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Effective derivation of ventricular cardiomyocytes from hPSCs using ascorbic acid-containing maturation medium Effective derivation of ventricular cardiomyocytes from hPSCs using ascorbic acid-containing...
Kim, Ji-eun; Kim, Eun-Mi; Lee, Hyang-Ae; Kim, Ki-Suk
2023-12-11 00:00:00
ANIMAL CELLS AND SYSTEMS 2023, VOL. 27, NO. 1, 82–92 https://doi.org/10.1080/19768354.2023.2189932 Effective derivation of ventricular cardiomyocytes from hPSCs using ascorbic acid-containing maturation medium a b b b Ji-eun Kim , Eun-Mi Kim , Hyang-Ae Lee and Ki-Suk Kim a b Dongguk University, Seoul, Republic of Korea; Korea Institute of Toxicology, Daejeon, Republic of Korea ABSTRACT ARTICLE HISTORY Received 13 October 2022 Cardiomyocytes derived from human pluripotent stem cells (hPSCs) can be used in various Revised 5 February 2023 applications including disease modeling, drug safety screening, and novel cell-based cardiac Accepted 10 February 2023 therapies. Here, we report an optimized selection and maturation method to induce maturation of cardiomyocytes into a specific subtype after differentiation driven by the regulation of Wnt KEYWORDS signaling. The medium used to optimize selection and maturation was in a glucose starvation Cardiomyocyte; ascorbic conditions, supplemented with either a nutrition complex or ascorbic acid. Following optimized acid; albumin; Wnt signaling selection and maturation, more cardiac Troponin T (cTnT)-positive cardiomyocytes were detected using albumin and ascorbic acid than B27. In addition, ascorbic acid enriched maturation of ventricular cardiomyocytes. We compared cardiomyocyte-specific gene expression patterns under different selection and maturation conditions by next-generation sequencing (NGS) analysis. Our optimized conditions will enable simple and efficient maturation and specification of the desired cardiomyocyte subtype, facilitating both biomedical research and clinical applications. Introduction pathway by small molecules leads to nuclear accumu- Human pluripotent stem cells (hPSCs) are a potentially lation of β-catenin, which is associated with Tcf/Lef (T- an unlimited source of desired cell types, including car- cell factor/lymphoid enhancer-binding factor) and acti- diomyocytes (Takahashi et al. 2007; Burridge et al. 2012; vation of cardiac-related gene transcription, resulting Tohyama et al. 2013). hPSC-derived cardiomyocytes can in efficient differentiation of hPSCs into cardiomyocytes be used in diverse applications, including disease mod- (Lian et al. 2012; Minami et al. 2012; Kim et al. 2022; Jung eling, cardiotoxicity screening, drug discovery, and et al. 2022). Maturation into specific cell subtypes and basic research (Matsa et al. 2014). These applications purification of the resultant cells are critical steps for require scalable, cost-effective, and reproducible pro- the successful use of hPSC-derived cardiomyocytes in duction of cells under chemically defined conditions applications such as high-throughput drug screening (Burridge et al. 2014) and feasible protocols for differen- and toxicity testing. tiating and maturing specific cardiac cell types (Liang Ascorbic acid is a small molecule that promotes et al. 2013; Landgren and Sartipy 2014). Over the past induction of hPSC-derived cardiomyocytes (Takahashi few years, several methods have been developed for et al. 2003; Fonoudi et al. 2015; Ivanyuk et al. 2015). generating cardiomyocytes from hPSCs, including Specifically, the addition of ascorbic acid during the embryoid body-based (Kattman et al. 2011) and mono- maturation and purification of cardiomyocytes induces layer-based differentiation protocols directed by a unique subtype of cells that express cardiac-related addition of factors such as bone morphogenic protein genes, but not mesodermal markers (Cao et al. 2012). 4 (BMP4), fibroblast growth factor 2 (FGF2), vascular In other methods, differentiation by manipulation of endothelial growth factor (VEGF), and dickkopf-related the Wnt pathway in the presence of ascorbic acid protein 1 (DKK-1) (Laflamme et al. 2007; Paige et al. yields a heterogeneous cell population that includes 2010). However, these methods have variable results non-cardiac cell types such as fibroblasts, smooth and low efficiency in several cell lines and experimental muscle cells, and endothelial cells (Birket et al. 2013). replications (Paige et al. 2010). Regulation of the Wnt Cardiomyocytes use glucose, fatty acids, and lactate as CONTACT Ki-Suk Kim idkks@kitox.re.kr Korea Institute of Toxicolgoy, 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, Republic of Korea Supplemental data for this article can be accessed online at https://doi.org/10.1080/19768354.2023.2189932 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ANIMAL CELLS AND SYSTEMS 83 their main energy sources; in particular, the fetal heart Immunostaining uses lactate as its main energy source, whereas the Cells were fixed with 4% (vol/vol) paraformaldehyde for adult heart uses fatty acids (Lopaschuk et al. 1992). 15 min, permeabilized with 0.5% (vol/vol) Triton X-100 in Exposing differentiated cells to abundant lactate and PBS for 5 min, blocked with 2% (wt/vol) BSA in PBS con- glucose-depletion selectively purify cardiomyocytes taining 0.1% (vol/vol) Triton X-100 for 20 min at room (Tohyama et al. 2013). temperature, and then incubated overnight at 4°C with In this study, we produced hPSC-derived cardiomyo- primary antibodies diluted in PBS with 0.1% (vol/vol) cytes by modulating the Wnt pathway with a small mol- Triton X-100 and 2% (wt/vol) BSA. Immunoreactive ecule inhibitor, and then purifying and maturing cells were detected using Alexa Fluor 488–conjugated cardiomyocytes by taking advantage of their specific secondary antibody. Nuclear DNA was stained using metabolic properties. We found that ascorbic acid pro- Gold Antifade Reagent with DAPI (Invitrogen, U.S.A). moted both structural and functional maturation of car- diomyocytes, as well as their differentiation from hPSCs. In addition, we analyzed gene expression patterns in the Flow cytometry presence of ascorbic acid, either alone or in combination Cultures were dissociated into single cells and then fixed with other factors. This reproducible and efficient with 4% (vol/vol) paraformaldehyde for 20 min at room method, in which lactate-enriched conditions are used temperature, permeabilized with Perm buffer III (BD Bio- to selectively purify cardiomyocytes and ascorbic acid sciences, U.S.A) for 20 min at 4°C, and stained with is used to promote maturation simultaneously, will facili- primary and secondary antibodies in PBS plus 0.5% tate clinical and pharmaceutical applications of hPSC- (vol/vol) FBS (Invitrogen, U.S.A) and 0.1% (wt/vol) derived cardiomyocytes. sodium azide (Sigma-Aldrich, U.S.A). Data were collected on a FACSCalibur flow cytometer (Becton Dickinson, U.S.A) and analyzed using FlowJo (FlowJo, LLC). Materials and methods Culture and differentiation of hPSCs into cardiomyocytes Electron microscopy hPSCs (H9) were maintained in E8 medium on vitronec- Differentiated cells were plated on gelatin-coated glass tin-coated plates. Cells were dissociated and plated coverslips. The clusters were fixed overnight at 4°C in using 0.5 mM EDTA in the presence of ROCK (Rho-associ- 2.5% (vol/vol) glutaraldehyde, and then postfixed with ated kinase) inhibitor (Stemgent, U.S.A). To differentiate 1% (wt/vol) osmium tetroxide. Samples were dehy- the cells into cardiomyocytes, the hPSCs were main- drated using an ethanol gradient and substituted with tained on Matrigel-coated plates. Upon reaching 100% (wt/vol) propylene oxide for 30 min, and then almost complete confluence (∼99%), the cells were embedded with propylene oxide and Epon 812. Ultra- treated for 24 h with 10 µM CHIR99021 (Selleckchem, thin 70 nm sections were stained with uranyl acetate U.S.A) in RPMI/B27-insulin (day 0 to day 1). To inhibit and lead citrate. Samples were visualized on a Tecnai Wnt signaling, 5 μM IWP4 (Stemgent, U.S.A) was added G2 Spirit scanning transmission electron microscope on day 3 and removed during the medium change on (FEI) at 120 kV. Images were acquired on a US4000 day 5. Beating cells were observed starting on day 8–9, camera (Gatan, Inc.). and the medium was replaced every other day. Whole-cell patch-clamp recording for action Selection and maturation of cardiomyocytes potential analysis To select and maturate cardiomyocytes, the differentiated Action potentials (APs) were recorded using the whole- cells were cultured for 10 days under one of two con- cell patch-clamp technique with an Axopatch200B ditions: RPMI1640 without glucose (Life Technologies, amplifier, an A/D converter, a Digidata 1440 digitizer, U.S.A) supplemented with 4 mM sodium DL-lactate and pClamp10.2 software (Axon Instruments for Molecu- (Sigma-Aldrich, U.S.A) and B27-insulin (GIBCO, U.S.A); or lar Devices, USA). Patch pipettes were made from boro- RPMI1640 without glucose (Life Technologies, U.S.A) sup- silicate glass capillaries (Clark Electromedical plemented with 5 mM sodium DL-lactate (Sigma-Aldrich, Instruments, UK) using a pipette puller (PP-830, Nar- U.S.A), 500 µg/mL human albumin (Sigma-Aldrich, U.S.A), ishige, Japan). Resistance was 5–7MΩ when filled with and 211 µg/mL L-ascorbic acid (Sigma-Aldrich, U.S.A). The pipette solution. After 2 weeks of cardiac differentiation, medium was replaced every other day. hPSC-derived cardiomyocytes were transferred to four- 84 J.-E. KIM ET AL. well culture plates containing 0.1% gelatin-coated glass After 2 days with IWP4, this generated cardiac progeni- coverslips and maintained in a culture incubator at 37 °C tor cells (CPCs) (Sharma et al. 2015). The number of for 2 weeks to allow further maturation. CPCs increased substantially after withdrawal of IWP4, For recording, a coverslip with adherent cells was and we observed beating cells after 8–9 days. placed into the recording chamber and perfused with an extracellular solution containing the following (in Ascorbic acid with albumin promotes mM): 137 NaCl, 5.4 KCl, 1.8 CaCl , 0.5 MgCl , 10 HEPES, 2 2 cardiomyocyte differentiation and 10 glucose (adjusted to pH 7.4 with NaOH). The internal solution contained (in mM) 120 K-Asp, 20 KCl, To optimize the selection and maturation of cardiomyo- 5 NaCl, 2 CaCl , 10 HEPES, 5 EGTA, and 5 Mg-ATP (pH cyte, differentiated cells (day 10 from onset of differen- 7.25). Typical APs in hPSC-derived cardiomyocytes tiation) were exposed to glucose-depleted, lactate- were recorded in I = 0 mode. The spontaneous beating enriched RPMI medium with either B27 or albumin activity of single hPSC-derived cardiomyocytes was with ascorbic acid for 10 days, and then the selected recorded, and only hPSC-derived cardiomyocytes that and maturated cells were cultured in RPMI with could beat stably were included in the analysis. Follow- glucose containing B27 (Figure 2(A)). We observed no ing stabilization of the AP waveforms, the average of difference in cell morphology between the two con- five recorded APs was analyzed. ditions on days 20 and 30 (Figure 2(B)). To compare the yields of cardiac differentiation, we analyzed the population of cTnT-positive cells by flow cytometry RNA isolation (Figure 2(C), left side). cTnT-positive cells constituted more than 70% of the total populations under both con- Total RNA was extracted from different samples using ditions, indicating that the identity of the supplement TRIzol (Invitrogen, U.S.A). The harvested samples were had no effect on the efficiency of cardiomyocyte selec- homogenized by several passages through a syringe tion and maturation (supplementary Figure 3). needle in the presence of TRIzol solution. The resultant However, we did observe a difference in expression of lysate was mixed with chloroform at a 1:5 ratio and cen- the two major myosin light-chain 2 isoforms (MLC2a trifuged for 15 min at 12,000 ×g at 4°C. The aqueous and MLC2v) in cTnT-positive cells on day 30 (Figure 2 phase was transferred to a new tube and mixed with iso- (C), right side), indicating diversity and maturity in the propanol at a 1:1 ratio. After centrifugation for 10 min at cardiomyocytes (Kubalak et al. 1994; Franco et al. 1999; 12,000 ×g at 4°C, total RNA was obtained after ethanol Segev et al. 2005). In RPMI without glucose, with dehydration and dissolution by DEPC-D.W. ascorbic acid and with albumin, more cTnT-positive cells expressed MLC2v, a marker of mature ventricular Results cardiomyocytes, than MLC2c, a marker of mature atrial cardiomyocytes (Figure 2(C), right side and lower Cardiomyocytes differentiated from hPSCs via panel). On days 40 and 60 after differentiation, we quan- temporal regulation of Wnt signaling titatively assessed the differential expression of MLC2a To induce cardiomyocyte differentiation, hPSCs were and MLC2v (Figure 3). The percentage of MLC2v-positive cultured on Matrigel-coated plates to confluence. cells was higher in the presence of both ascorbic acid Before exposure to differentiation protocol, the hPSCs and albumin than B27 supplement alone (Figure 3(A, consistently maintained pluripotency (Supplementary B)). The percentage of MLC2v-positive cells was higher Figure 1). We attempted differentiation with a number on day 60 than on day 40, whereas the proportion of of cell types to establish appropriate conditions (Sup- MLC2a-positive cells decreased under both conditions plementary Figure 2). To inhibit GSKα/β signaling, (Figure 3(A,B), right side). Morphology was rod-shaped, hPSCs were exposed to CHIR99021 for 24 h in RPMI corresponding to ventricular cardiomyocytes, or round, medium with B27-insulin (Figure 1(A)) (Burridge et al. corresponding to atrial cardiomyocytes (Figure 3(C,D)). 2014; Fonoudi et al. 2015). Cells exposed to CHIR99021 Thus, we confirmed that maturation of cardiomyocytes exhibited a change in overall morphology (Figure 1(B)). can be manipulated by addition of diverse supplements. Inhibition of GSKα/β signaling also induced the acti- vation of the Tcf/Lef promoter, leading to genetically Structural and functional characterization of and consequentially mesoderm (Lian et al. 2012). After hPSC-derived cardiomyocytes culture for an additional 2 days in RPMI medium with B27-insulin to produce precardiac mesoderm, we To functionally characterize the cardiomyocytes selected exposed the cells to IWP4, an inhibitor of Wnt signaling. and matured in glucose-depleted and lactate-enriched ANIMAL CELLS AND SYSTEMS 85 Figure 1. Cardiomyocyte differentiated from hPSC via temporal regulation of Wnt signaling. (A) Schematic representation of cardiac differentiation from hPSC via regulation of temporal Wnt signaling. (B) Cell morphology for differentiation process by date was assessed by phase contrast imaging. (Scale bar, 200 μm). medium including ascorbic acid with albumin, we con- shown in Figure 5. Most cells exhibited ventricular-type ducted immunostaining for subunits of ion channels, APs in both the B27 and ascorbic acid with albumin which are critical for cardiac function (Figure 4(A)). On groups; the percentage of ventricular-type APs was day 30, this immunostaining revealed expression of higher in the ascorbic acid with albumin group. Nodal- Nav1.5 (a cardiac sodium channel) (Black and Waxman type APs were observed only in the B27 group, but the 2013), Cav1.2 (a cardiac calcium channel) (He et al. atrial-type APs were observed in both groups, with a 2013), Kv7.1 (a delayed rectifier potassium channel), higher percentage in the B27 group. Ventricular-type hERG (a cardiac potassium channel) (Aimond et al. 2005; cells could be distinguished by their more negative Abbott et al. 2007;Heet al. 2013), and Kir2.1 (a cardiac maximum diastolic membrane potential (MDP) and inward rectifying potassium channel) (Lange et al. 2003) rapid AP upstroke with long plateau phase. The in the cardiomyocytes. In addition, we assessed cardiac absence of a prominent plateau phase is a characteristic sarcomere organization by electron microscopy (Figure of atrial-type APs, resulting in shorter AP duration 4(B)). We observed myofibrillar bundles, transverse Z- (100 ms < AP duration at 90% repolarization (APD90) < bands, and enriched mitochondria, all of which are 250 ms) than in ventricular-type APs (250 ms < APD90). typical structural features of cardiomyocytes. Nodal-type APs had less negative MDP and slower AP upstroke. Table 1 compares the ventricular-type AP characteristics of hPSC-derived CMs in the B27 group Electrophysiological characterization of selected and ascorbic acid with albumin group, alongside data hPSC-derived cardiomyocytes for native human ventricular myocytes (hVMs) taken Using the patch-clamp technique, we measured spon- from the literature (Magyar et al. 2000). taneous APs in contracting hPSC-derived cardiomyo- Next, we analyzed MDP, maximum upstroke velocity cytes. A schematic overview of APs and representative (dV/dt ), AP amplitude (APA), and APD90 in ventricu- max traces recorded in hPSC-derived cardiomyocytes are lar-type APs of H9-derived cardiomyocytes from the 86 J.-E. KIM ET AL. Figure 2. Ascorbic acid and albumin promote cardiovascular differentiation. (A) Schematic representation of cardiovascular differen- tiation via selection and maturation with or without ascorbic acid and albumin. (B) Phase contrast images show cell morphology at days 20 and 30 of selection and maturation medium. (Upper panel: Scale bar, 100 μm and lower panel: Scale bar, 200 µm) (C) The percentage of positive cells for MLC-2v, a marker for ventricular specification, in population of cTnT-positive cells maturated by medium containing albumin and ascorbic acid is higher than that in population of cTnT-positive cells by medium containing B27. B27 group (n = 24 cells) and ascorbic acid with albumin three different formulations: albumin, ascorbic acid group (n = 16 cells). The MDPs of ventricular-type CMs with albumin, and ascorbic acid in RPMI1640 without in the ascorbic acid with albumin group were still glucose (Figure 6(A)). The percentage of MLC2v-positive depolarized relative to the native hVMs, but still more cells was highest in the ascorbic acid only group (Figure negative than in the B27 group. dV/dt was lower in 6(B), middle panel), whereas the percentage of cTnT- max the ascorbic acid with albumin group (94.2 ± 16.5 V/s) positive cells was highest in the albumin condition than in the native hVMs (215 ± 33 V/s), but much (Figure 6(B), left panel). Thus, ascorbic acid promotes higher than in the B27 group (14.9 ± 3.1 V/s). AP ampli- specification of ventricular cardiomyocytes to a greater tudes were higher in the ascorbic acid + albumin extent than albumin alone (Figure 6(B), middle and group (115.8 ± 1.8 mV) than in hVMs (106.7 ± 1.4 mV) right panels). In addition, ascorbic acid promotes specifi- or the B27 group (100.6 ± 1.1 mV). APD90 was compar- cation of MLC2v-positive cells (i.e. ventricular cardio- able to each other between the ascorbic acid with myocytes) among cTnT-positive cells. albumin group (302.5 ± 32.8 ms) and the B27 group (306.6 ± 20.6 ms), but slightly higher in native hVMs (351 ± 14 ms). Comparison of gene expression in cardiovascular differentiation via selection and maturation with or without ascorbic acid Ascorbic acid promotes differentiation of We then performed next-generation sequence (NGS) to ventricular cardiomyocytes compare gene expression among the albumin alone, We investigated whether ascorbic acid promotes differ- ascorbic acid with albumin, and ascorbic acid alone con- entiation of ventricular cardiomyocytes by examining ditions. We analyzed gene expressions of ANIMAL CELLS AND SYSTEMS 87 Figure 3. Quantitative analysis of hPSC-induced cardiomyocyte depending on the selection and maturation medium. (A and B) The differentiated cells were analyzed for MLC-2a and MLC-2v using flow cytometry at the indicated time points. The percentage of MLC2v-positive cells was higher in the presence of both ascorbic acid and albumin than B27 supplement alone. The percentage of MLC2v-positive cells was higher on day 60 than on day 40, whereas the proportion of MLC2a-positive cells decreased under both conditions. (C and D) The phase contrast images of the selected and matured cells depending on condition. Yellow arrow indi- cates the morphology of round shaped as atrial cardiomyocyte and red arrow indicates rod shaped as ventricular cardiomyocyte. cardiomyocytes on 30 days after differentiation. Among were down-regulated in the condition without ascorbic the conditions compared, 34 genes were up-regulated in acid. Also, 10 genes were up-regulated and 18 genes the albumin with ascorbic acid condition and 33 genes were down-regulated in the only ascorbic acid condition Figure 4. Structural and functional characterization of hPSC derived cardiomyocyte via Wnt signaling. (A) Immunostaining to various ion channels (Nav1.5, Cav 1.2, hERG, Kv7.1 and Kir2.1) reveals sarcomere organization. Nuclei and cTnT are shown in blue and green, respectively. (Scale bar, 200 μm) (B) An images of transmission electron microscopic shows myofibrils (red arrow) with Z-bands (black arrow) and mitochondria (blue arrow). (Scale bar, 0.5 μm). 88 J.-E. KIM ET AL. Figure 5. Action potentials (APs) of hPSC-derived cardiomyocytes (A) Representative images of three major types of AP traces observed in hPSC-derived cardiomyocytes. Nodal- (top), atrial- (middle), and ventricular-type (bottom) APs in hPSC-derived CMs. APs were recorded using intracellular sharp microelectrode recordings of single cells within monolayers. (B) Distribution of three different traces observed in B27 only (n = 24 cells) and ascorbic acid with albumin (n = 16 cells). (Figure 7(A–C)). This consequently yielded a list of up- In this study, we demonstrated differentiation of regulated genes in selection and maturation medium hPSC-derived cardiomyocyte via regulation of canonical containing albumin with ascorbic acid (Table 2). Wnt signaling without addition of growth factors. This protocol enabled hPSCs to differentiate into a popu- lation containing more than 70% cTnT-positive cells, Discussion indicating efficient and reproducible mesodermal induc- tion through activation of Wnt/β-catenin signaling via Cardiomyocytes derived from human pluripotent stem Gsk3 inhibition (in this case, CHIR99021) (Lian et al. cells are useful in vitro cardiac models for toxicity screen- 2012; Burridge et al. 2014). Canonical Wnt signaling, a ing and drug evaluations (Liang et al. 2013; Wang et al. major regulator of hPSC-derived cardiomyocytes, trig- 2014; Sharma et al. 2017). Accordingly, a reproducible, gers expression of developmental factors related to car- robust, and large-scale protocol for generating hPSC- diomyocyte differentiation (Hoppler et al. 2014). After derived cardiomyocytes is required for further develop- activation of Wnt signaling, inhibition of Wnt signaling ment of pharmacology and toxicology platforms. with IWP4 induced highly efficient cardiomyocyte differ- entiation (Sharma et al. 2015). We achieved a cardiomyo- Table 1. Action potential parameters in ventricular-type cyte yield of up to 70% when purification was conducted cardiomyocytes in native human, B27 + Lactate group and using lactate-enriched and glucose-starved conditions. albumin + ascorbic acid + lactate group. dV/dt Unlike selection by sorting, medium with lactate as the max Cells MDP (mV) (V/s) APA (mV) APD90 (ms) energy source allowed us to select cardiomyocyte in a Human VMs −81.8 ± 3.3 215 ± 33 106.7 ± 1.4 351 ± 14 simple and cost-efficient manner, taking advantage of (Magyar et al. the metabolic properties of these cells (Sharma et al. 2000) hPSC-derived −59.8 ± 0.8 14.9 ± 3.1 100.6 ± 1.1 306.6 ± 20.6 2015). Selection and maturation of cardiomyocytes was VM achieved using B27 or albumin with ascorbic acid with hPSC-derived −62.4 ± 2.4 94.2 ± 16.5 115.8 ± 1.8 302.5 ± 32.8 VM conversion of the energy source. The percentage of Note: The ventricular-type action potential parameters of native human in cTnT-positive cells was higher in medium containing literature and both of B27 + lactate group and albumin + ascorbic acid albumin with ascorbic acid than in medium including + lactate group are summarized (mean ± SEM). VMs, ventricular myo- cytes;MDP, maximum diastolic membrane potential; maximum upstroke B27 alone. In addition, the percentage of ventricular car- velocity (dV/dt );action potential amplitude (APA);APD , AP duration max 90 1 2 diomyocytes among the cTnT-positive population was at 90_ % repolarization. B27 + lactate group, albumin + ascorbic acid + lactate group. 3.3-fold higher in the albumin with ascorbic acid group ANIMAL CELLS AND SYSTEMS 89 Figure 6. Ascorbic acid promotes differentiation of ventricular cardiomyocyte. (A) Schematic representation of cardiovascular differ- entiation via selection and maturation with or without ascorbic acid. (B) The percentage of positive cells for MLC-2v, a marker for cardiomyocyte and ventricular specification, is higher in the culture using a medium containing only ascorbic acid is higher than in other cultures of albumin or both. Lactate is added into all culture types. Figure 7. Heat map representing differentially expressed genes in conditions for selection and maturation of hPSC-derived cardio- myocyte. Blue indicates up-regulated expression, and red indicates down-regulated expression. 90 J.-E. KIM ET AL. Table 2. Genes up-regulated in hPSC-derived cardiomyocytes pathway with a small molecule inhibitor and exposing cultured in selection and maturation medium containing cells to glucose starvation. hPSC-derived cells generated alubumin + ascorbic acid. with this technique expressed the specific markers of Genes Full name cardiomyocytes, as measured by FACS analysis, as well FHL2 Four and a half LIM domains 2 as other structural and functional properties of cardio- MYL2 myosin, light chain 2 DES desmin myocytes, including appropriate ion channel expression ANGPT1 angiopoietin 1 and typical cardiomyocyte organization. These hPSC- KIF1A kinesin family member 1A derived cardiomyocytes, selected and matured under NDRG1 N-myc downstream regulated 1 SMIM1 small integral membrane protein 1 conditions containing ascorbic acid, also exhibited APOA4 Apolipoprotein 4 normal electrophysiological properties, including NMRK2 nicotinamide riboside kinase 2 COX6A2 cytochrome c oxidase subunit VIa polypeptide 2 different proportions of ventricular-type and atrial-type RASL12 RAS-like, family 12 APs, but not nodal-type AP morphology. In an NGS C14orf180 chromosome 14 open reading frame 180 COL14A1 collagen, type XIV, alpha 1 analysis, we demonstrated that genes related to cardio- TBX3 T-box 3 vascular development and muscle system processes IGFBP1 insulin-like growth factor binding protein-1 were upregulated in conditions containing ascorbic Note: The selected cardiomyocytes expressed genes related to cardiovascu- lar development (ANGPT1, MYL2, COL14A, FHL2, SMIM1, IGFBP1 and TBX3) acid. This study not only demonstrates the role of and muscle system processes (DES, KIF1A, RASL12, C14orf180 and NMRK2). ascorbic acid in maturation and purification of hPSC- derived cardiomyocytes, but also provides tools for drug evaluation related to cardiotoxicity. than in the B27 group. Albumin helps cells to survive the process of selection and maturation (Francis 2010), and ascorbic acid promotes proliferation of CPCs (Takahashi Author contributions et al. 2003; Cao et al. 2012) and large-scale generation of cardiomyocytes (Takahashi et al. 2003). Maturation into E.-M.K., H.-A.L. and K.-S.K designed the experiments. J.-E.K., E.- an adult-like phenotype and specific cardiac subtypes M.K. and H.-A.L. conducted the experiments and wrote the paper. of hPSC-derived cardiomyocytes is necessary for toxicity evaluations using these cells (Yang et al. 2014). Ascorbic acid encourages the maturation of hPSC-derived cardio- Disclosure statement myocytes by promoting accumulation of ECM com- ponents (Baharvand et al. 2005). In addition, the No potential conflict of interest was reported by the author(s). albumin with ascorbic acid condition enriched the ven- tricular subtype of cardiomyocytes during long-term culture. Our selected cardiomyocytes expressed cardiac Funding ion channels and exhibited sarcomere organization, This research was funded by the Ministry of Trade, Industry and which are functional and structural features of mature Energy (20009748) and the Korea Institute of Toxicology, cardiomyocytes, and 94% of their APs were ventricular- Republic of Korea (1711159826), the National Research Foun- dation of Korea (NRF) grant funded by the Korea Government like. Under lactate-enriched conditions, specification of (MSIT) (NO. 2018R1D1A1B07045689) and Korea Environment the ventricular subtype was stronger in medium con- Industry and Technology Institute (KEITI) through the Environ- taining only ascorbic acid. Ventricular cardiomyocytes, ment Health Action Program, funded by Korea Ministry of which are the major source of cardiac contractile Environment (MOE) (2018001360003). forces, are important tools for in vitro assessment of drug toxicity (Cavero and Holzgrefe 2014; Chang et al. 2017). Accordingly, ascorbic acid promotes the specifica- ORCID tion of ventricular cardiomyocytes, as well as the pro- Ki-Suk Kim http://orcid.org/0000-0002-0931-0924 liferation of CPCs. Furthermore, selection and maturation medium containing ascorbic acid induced upregulation of genes related to cardiovascular develop- References ment and muscle system processes. Abbott GW, Xu X, Roepke TK. 2007. Impact of ancillary subunits on ventricular repolarization. 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