cheng Kung University, Tainan, Taiwan), the IK channel remained Ca Fluorescence intensity functionally active and was sensitive to suppression by TRAM-34, but not by verruculogen. Differentiated AFSCs hESC-CMs 2+ cTnT 95.94 ± 5.82 83.27 ± 3.43 Characterization of voltage-gated L-type Ca current MLC2a N/A 81.84 ± 5.13 (I ) in differentiated AFSCs Ca,L Although the activities of BK and IK channels were Ca Ca MLC2v 11.87 ± 2.56 72.11 ± 2.25 significantly reduced in differentiated AFSCs, we detected AFSC indicated amniotic fluid derived stem cell. Data were expressed as mean ± SEM the presence of I in differentiated AFSCs (Fig. 6a). Ca,L cTnT cardiac troponin T, hESC-CMs human embryonic stem cell-derived cardiomyocytes, MLC myosin light chain, N/A not available Additionally, the properties of I caused by rapid Na Fig. 3 Immunofluorescence staining in amniotic fluid derived stem cells (AFSCs) and human embryonic stem cells (hESCs) following cardiac differentiation. Immunostaining for a cardiac troponin T (cTnT), b myosin light chain (MLC)2v, and c MLC2a detection in differentiated AFSCs and hESC-derived cardiomyocytes (hESC-CMs). The differentiated AFSCs expressed cTnT and MLC2v but not MLC2a. This finding was compatible with the gene expression results. These results suggested that AFSCs after 14 days of cardiac differentiation were cardiac ventricular-like cells. Scale bars represent 200 µm Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 6 of 11 Fig. 4 Characterization of BK -channel activity recorded from undifferentiated amniotic fluid-derived stem cells (AFSCs). Cells were bathed Ca + 2+ in a high K solution containing 0.1 μMCa . a Original BK currents for different levels of membrane potential (indicated on the upper part of each Ca trace). The upward deflection indicates an opening event of the BK channel. b Based on the I–V relationship of BK channels, the single-channel Ca Ca conductance in these undifferentiated AFSCs was 185 ± 4 pS (n = 11). Note that the dashed line indicates the reverse potential (i.e., 0 mV). c This bar graph summarized the effects of GMQ (10 μM), verruculogen (Verr, 1 μM), or TRAM-34 (1 μM) on the BK -channel open probability measured at + 60 Ca mV (mean ± S.E.M.; n = 9 for each bar). The addition of 10 μM GMQ significantly increased the probability of channel openings (control vs. GMQ: 0.019 ± 0.004 vs. 0.039 ± 0.007, n = 9/group, p = 0.008), whereas 1 μM verruculogen effectively decreased open BK channels (control vs. Ca verruculogen: 0.019 ± 0.004 vs. 0.002 ± 0.001, n = 9/group, p= 0.004). d–g Each large current downward deflection, with an amplitude of 3.1 ± 0.1 pA (n = 12), indicated the opening of a single BK channel. We simultaneously measured the open channel probability (d) and the waveform height (e) Ca of the membrane potential in differentiated AFSCs. Each circled symbol in e indicates detection of the depolarizing waveform. f The probability of channel openings tended to be positively correlated with changes in the amplitude of depolarizing waveforms. The slope between the open channel probability and the waveform height was 0.44. g This graph showed the cross-correlation plot (i.e., correlation coefficient versus time lag) with a time lag of approximate 5.0 ms (indicated by an asterisk). Significantly different from the control (p< 0.05) membrane depolarization could be clearly identified. The Discussion 29 + depolarizing step also gives rise to I . Although 0.1 In this study, we showed that Oct 3/4 AFSC differ- Ca,L μM isoproterenol (Sigma-Aldrich, St. Louis, MA, USA) entiation into functional cardiomyocytes was not possible could effectively increase the peak I amplitude using a well-established direct cardiac differentiation Ca,L (Fig. 6b), there was no significant change in the overall protocol, despite the expression of cardiac-specific genes I–V relationship of I or the current inactivation pro- and proteins in differentiated AFSCs. Connexin 43 has Ca,L cess when differentiated AFSCs were treated with iso- been detected in the cell junctions between cardiac- 30,31 proterenol. These differentiated AFSCs expressed cardiac- differentiated AFSCs. However, we found that these specific biomarkers and had I and I activity but these differentiated AFSCs could not spontaneously contract. Na Ca,L cells did not have significant BK and IK channel We further characterized the electrophysiological prop- Ca Ca activity, which may explain why these differentiated erties of hESC-CMs and differentiated AFSCs using var- AFSCs had cardiac sarcomeric protein expression but ious configurations of the patch-clamp technique. Under could not spontaneously contract. cell-attached voltage- or whole-cell current-clamp modes, Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 7 of 11 Fig. 5 Characterization of IK -channel activity in undifferentiated amniotic fluid-derived stem cells (AFSCs). a Original IK -channel currents Ca Ca were obtained at 0 mV relative to the bath. The downward deflection indicates the opening event of the undifferentiated AFSC IK channel. b A Ca single IK -channel amplitude (n = 9 for each point) was plotted as a function of potential (i.e., Δvoltage). Notably, as the patch potential is the sum of Ca the resting potential and the pipette potential, these inward currents reverse at approximately +65 mV. As different levels of membrane potential were applied to the undifferentiated AFSCs, the I–V relationship for IK channels (i.e., single-channel amplitude versus Δvoltage) was established. The Ca single-channel conductance was 28.1 ± 1.1 pS (n = 9). Moreover, the dashed line indicates the reversal of potential (i.e.,+ 65 mV). c The effects of TRAM-34 (1 μM), TRAM-39 (1 μM), or verruculogen (Verr, 1 μM) on the probability of IK -channel openings were investigated (n = 9 for each bar). The Ca opening events of IK channels were measured at 0 mV relative to the bath solution. The addition of either TRAM-34 (1 μM) or TRAM-39 (1 μM) Ca significantly decreased the probability of IK -channel openings (control vs. TRAM-39: 0.011 ± 0.001 vs. 0.0015 ± 0.0005, p = 0.001), while verruculogen Ca (1 μM) did not have any effect (control vs. verruculogen: 0.011 ± 0.001 vs. 0.0011 ± 0.0014, p = 0.06) Significantly different from the control (p< 0.05). 1-[(2-Chlorophenyl)diphenylmethyl]- H-pyrazole indicates TRAM-34; 2-chloro-α,α-diphenyl benzeneacetonitrile, TRAM-39 2+ Fig. 6 Properties of the voltage-gated L-type Ca current (I ) recorded from differentiated amniotic fluid-derived stem cells (AFSCs). Ca,L The differentiated AFSCs were immersed in a bath solution containing 1 μM tetrodotoxin and 10 μM tetraethylammonium chloride, and the recording pipettes were filled with a Cs -containing solution. a Superimposed current traces elicited in response to depolarizing pulses (the upper part). We detected the presence of I in differentiated AFSCs and the properties of I caused by rapid membrane depolarization was clearly Ca,L Na identified. b Average I–V relations of peak I in the absence (■) or presence (□) of 0.1 μM isoproterenol (n = 8 for each point). Cells exposed to Ca,L isoproterenol had an increasing peak I but there was no significant change in the overall I–V relationship of I or the current inactivation process Ca,L Ca,L when differentiated AFSCs were treated with isoproterenol Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 8 of 11 we recorded spontaneous ACs or APs in hESC-CMs but significantly diminished, though these differentiated not in differentiated AFSCs. Moreover, both BK and AFSCs exhibited I and I activity. The functional Ca Na Ca,L IK channel activity were detected in hESC-CMs but expression of BK and IK channels was reduced during Ca Ca Ca were significantly decreased in differentiated AFSCs. differentiation. This novel finding may explain, in part, + - In previous studies, c-kit Oct 3/4 AFSCs did not why differentiated AFSCs expressed cardiac-specific 30,32 efficiently differentiate into cardiomyocytes. Oct 3/4 markers but could not spontaneously contract. 33–36 is the gatekeeper for stem cell pluripotency and is expected to be an important factor for cardiac differ- Conclusion 37–39 entiation. In this study, AFSCs expressed the tran- AFSCs are viewed as a promising cell source for scription factors Oct 3/4, Nanog, and SSEA, indicating regenerative medicine. However, our results showed self-renewal ability and stem cell pluripotency. Therefore, that Wnt signaling modulation could not efficiently using a well-defined direct cardiomyocyte differentiation induce functional cardiomyocyte differentiation from protocol, we proposed that Oct 3/4 AFSCs could differ- AFSCs. Differentiated AFSCs expressed cardiac-specific entiate into cardiomyocytes. Indeed, the expression of genes and proteins but did not spontaneously contract. cardiac-specific genes (i.e., cTnT and MLC2v) could be Our electrophysiological analysis revealed that the detected on differentiation day 5 and increased sig- decreased activity of both BK and IK channels in Ca Ca nificantly thereafter (Fig. 2a, b). It is worth mentioning differentiated AFSCs might lead to a lack of sponta- that compared to hESC-CMs, although day-14 differ- neous ACs and APs, explaining the absence of sponta- entiated AFSCs expressed cTnT and MLC2v, a bimodal neous contraction. distribution of cTnT expression was not observed (Fig. 2c) and only a relatively small percentage of differentiated Methods and materials AFSCs were positive for MLC2v. Importantly, no spon- Cell culture conditions taneous contraction was observed during the 2-week Under a 5% CO atmosphere at 37 °C, both human differentiation period. Therefore, our results indicate that AFSCs (a gift from Dr. Shiaw-Min Hwang, National these differentiated AFSCs might be ventricular-like cells Health Research Institute Cell Bank, Bioresource Collec- but are not real ventricular cardiomyocytes. tion and Research Center, Food Industry Research and Because these differentiated AFSCs expressed connexin Development Institute, Hsinchu, Taiwan) and hESCs 43 but could not spontaneously contract, we were inter- (RUES2 cells, a gift from Dr. Patrick C.H. Hsieh and Dr. ested in their electrophysiological characteristics. Based Jean Lu, Institute of Biomedical Sciences, Academia on patch-clamp electrophysiological studies, hESC-CMs Sinica, Taipei, Taiwan) were maintained in α-Minimum showed spontaneous ACs or APs, which could be sup- Essential Media (MEM) (11900-024; Gibco, Waltham, pressed by 10 μM ranolazine (Supplemental Figure 2). MA, USA) containing 15% fetal bovine serum (FBS, When hESC-CMs were treated with tefluthrin (10 μM), a SH30087.03; HyClone, Boston, MA, USA), 1% glutamine synthetic type-I pyrethroid, there were increases in the (GlutaMAX Supplement, 35050061; Gibco), and 1% amplitude and frequency of ACs. penicillin/streptomycin (15140148; Gibco) for further Moreover, these hESC-CMs displayed periodic rhythms in vitro experiments. The experimental protocol was (Supplemental Figure 1) over several minutes in vitro; approved by the Institutional Review Boards of National accordingly, it is important to use an electrophysiological Cheng Kung University Hospital, Tainan, Taiwan (IRB system that does not interfere with cell behavior to record No. A-EX-105-034). the firing patterns of these contracting cells. Cell-attached Undifferentiated human AFSCs were expanded using α- current-clamp or voltage-clamp recordings may accom- MEM supplemented with 4 ng/ml basic fibroblast growth plish this objective with minimal effort and without factor (bFGF, 233-FB; R&D Systems, Minneapolis, MN, 26,27,40 causing significant damage to cells. The openings of USA). hESCs were used as a positive control for cardio- single hESC-CM BK channels could potentially trigger myocyte differentiation experiments. Using a well- Ca fluctuations in membrane potential. Based on these established direct differentiation protocol (Murry Lab, results, we proposed that the random opening and closure Institute for Stem Cell and Regenerative Medicine, Uni- 5,14–16 of BK channels could lead to the stochastic triggering of versity of Washington, Seattle, WA, USA) , the dif- Ca depolarizing waveforms of cardiomyocytes. In hESC-CMs ferentiation of both AFSCs and hESCs into with high input resistance, current relaxation resulting cardiomyocytes was evaluated (Fig. 1a; Table 1). from the opening of BK channels can depolarize cells. Ca After the temporal or spatial summation of depolarizing Immunocytochemical staining currents, APs were generated. hESC-CMs exhibited BK Differentiated AFSCs and hESCs were fixed in 4% par- Ca activity and were electrically coupled. However, the BK aformaldehyde and then washed with phosphate-buffered Ca and IK channel activity of differentiated AFSCs was saline (PBS). Fixed cells were treated with 1.5% normal Ca Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 9 of 11 goat serum (X0907; Dako, Santa Clara, CA, USA) for 1 h cell suspension was placed on a recording chamber that at room temperature (22–26 °C) and incubated with pri- was tightly affixed to an inverted fluorescence microscope mary antibodies overnight at 4°C. Antibodies (Supple- stage (CKX-41; Olympus). The microscope was equipped mental Table 1) used in this study included mouse anti-α- with a digital video system (DCR-TRV30; Sony, Tokyo, actinin, mouse anti-myosin light chain (MLC)2v, and Japan) with a maximum magnification of 1500×. During mouse anti-cardiac troponin T (cTnT). After rinsing with the recordings, cells were immersed at room temperature in normal Tyrode’s solution containing 1.8 mM CaCl PBS, samples were incubated with secondary antibodies and (Goat anti mouse Alexa Fluor 488, ab150117 and Goat were identified visually using a microscope equipped with anti rabbit Alexa Fluor 568, ab175471; Abcam, Cam- differential interference contrast optics and a 40× objec- bridge, UK). Fluorescent images were acquired (BX51; tive lens. The electrodes were fabricated from Kimax-51 OLYMPUS, Tokyo, Japan). The confocal images were capillaries with an external diameter of 1.5 mm (34500; analyzed and quantified by using the Adobe Photoshop. Kimble Chase, Vineland, NJ, USA). These electrodes had a tip resistance of 3–5MΩ. Additionally, an anti-vibration Flow cytometry air table was used to avoid mechanical noise. Using an At day 14 of cardiac differentiation, the dissociated cells RK-400 (Bio-Logic, Claix, France) or Axopatch 200B were stained for cTnT, MLC2a, and MLC2v. Fluorescence (Molecular Devices, Sunnyvale, CA, USA) amplifier, characterization and analyses were performed using a BD patch-clamp recording experiments were performed in FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). the cell-attached, inside-out, or whole-cell configura- tion. The recordings were commonly achieved by Quantitative reverse transcription polymerase chain advancing a pipette until it was observed to distort the reaction (qRT-PCR) cell; next, negative pressure by gentle suction of the cell RNA was extracted from undifferentiated/differentiated membrane was applied to form a high resistance seal. AFSCs and hESC-CMs and reverse-transcribed into Using cell-attached clamp recording, action currents cDNA. qRT-PCR was performed using all samples with (ACs) and action potentials (APs) of hESC-CMs and oligonucleotide primers (Supplemental Table 2) in tripli- undifferentiated and differentiated AFSCs were mea- 26,27,40 cate. Target gene expression levels were normalized sured. The amplitude and frequency of ACs were against GAPDH expression. Gene expression was quan- evaluated using Mini-Analysis (Synaptosoft, Leonia, NJ, tified with SYBR Green Master Mix (4309155; Applied USA). During voltage-clamp recordings, the potential was Biosystems, Waltham, MA, USA) and detected using maintained at approximately −65 mV. AC measurements Applied Biosystems Step One Plus. Log2-fold changes enabled the quantification of AP frequency. AC wave- were evaluated by the ΔΔCT method, using values for the forms were mainly due to the capacitive current when a undifferentiated AFSC group as a benchmark. cell fired an AP and emerged as a brief spike in the downward deflection. Chemicals and solutions for electrophysiological analyses Signals were obtained using a digital oscilloscope Normal Tyrode’s solution was used as the bath solution (model 1602; Gould, Chandler, AZ, USA). Data were for the electrophysiological analysis. To measure macro- stored in a laptop at 10 kHz using an acquisition interface scopic K currents as well as changes in membrane (Digidata-1440; Molecular Devices) and analyzed using potential and to eliminate contamination by Cl currents, either pCLAMP 10.2 (Molecular Devices) or 64-bit Ori- a patch pipette was filled with a solution containing 130 ginPro 2016 (OriginLab, Northampton, MA, USA). mM K-aspartate, 20 mM KCl, 1 mM KH PO ,1mM Through digital-to-analog conversion, the voltage-step 2 4 MgCl , 3 mM Na ATP, 0.1 mM Na GTP, 0.1 mM EGTA, protocol generated by pCLAMP was implemented to 2 2 2 and 5 mM HEPES-KOH buffer, pH 7.2. To record the determine the I–V relationships for membrane ion cur- + 2+ voltage-gated Na current (I ) and L-type Ca current rents, such as I or I . The activation or inactivation Na Na Ca,L + + (I ), equimolar Cs ions were replaced with K ions in time constants of I or I elicited by membrane Ca,L Na Ca,L the pipette solution and CsOH was used to adjust the pH depolarizations were appropriately estimated by fitting to 7.2. To measure BK -channel activity, a high K current trajectories to a single or double exponential Ca bathing solution was used (145 mM KCl, 0.53 mM MgCl , function with the non-linear least-squares procedure. and 5 mM HEPES-KOH buffer, pH 7.4) and the recording pipette was filled with a solution containing 145 mM KCl, Single-channel analyses 2 mM MgCl and 5 mM HEPES-KOH buffer, pH 7.2. Ion channel activity was analyzed using pCLAMP 10.2. Multi-Gaussian adjustments of the amplitude dis- Electrophysiological measurements tributions among channels were used to determine the Shortly prior to each experiment, a 1% trypsin/EDTA channel opening events. The number of active channels solution was used to dissociate cells and an aliquot of the was defined as the maximum number of simultaneously Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 10 of 11 Received: 2 November 2018 Revised: 31 December 2018 Accepted: 8 open channels when the maximal channel open prob- January 2019 ability was achieved. The open state probabilities were computed using an iterative process to minimize esti- mated χ values. Single-channel conductance, such as the 2+ + large-conductance Ca -activated K channel (BK )or Ca 2+ + References intermediate-conductance Ca -activated K channel 1. Senyo, S. E. et al. Mammalian heart renewal by pre-existing cardiomyocytes. (IK ), was determined by linear regression using mean Ca Nature 493,433–436 (2013). values of current amplitudes at different potential levels. 2. Gaudette,G.R.&Cohen, I. S. 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The biochemical and electrophysiological profiles of amniotic fluid-derived stem cells following Wnt signaling modulation cardiac differentiation

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Copyright © 2019 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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Abstract

Owing to the beneficial properties of amniotic fluid-derived stem cells (AFSCs), including pluripotency and the lack of ethical issues associated with embryonic stem cells (ESCs), they should be a promising cell source for regenerative medicine. However, how to differentiate AFSCs into contracting cardiomyocytes has not been established. In this study, a well-established, direct cardiac differentiation protocol involving the modulation of Wnt signaling was used to differentiate Oct 3/4 AFSCs into cardiomyocytes. By day 14 of cardiomyocyte differentiation, these AFSCs expressed cardiac-specific genes (i.e., cardiac troponin T and myosin light chain 2v) and proteins but could not spontaneously contract. Using the patch-clamp technique, we further characterized the electrophysiological properties of human ESC-derived cardiomyocytes (hESC-CMs) and differentiated AFSCs. We used different configurations to investigate membrane potentials and ion currents in differentiated AFSCs and hESC-CMs. Under cell-attached voltage- or whole- cell current-clamp modes, we recorded spontaneous action currents (ACs) or action potentials (APs) in hESC-CMs but not in differentiated AFSCs. Compared to hESC-CMs, differentiated AFSCs showed significantly diminished activity of both BK and IK channels, which might lead to a lack of spontaneous ACs and APs in differentiated AFSCs. These Ca Ca results indicated that this well-established Wnt signaling modulating cardiac differentiation protocol was insufficient to induce the differentiation of functional cardiomyocytes from Oct 3/4 AFSCs. Therefore, AFSC may not be an ideal candidate for cardiomyocyte differentiation. Introduction tissues with functional stem cell-derived cardiomyocytes. After severe myocardial injury, such as myocardial It was reported that exogenous bone-marrow-derived c- + 3 + infarction, the regenerative ability of mammalian hearts is kit hematopoietic stem cells and endogenous c-kit 1 4 very limited, which may lead to impaired cardiac systolic cardiac progenitor cells restored the infarcted myo- function, heart failure or even death. Ideally, post-infarct cardium, supporting the concept that stem cells may be cardiac contractility could be restored by replacing scar effective for cardiac regeneration. However, several stu- dies have shown that c-kit stem cells, including hema- topoietic stem cells and cardiac progenitor cells, do not 5–7 Correspondence: Y-W. Liu (wen036030@gmail.com) or S-N. Wu (snwu@mail. efficiently differentiate into cardiomyocytes. Addition- ncku.edu.tw) ally, over the last decade, hundreds of patients have Division of Cardiology, Department of Internal Medicine, National Cheng received c-kit stem cell therapy, with conflicting results Kung University Hospital, College of Medicine, National Cheng Kung University, 8–13 138 Sheng-Li Rd. North District, Tainan 70403, Taiwan regarding the improvement in cardiac function. Institute of Clinical Medicine, National Cheng Kung University Hospital, Human embryonic stem cells (hESCs) are pluripotent. College of Medicine, National Cheng Kung University, Tainan, Taiwan There is no doubt that using a well-established cardiac Full list of author information is available at the end of the article. Edited by M. V. Nikilson Chirou © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Liu et al. Cell Death Discovery (2019) 5:59 Page 2 of 11 differentiation protocol, hESCs can differentiate into undifferentiated cells (undifferentiated AFS cells: 1 ± 0.25; 14–16 contracting cardiomyocytes. hESC-derived cardio- day 0: 0.13 ± 0.01; day 5: 8.24 ± 1.67; day 10: 11.38 ± 2.7; myocytes (hESC-CMs) can sufficiently repair damaged day 14: 18.67 ± 2.52; undifferentiation vs. day 5: p = 14–19 cardiac tissues and result in favorable cardiac repair. 0.0016; undifferentiation vs. day 10: p = 0.0012; undiffer- Although cardiac regeneration using hESC-CMs is pro- entiation vs. day 14: p < 0.001; Fig. 2c). mising, significant obstacles limit their clinical applica- At differentiation day 14, we performed flow cytometry using both differentiated AFSCs and hESC-CMs to tion. For example, after hESC-CM transplantation, the recipients will need the life-long use of strong immuno- evaluate expression changes in cardiac-specific sarco- suppressive drugs to prevent rejection of these trans- meric proteins (i.e., cTnT, MLC2a, and MLC2v) (Fig. 2d). planted cells ; nevertheless, these drugs may cause These differentiated cells were not positive for MLC2a, several major adverse events, such as kidney injury, ser- indicating that they were not atrial cardiomyocytes. ious infection, and malignancy. Additionally, the use of Compared to undifferentiated AFSCs, these differentiated hESCs for research or therapy has complex social and AFSCs had significantly higher expression levels of ven- ethical issues. tricular cardiomyocyte markers, i.e., cTnT and MLC2v Amniotic fluid-derived stem cells (AFSCs) express the (Table 1;Fig. 2d). The percentages of differentiated transcription factor Oct-4, indicating that they should be AFSCs expressing cTnT and MLC2v were 60.2 and 21,22 pluripotent. Importantly, owing to low major histo- 27.5%, respectively. Although the percentage of differ- compatibility complex (MHC) class I antigen expression entiated AFSCs expressing cTnT was 60.2%, a typical and the absence of MHC class II antigens, AFSCs may bimodal distribution of cardiomyocytes was not detected 21–23 have immune privilege. Moreover, unlike hESCs, (Fig. 2d), indicating that these differentiated AFSCs were using AFSCs for research does not have any major ethical not true ventricular cardiomyocytes or were immature issues. Owing to these beneficial properties, AFSCs should ventricular cardiomyocytes. Because the expression pat- be a good candidate for regenerative medicine research. tern of MLC2v may provide information regarding the 24,25 Accordingly, we aimed to investigate whether AFSCs maturity of differentiated cells, we used flow cyto- could be differentiated into contracting cardiomyocytes metry to evaluate MLC2v expression and found a low in vitro. percentage of MLC2v expression in differentiated AFSCs. Moreover, we performed and quantified immuno- Results fluorescence staining on day 14 of cardiac differentiation (Table 2; Fig. 3). Obvious cTnT expression was observed AFSC characteristics Undifferentiated AFSCs predominantly exhibited a in differentiated AFSCs and hESC-CMs (Fig. 3a), but low fibroblast-like morphology (Fig. 1a). Flow cytometry expression of MLC2v was noted in differentiated AFSCs indicated that undifferentiated AFSCs and hESCs (Fig. 3b). Furthermore, differentiated AFSCs did not expressed the pluripotent stem cell markers, i.e., Nanog, express MLC2a (Fig. 3c). These immunofluorescence Oct3/4, and SSEA4 (Table 1; Fig. 1b). At cardiac differ- staining results were compatible with the flow cytometry entiation day 14, the expression of these 3 pluripotent results. Our results indicated that the differentiated stem cell markers significantly reduced in both differ- AFSCs were not atrial cardiomyocytes and were likely entiated AFSCs and hESC-CMs (Table 1; Fig. 1b). This immature ventricular cardiomyocytes or cardiomyocyte- finding indicated that ASFCs possessed pluripotent like cells. characteristics, similar to those of hESCs and induced pluripotent stem cells. Characterization of the electrophysiological properties of differentiated AFSCs and hESC-CMs Cardiac differentiation of AFSCs Although differentiated AFSCs expressed cardiac- Using the direct cardiac differentiation protocol based specific markers (i.e., cTnT and MLC2v), they did not on the Wnt signaling pathway (Fig. 2a), differentiated contract spontaneously. Therefore, it is necessary to AFSCs were elongated and larger in size than undiffer- identify the difference in electrophysiological character- entiated cells (Fig. 1a). During the differentiation period, istics between differentiated AFSCs and hESC-CMs. By significant changes in cardiac gene expression, i.e., posi- using whole-cell current-clamp recordings, we evaluated tive expression of both cardiac troponin T (cTnT) and membrane potential in differentiated AFSCs and hESC- myosin light chain (MLC) 2v, were observed since dif- CMs. A periodic change in membrane potential was ferentiation day 10 (Fig. 2b). A quantitative reverse tran- clearly detected in hESC-CMs (Supplementary figure 1) scription polymerase chain reaction (qRT-PCR) analysis but not in differentiated AFSCs. The hESC-CM ampli- of cTnT expression was performed on days 0, 5, 10, and tude, firing frequency, and initial rate of increase in 14. Relative cTnT gene expression was significantly higher spontaneous APs were 64 ± 4 mV, 0.29 ± 0.03 Hz, and on days 5, 10, and 14 in differentiated ASFCs than in 0.72 mV/ms (n = 9), respectively. To study spontaneous Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 3 of 11 Fig. 1 Characterization of undifferentiated and differentiated amniotic fluid-derived stem cells (AFSCs). a Representative images showed the appearance of undifferentiated and differentiated AFSCs, human embryonic stem cell (hESC) and hESC-derived cardiomyocytes (hESC-CMs). Undifferentiated AFSCs exhibited a heterogeneous morphology with a preponderance of fibroblastoid, mesenchymal-like cell shapes. After 14 days of differentiation, the morphology of AFSCs exhibited a rod-like appearance, different from that of human embryonic stem cell-derived cardiomyocytes. Scale bar, 200 µm. b Undifferentiated AFSCs and human embryonic stem cells (hESCs) expressed the pluripotent stem cell markers Nanog, Oct3/4, and SSEA4. At cardiac differentiation day 14, the expression of these 3 pluripotent stem cell markers significantly reduced in both differentiated AFSCs and hESC–derived cardiomyocytes (hESC-CMs) 2+ + ACs in hESC-CMs, we utilized cell-attached current Characterization of large-conductance Ca -activated K 26,27 recordings. The resting membrane potential of these (BK ) channel activity in AFSCs Ca hESC-CMs was approximately −65 mV. Biphasic current Because we were unable to detect the APs and ACs of waveforms (i.e., ACs) induced across the membrane patch differentiated AFSCs, we were interested in ion channel by intracellular APs were observed in hESC-CMs (Sup- expression. Thus, we characterized membrane ion cur- plementary figure 2) but not in differentiated AFSCs. rents in undifferentiated and differentiated AFSCs. These These currents with irregular amplitudes are thought to AFSCs were immersed in a high K solution (145 mM) 2+ be electrical manifestations of APs as cell-attached vol- containing 0.1 μMCa . When inside-out current tage-clamp recordings are achieved. The spontaneous recordings were established, BK channel activity was Ca ACs of hESC-CMs with a firing frequency of 0.36 ± 0.01 observed at different levels of holding potential (Fig. 4a). Hz (n = 8) were measured. Notably, the emergence of Unexpectedly, in undifferentiated AFSCs, but not in dif- ACs appears when a spike in the downward deflection ferentiated AFSCs, we detected high activity of BK Ca occurs. channels, as previously reported in human cardiac Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 4 of 11 Table 1 Median fluorescence intensity (MFI) for surface markers of amniotic fluid derived stem cells and human embryonic stem cells MFI Undifferentiated Differentiated hESCs hESC- AFSCs AFSCs CMs Nanog 648 168 632 108 Oct 3/4 551 127 5048 170 SSEA4 1512 146 5622 136 cTnT 89 1319 72 806 MLC2a 182 464 101 320 MLC2v 167 1230 111 475 AFSC indicated amniotic fluid derived stem cell cTnT cardiac troponin T, hESC-CMs human embryonic stem cell derived cardiomyocytes, MFI median fluorescence intensity, MLC myosin light chain, Oct 3/4 octamer-binding transcription factor 3/4, SSEA4 stage-specific embryonic antigen-4 fibroblasts. Based on the I–V relationship of these channels (Fig. 4b), the single-channel conductance in these undifferentiated AFSCs was 185 ± 4 pS (n = 11). Moreover, the addition of 10 μM 2-guanidine-4-methyl- quinazoline (GMQ) significantly increased the probability of channel openings (control vs. GMQ: 0.019 ± 0.004 vs. 0.039 ± 0.007, n = 9/group, p = 0.008), whereas 1 μM verruculogen effectively decreased open BK channels Ca (control vs. verruculogen: 0.019 ± 0.004 vs. 0.002 ± 0.001, Fig. 2 Gene and protein expression in amniotic fluid-derived n = 9/group, p = 0.004) (Fig. 4c). However, neither GMQ stem cells (AFSCs) following cardiac differentiation. a Schematic nor verruculogen was capable of modifying the single- representation of the cardiac differentiation protocol involving the modulation of Wnt signaling. b Gene expression of cardiac troponin T channel conductance of these channels. (cTnT) and myosin light chain 2 v (MLC2v) in those differentiating Each large current downward deflection, with an AFSCs since day 10 of cardiac differentiation was confirmed by reverse amplitude of 3.1 ± 0.1 pA (n = 12), indicated the opening transcription-polymerase chain reaction (RT-PCR). P, positive control of a single BK channel. Notably, the current deflection Ca (human embryonic stem cell-derived cardiomyocytes, hESC-CMs); Un, coincided with the emergence of a depolarizing waveform, undifferentiated AFSCs; 0, differentiation day 0; 5, differentiation day 5; 10, differentiation day 10; 14, differentiation day 14. c Relative gene observed as an upward deflection. A change in the expression of cTnT was measured by quantitative RT-PCR (qRT-PCR). depolarizing waveform may result from the opening tra- Compared to undifferentiated AFSCs, cTnT gene expression was jectory of the channel, which was distorted with a current significantly increased since day 5 of cardiac differentiation. d Flow relaxation of approximately 14 ms. When the open state cytometry analysis of cell surface markers staining determined the probability of a single BK channel was derived and percentage of cTnT-positive, MLC2a-positive, and MLC2v-positive cells Ca after 14-day cardiac differentiation. Isotype controls are in blue and plotted over time (Fig. 4d, e), the probability of channel the surface markers are in red. Expression of each surface marker or openings tended to be positively correlated with changes isotype control was analyzed on 100,000 cells. The percentages of in the amplitude of depolarizing waveforms (Fig. 4f). The hESC-CMs expressing cTnT, MLC2a, and MLC2v were 93.2, 41, and time lag was 4.9 ± 0.2 ms (n = 12) (Fig. 4g). 74.2%, respectively. The percentages of differentiated AFSCs expressing cTnT, MLC2a, and MLC2v were 60.2, 0.1, and 27.5%, respectively. Compared to hESC-CMs, the differentiated AFSCs did not Characterization of intermediate-conductance 2+ + show a typical bimodal distribution of cardiac differentiation Ca -activated K (IK ) channel activity in AFSCs Ca When the membrane was maintained at 0 mV relative to the bath solution, we could detect the IK channel Ca activity of undifferentiated AFSCs (Fig. 5a). Again, the channels (i.e., single-channel amplitude versus Δvoltage) IK activity of differentiated AFSCs was not detected. As was established (Fig. 5b). The single-channel conductance Ca different levels of membrane potential were applied to the was 28.1 ± 1.1 pS (n = 9). Moreover, the addition of either undifferentiated AFSCs, the I–V relationship for IK 1-[(2-chlorophenyl)diphenylmethyl]- H-pyrazole Ca Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 5 of 11 (TRAM-34) (1 μM) or 2-chloro-α,α-diphenyl benzenea- probability of IK -channel openings (control vs. TRAM- Ca cetonitrile (TRAM-39) (1 μM) significantly decreased the 39: 0.011 ± 0.001 vs. 0.0015 ± 0.0005, n = 9/group, p = 0.001), while verruculogen (1 μM) did not have any effect (control vs. verruculogen: 0.011 ± 0.001 vs. 0.0011 ± Table 2 Quantification of the fluorescence intensity for 0.0014, n = 9/group, p = 0.06) (Fig. 5c). When the pipette immunofluorescent staining in differentiated amniotic solution included 1 μM chlorotoxin (a Cl channel fluid derived stem cells and human embryonic stem cell- blocker, a gift from Dr. Woei-Jer Chuang, Department of derived cardiomyocytes Biochemistry, College of Medicine, National Cheng Kung University, Tainan, Taiwan), the IK channel remained Ca Fluorescence intensity functionally active and was sensitive to suppression by TRAM-34, but not by verruculogen. Differentiated AFSCs hESC-CMs 2+ cTnT 95.94 ± 5.82 83.27 ± 3.43 Characterization of voltage-gated L-type Ca current MLC2a N/A 81.84 ± 5.13 (I ) in differentiated AFSCs Ca,L Although the activities of BK and IK channels were Ca Ca MLC2v 11.87 ± 2.56 72.11 ± 2.25 significantly reduced in differentiated AFSCs, we detected AFSC indicated amniotic fluid derived stem cell. Data were expressed as mean ± SEM the presence of I in differentiated AFSCs (Fig. 6a). Ca,L cTnT cardiac troponin T, hESC-CMs human embryonic stem cell-derived cardiomyocytes, MLC myosin light chain, N/A not available Additionally, the properties of I caused by rapid Na Fig. 3 Immunofluorescence staining in amniotic fluid derived stem cells (AFSCs) and human embryonic stem cells (hESCs) following cardiac differentiation. Immunostaining for a cardiac troponin T (cTnT), b myosin light chain (MLC)2v, and c MLC2a detection in differentiated AFSCs and hESC-derived cardiomyocytes (hESC-CMs). The differentiated AFSCs expressed cTnT and MLC2v but not MLC2a. This finding was compatible with the gene expression results. These results suggested that AFSCs after 14 days of cardiac differentiation were cardiac ventricular-like cells. Scale bars represent 200 µm Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 6 of 11 Fig. 4 Characterization of BK -channel activity recorded from undifferentiated amniotic fluid-derived stem cells (AFSCs). Cells were bathed Ca + 2+ in a high K solution containing 0.1 μMCa . a Original BK currents for different levels of membrane potential (indicated on the upper part of each Ca trace). The upward deflection indicates an opening event of the BK channel. b Based on the I–V relationship of BK channels, the single-channel Ca Ca conductance in these undifferentiated AFSCs was 185 ± 4 pS (n = 11). Note that the dashed line indicates the reverse potential (i.e., 0 mV). c This bar graph summarized the effects of GMQ (10 μM), verruculogen (Verr, 1 μM), or TRAM-34 (1 μM) on the BK -channel open probability measured at + 60 Ca mV (mean ± S.E.M.; n = 9 for each bar). The addition of 10 μM GMQ significantly increased the probability of channel openings (control vs. GMQ: 0.019 ± 0.004 vs. 0.039 ± 0.007, n = 9/group, p = 0.008), whereas 1 μM verruculogen effectively decreased open BK channels (control vs. Ca verruculogen: 0.019 ± 0.004 vs. 0.002 ± 0.001, n = 9/group, p= 0.004). d–g Each large current downward deflection, with an amplitude of 3.1 ± 0.1 pA (n = 12), indicated the opening of a single BK channel. We simultaneously measured the open channel probability (d) and the waveform height (e) Ca of the membrane potential in differentiated AFSCs. Each circled symbol in e indicates detection of the depolarizing waveform. f The probability of channel openings tended to be positively correlated with changes in the amplitude of depolarizing waveforms. The slope between the open channel probability and the waveform height was 0.44. g This graph showed the cross-correlation plot (i.e., correlation coefficient versus time lag) with a time lag of approximate 5.0 ms (indicated by an asterisk). Significantly different from the control (p< 0.05) membrane depolarization could be clearly identified. The Discussion 29 + depolarizing step also gives rise to I . Although 0.1 In this study, we showed that Oct 3/4 AFSC differ- Ca,L μM isoproterenol (Sigma-Aldrich, St. Louis, MA, USA) entiation into functional cardiomyocytes was not possible could effectively increase the peak I amplitude using a well-established direct cardiac differentiation Ca,L (Fig. 6b), there was no significant change in the overall protocol, despite the expression of cardiac-specific genes I–V relationship of I or the current inactivation pro- and proteins in differentiated AFSCs. Connexin 43 has Ca,L cess when differentiated AFSCs were treated with iso- been detected in the cell junctions between cardiac- 30,31 proterenol. These differentiated AFSCs expressed cardiac- differentiated AFSCs. However, we found that these specific biomarkers and had I and I activity but these differentiated AFSCs could not spontaneously contract. Na Ca,L cells did not have significant BK and IK channel We further characterized the electrophysiological prop- Ca Ca activity, which may explain why these differentiated erties of hESC-CMs and differentiated AFSCs using var- AFSCs had cardiac sarcomeric protein expression but ious configurations of the patch-clamp technique. Under could not spontaneously contract. cell-attached voltage- or whole-cell current-clamp modes, Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 7 of 11 Fig. 5 Characterization of IK -channel activity in undifferentiated amniotic fluid-derived stem cells (AFSCs). a Original IK -channel currents Ca Ca were obtained at 0 mV relative to the bath. The downward deflection indicates the opening event of the undifferentiated AFSC IK channel. b A Ca single IK -channel amplitude (n = 9 for each point) was plotted as a function of potential (i.e., Δvoltage). Notably, as the patch potential is the sum of Ca the resting potential and the pipette potential, these inward currents reverse at approximately +65 mV. As different levels of membrane potential were applied to the undifferentiated AFSCs, the I–V relationship for IK channels (i.e., single-channel amplitude versus Δvoltage) was established. The Ca single-channel conductance was 28.1 ± 1.1 pS (n = 9). Moreover, the dashed line indicates the reversal of potential (i.e.,+ 65 mV). c The effects of TRAM-34 (1 μM), TRAM-39 (1 μM), or verruculogen (Verr, 1 μM) on the probability of IK -channel openings were investigated (n = 9 for each bar). The Ca opening events of IK channels were measured at 0 mV relative to the bath solution. The addition of either TRAM-34 (1 μM) or TRAM-39 (1 μM) Ca significantly decreased the probability of IK -channel openings (control vs. TRAM-39: 0.011 ± 0.001 vs. 0.0015 ± 0.0005, p = 0.001), while verruculogen Ca (1 μM) did not have any effect (control vs. verruculogen: 0.011 ± 0.001 vs. 0.0011 ± 0.0014, p = 0.06) Significantly different from the control (p< 0.05). 1-[(2-Chlorophenyl)diphenylmethyl]- H-pyrazole indicates TRAM-34; 2-chloro-α,α-diphenyl benzeneacetonitrile, TRAM-39 2+ Fig. 6 Properties of the voltage-gated L-type Ca current (I ) recorded from differentiated amniotic fluid-derived stem cells (AFSCs). Ca,L The differentiated AFSCs were immersed in a bath solution containing 1 μM tetrodotoxin and 10 μM tetraethylammonium chloride, and the recording pipettes were filled with a Cs -containing solution. a Superimposed current traces elicited in response to depolarizing pulses (the upper part). We detected the presence of I in differentiated AFSCs and the properties of I caused by rapid membrane depolarization was clearly Ca,L Na identified. b Average I–V relations of peak I in the absence (■) or presence (□) of 0.1 μM isoproterenol (n = 8 for each point). Cells exposed to Ca,L isoproterenol had an increasing peak I but there was no significant change in the overall I–V relationship of I or the current inactivation process Ca,L Ca,L when differentiated AFSCs were treated with isoproterenol Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 8 of 11 we recorded spontaneous ACs or APs in hESC-CMs but significantly diminished, though these differentiated not in differentiated AFSCs. Moreover, both BK and AFSCs exhibited I and I activity. The functional Ca Na Ca,L IK channel activity were detected in hESC-CMs but expression of BK and IK channels was reduced during Ca Ca Ca were significantly decreased in differentiated AFSCs. differentiation. This novel finding may explain, in part, + - In previous studies, c-kit Oct 3/4 AFSCs did not why differentiated AFSCs expressed cardiac-specific 30,32 efficiently differentiate into cardiomyocytes. Oct 3/4 markers but could not spontaneously contract. 33–36 is the gatekeeper for stem cell pluripotency and is expected to be an important factor for cardiac differ- Conclusion 37–39 entiation. In this study, AFSCs expressed the tran- AFSCs are viewed as a promising cell source for scription factors Oct 3/4, Nanog, and SSEA, indicating regenerative medicine. However, our results showed self-renewal ability and stem cell pluripotency. Therefore, that Wnt signaling modulation could not efficiently using a well-defined direct cardiomyocyte differentiation induce functional cardiomyocyte differentiation from protocol, we proposed that Oct 3/4 AFSCs could differ- AFSCs. Differentiated AFSCs expressed cardiac-specific entiate into cardiomyocytes. Indeed, the expression of genes and proteins but did not spontaneously contract. cardiac-specific genes (i.e., cTnT and MLC2v) could be Our electrophysiological analysis revealed that the detected on differentiation day 5 and increased sig- decreased activity of both BK and IK channels in Ca Ca nificantly thereafter (Fig. 2a, b). It is worth mentioning differentiated AFSCs might lead to a lack of sponta- that compared to hESC-CMs, although day-14 differ- neous ACs and APs, explaining the absence of sponta- entiated AFSCs expressed cTnT and MLC2v, a bimodal neous contraction. distribution of cTnT expression was not observed (Fig. 2c) and only a relatively small percentage of differentiated Methods and materials AFSCs were positive for MLC2v. Importantly, no spon- Cell culture conditions taneous contraction was observed during the 2-week Under a 5% CO atmosphere at 37 °C, both human differentiation period. Therefore, our results indicate that AFSCs (a gift from Dr. Shiaw-Min Hwang, National these differentiated AFSCs might be ventricular-like cells Health Research Institute Cell Bank, Bioresource Collec- but are not real ventricular cardiomyocytes. tion and Research Center, Food Industry Research and Because these differentiated AFSCs expressed connexin Development Institute, Hsinchu, Taiwan) and hESCs 43 but could not spontaneously contract, we were inter- (RUES2 cells, a gift from Dr. Patrick C.H. Hsieh and Dr. ested in their electrophysiological characteristics. Based Jean Lu, Institute of Biomedical Sciences, Academia on patch-clamp electrophysiological studies, hESC-CMs Sinica, Taipei, Taiwan) were maintained in α-Minimum showed spontaneous ACs or APs, which could be sup- Essential Media (MEM) (11900-024; Gibco, Waltham, pressed by 10 μM ranolazine (Supplemental Figure 2). MA, USA) containing 15% fetal bovine serum (FBS, When hESC-CMs were treated with tefluthrin (10 μM), a SH30087.03; HyClone, Boston, MA, USA), 1% glutamine synthetic type-I pyrethroid, there were increases in the (GlutaMAX Supplement, 35050061; Gibco), and 1% amplitude and frequency of ACs. penicillin/streptomycin (15140148; Gibco) for further Moreover, these hESC-CMs displayed periodic rhythms in vitro experiments. The experimental protocol was (Supplemental Figure 1) over several minutes in vitro; approved by the Institutional Review Boards of National accordingly, it is important to use an electrophysiological Cheng Kung University Hospital, Tainan, Taiwan (IRB system that does not interfere with cell behavior to record No. A-EX-105-034). the firing patterns of these contracting cells. Cell-attached Undifferentiated human AFSCs were expanded using α- current-clamp or voltage-clamp recordings may accom- MEM supplemented with 4 ng/ml basic fibroblast growth plish this objective with minimal effort and without factor (bFGF, 233-FB; R&D Systems, Minneapolis, MN, 26,27,40 causing significant damage to cells. The openings of USA). hESCs were used as a positive control for cardio- single hESC-CM BK channels could potentially trigger myocyte differentiation experiments. Using a well- Ca fluctuations in membrane potential. Based on these established direct differentiation protocol (Murry Lab, results, we proposed that the random opening and closure Institute for Stem Cell and Regenerative Medicine, Uni- 5,14–16 of BK channels could lead to the stochastic triggering of versity of Washington, Seattle, WA, USA) , the dif- Ca depolarizing waveforms of cardiomyocytes. In hESC-CMs ferentiation of both AFSCs and hESCs into with high input resistance, current relaxation resulting cardiomyocytes was evaluated (Fig. 1a; Table 1). from the opening of BK channels can depolarize cells. Ca After the temporal or spatial summation of depolarizing Immunocytochemical staining currents, APs were generated. hESC-CMs exhibited BK Differentiated AFSCs and hESCs were fixed in 4% par- Ca activity and were electrically coupled. However, the BK aformaldehyde and then washed with phosphate-buffered Ca and IK channel activity of differentiated AFSCs was saline (PBS). Fixed cells were treated with 1.5% normal Ca Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 9 of 11 goat serum (X0907; Dako, Santa Clara, CA, USA) for 1 h cell suspension was placed on a recording chamber that at room temperature (22–26 °C) and incubated with pri- was tightly affixed to an inverted fluorescence microscope mary antibodies overnight at 4°C. Antibodies (Supple- stage (CKX-41; Olympus). The microscope was equipped mental Table 1) used in this study included mouse anti-α- with a digital video system (DCR-TRV30; Sony, Tokyo, actinin, mouse anti-myosin light chain (MLC)2v, and Japan) with a maximum magnification of 1500×. During mouse anti-cardiac troponin T (cTnT). After rinsing with the recordings, cells were immersed at room temperature in normal Tyrode’s solution containing 1.8 mM CaCl PBS, samples were incubated with secondary antibodies and (Goat anti mouse Alexa Fluor 488, ab150117 and Goat were identified visually using a microscope equipped with anti rabbit Alexa Fluor 568, ab175471; Abcam, Cam- differential interference contrast optics and a 40× objec- bridge, UK). Fluorescent images were acquired (BX51; tive lens. The electrodes were fabricated from Kimax-51 OLYMPUS, Tokyo, Japan). The confocal images were capillaries with an external diameter of 1.5 mm (34500; analyzed and quantified by using the Adobe Photoshop. Kimble Chase, Vineland, NJ, USA). These electrodes had a tip resistance of 3–5MΩ. Additionally, an anti-vibration Flow cytometry air table was used to avoid mechanical noise. Using an At day 14 of cardiac differentiation, the dissociated cells RK-400 (Bio-Logic, Claix, France) or Axopatch 200B were stained for cTnT, MLC2a, and MLC2v. Fluorescence (Molecular Devices, Sunnyvale, CA, USA) amplifier, characterization and analyses were performed using a BD patch-clamp recording experiments were performed in FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). the cell-attached, inside-out, or whole-cell configura- tion. The recordings were commonly achieved by Quantitative reverse transcription polymerase chain advancing a pipette until it was observed to distort the reaction (qRT-PCR) cell; next, negative pressure by gentle suction of the cell RNA was extracted from undifferentiated/differentiated membrane was applied to form a high resistance seal. AFSCs and hESC-CMs and reverse-transcribed into Using cell-attached clamp recording, action currents cDNA. qRT-PCR was performed using all samples with (ACs) and action potentials (APs) of hESC-CMs and oligonucleotide primers (Supplemental Table 2) in tripli- undifferentiated and differentiated AFSCs were mea- 26,27,40 cate. Target gene expression levels were normalized sured. The amplitude and frequency of ACs were against GAPDH expression. Gene expression was quan- evaluated using Mini-Analysis (Synaptosoft, Leonia, NJ, tified with SYBR Green Master Mix (4309155; Applied USA). During voltage-clamp recordings, the potential was Biosystems, Waltham, MA, USA) and detected using maintained at approximately −65 mV. AC measurements Applied Biosystems Step One Plus. Log2-fold changes enabled the quantification of AP frequency. AC wave- were evaluated by the ΔΔCT method, using values for the forms were mainly due to the capacitive current when a undifferentiated AFSC group as a benchmark. cell fired an AP and emerged as a brief spike in the downward deflection. Chemicals and solutions for electrophysiological analyses Signals were obtained using a digital oscilloscope Normal Tyrode’s solution was used as the bath solution (model 1602; Gould, Chandler, AZ, USA). Data were for the electrophysiological analysis. To measure macro- stored in a laptop at 10 kHz using an acquisition interface scopic K currents as well as changes in membrane (Digidata-1440; Molecular Devices) and analyzed using potential and to eliminate contamination by Cl currents, either pCLAMP 10.2 (Molecular Devices) or 64-bit Ori- a patch pipette was filled with a solution containing 130 ginPro 2016 (OriginLab, Northampton, MA, USA). mM K-aspartate, 20 mM KCl, 1 mM KH PO ,1mM Through digital-to-analog conversion, the voltage-step 2 4 MgCl , 3 mM Na ATP, 0.1 mM Na GTP, 0.1 mM EGTA, protocol generated by pCLAMP was implemented to 2 2 2 and 5 mM HEPES-KOH buffer, pH 7.2. To record the determine the I–V relationships for membrane ion cur- + 2+ voltage-gated Na current (I ) and L-type Ca current rents, such as I or I . The activation or inactivation Na Na Ca,L + + (I ), equimolar Cs ions were replaced with K ions in time constants of I or I elicited by membrane Ca,L Na Ca,L the pipette solution and CsOH was used to adjust the pH depolarizations were appropriately estimated by fitting to 7.2. To measure BK -channel activity, a high K current trajectories to a single or double exponential Ca bathing solution was used (145 mM KCl, 0.53 mM MgCl , function with the non-linear least-squares procedure. and 5 mM HEPES-KOH buffer, pH 7.4) and the recording pipette was filled with a solution containing 145 mM KCl, Single-channel analyses 2 mM MgCl and 5 mM HEPES-KOH buffer, pH 7.2. Ion channel activity was analyzed using pCLAMP 10.2. Multi-Gaussian adjustments of the amplitude dis- Electrophysiological measurements tributions among channels were used to determine the Shortly prior to each experiment, a 1% trypsin/EDTA channel opening events. The number of active channels solution was used to dissociate cells and an aliquot of the was defined as the maximum number of simultaneously Official journal of the Cell Death Differentiation Association Liu et al. Cell Death Discovery (2019) 5:59 Page 10 of 11 Received: 2 November 2018 Revised: 31 December 2018 Accepted: 8 open channels when the maximal channel open prob- January 2019 ability was achieved. The open state probabilities were computed using an iterative process to minimize esti- mated χ values. Single-channel conductance, such as the 2+ + large-conductance Ca -activated K channel (BK )or Ca 2+ + References intermediate-conductance Ca -activated K channel 1. Senyo, S. E. et al. Mammalian heart renewal by pre-existing cardiomyocytes. (IK ), was determined by linear regression using mean Ca Nature 493,433–436 (2013). values of current amplitudes at different potential levels. 2. Gaudette,G.R.&Cohen, I. S. 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