Human induced pluripotent stem cell- derived cardiac myocytes and sympathetic royalsocietypublishing.org/journal/rstb neurons in disease modelling Ni Li1,2, Michael Edel3,4, Kun Liu1, Chris Denning5, Jacob Betts1, Oliver C. Neely1, Dan Li1,† and David J. Paterson1,† Research 1 Burdon Sanderson Cardiac Science Centre and BHF Centre of Research Excellence, Department of Physiology, Cite this article: Li N, Edel M, Liu K, Denning Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK 2 C, Betts J, Neely OC, Li D, Paterson DJ. 2023 Nuffield Department of Medicine, Chinese Academy of Medical Sciences Oxford Institute, Oxford OX3 7BN, UK 3 Faculty of Medicine, Unit of Anatomy and Embryology, Autonomous University of Barcelona, Barcelona 08193, Human induced pluripotent stem cell-derived Spain cardiac myocytes and sympathetic neurons in 4 Discipline of Medical Sciences and Genetics, School of Biomedical Sciences, University of Western Australia, disease modelling. Phil. Trans. R. Soc. B 378: Perth 6009, Australia 5 20220173. Faculty of Medicine & Health Sciences, University of Nottingham Biodiscovery Institute, Nottingham NG7 2RD, UK https://doi.org/10.1098/rstb.2022.0173 NL, 0000-0001-5696-6187; DL, 0000-0003-2230-9300; DJP, 0000-0002-7399-3256 Received: 1 September 2022 Human induced pluripotent stem cells (hiPSC) offer an unprecedented Accepted: 21 December 2022 opportunity to generate model systems that facilitate a mechanistic under- standing of human disease. Current differentiation protocols are capable of generating cardiac myocytes (hiPSC-CM) and sympathetic neurons One contribution of 23 to a theme issue ‘The (hiPSC-SN). However, the ability of hiPSC-derived neurocardiac co-culture heartbeat: its molecular basis and physiological systems to replicate the human phenotype in disease modelling is still in mechanisms’. its infancy. Here, we adapted current methods for efficient and replicable induction of hiPSC-CM and hiPSC-SN. Expression of cell-type-specific pro- Subject Areas: teins were confirmed by flow cytometry and immunofluorescence staining. The utility of healthy hiPSC-CM was tested with pressor agents to develop a physiology model of cardiac hypertrophy. Treatment with angiotensin II (AngII) resulted in: (i) cell and nuclear enlargement, (ii) enhanced fetal gene Keywords: expression, and (iii) FRET-activated cAMP responses to adrenergic hiPSC, cardiac myocytes, sympathetic neurons, stimulation. AngII or KCl increased intracellular calcium transients in neurocardiac co-culture hiPSC-SN. Immunostaining in neurocardiac co-cultures demonstrated anatomical innervation to myocytes, where myocyte cytosolic cAMP responses were enhanced by forskolin compared with monocultures. In con- Authors for correspondence: clusion, human iPSC-derived cardiac myocytes and sympathetic neurons Dan Li replicated many features of the anatomy and ( patho)physiology of these e-mail: dan.li@dpag.ox.ac.uk cells, where co-culture preparations behaved in a manner that mimicked David J. Paterson key physiological responses seen in other mammalian systems. This article is part of the theme issue ‘The heartbeat: its molecular basis e-mail: david.paterson@dpag.ox.ac.uk and physiological mechanisms’. 1. Introduction Stem cell science gained momentum when James Thomson in 1998 [1] grew the first embryonic stem cells in a dish. This set the platform for Christine Mum- mery in 2001 [2] to use stem cells to create beating heart cells outside the body. However, it was the reprogramming of fibroblasts back to their pluripo- † tent state in 2006 by Takahashi & Yamanaka that resulted in human induced Joint senior authors. pluripotent stem cell technology transforming biology and providing an oppor- tunity to study human disease in vitro [3,4]. In heart physiology, the induction Electronic supplementary material is available of cardiomyocytes from human induced pluripotent stem cells (hiPSC) was online at https://doi.org/10.6084/m9.figshare. rapidly realized by applying the standard embryoid bodies-based protocol c.6476237. © 2023 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. for embryonic stem cells [5,6], which was optimized Bochum, Bad Oeynhausen, Germany, registration no. 41/2008. 2 over time by more precise control of the embryonic ReBl-PATRYR2-WT isolation and use of patient fibroblasts were approved by the Nottingham Research Ethics Committee (License royalsocietypublishing.org/journal/rstb development signal [7–10]. To date, the speed to generate hiPSC has been enhanced and genetic stability improved 09/H0408/74) and sample collections are registered with the UK Clinical Research Network under project 8164. hiPSC were ampli- using novel modified synthetic mRNA transfection methods fied in Matrigel-coated (Corning, 356234) cell culture flasks in [11,12]. mTeSR medium (STEMCELL Technologies, 85850), then were While primary cells from patients with genetic-based frozen at 2 × 106 cells ml−1 in Cryostor (STEMCELL Technologies, arrhythmias are largely unavailable, hiPSC has emerged 07959) in liquid nitrogen for long-term storage. Frozen hiPSC as a tool to model inherited cardiac syndromes [13–16]. were thawed and passaged three times for amplification and revi- Human iPSC-induced cardiomyocytes were first applied in talization before differentiation commenced. For protocol a mechanistic study of long-QT syndrome (LQTS) in 2010 reproducibility, both cardiomyocyte and sympathetic neuron [13], where patient-derived cells were capable of replicating induction were repeated more than three times on SFC854-03-02 the electrical phenotype of the disease. Given the power of and iPS-OX1-19 lines. the sympathetic nervous system to trigger arrhythmia in Phil. Trans. R. Soc. B 378: 20220173 patients with cardiac channelopathies [17,18], attention has recently focused on developing neuronal–cardiac co-culture (b) hiPSC-CM differentiation systems with either animal cells or stem cells in order to Thawed iPSCs were passaged three times then seeded on Matrigel- facilitate the study of neurocardiac interactions [19–25]. coated 12-well plates and cultured in mTeSR medium until 85–90% This has met with some success. Oh et al. [21] were first to confluent. On day 0, the medium was changed to RPMI (Gibco, 11875093)/B27-insulin (Gibco, A1895601) (RB–) with 6 µM report evidence for functional coupling, where noradrenaline- CHIR99021 (Tocris Bioscience, 4423) for 48 h of incubation, and secreting sympathetic neurons in co-culture were capable of exchanged for RB– on day 2. On day 3, 2.5 µM Wnt-C59 driving heart rate in neonatal cardiac myocytes when stimu- (Tocris Bioscience, 5148) was applied in RB– for 48 h. From day lated pharmacologically or optogenetically. Similarly, others 5, cells were fed with RB– medium, which was changed every [23,24] showed selective induction of healthy human sympath- 2 days. Purification was conducted by changing the medium to etic neurons could enable precise control of cardiac myocyte RPMI-glucose (Gibco, 11879020)/B27 (Gibco, 17504044) plus beating. Development of a diseased cardiac–neural co-culture 5 mM sodium L-lactate (Sigma, 71718) to remove the glucose system has not been firmly established, although Winbo et al. nutrient supplement from day 11 to day 14. After purification, [26] recently reported that sympathetic-derived neurons in hiPSC-CM were kept in RPMI/B27 medium (RB+) from day 15. monoculture from an LQT1 patient (KCNQ1 loss-of-function For experiments, cells were dissociated with TrypLE enzyme variants) showed increased excitability and neurotransmission. (Gibco, 12604013), and re-seeded on FluoroDish tissue culture dishes (WPI, FD35PDL) or coverslips on around day 20. Matu- Whether an hiPSC cardiac–neural co-culture system can also ration medium (DMEM low glucose (Sigma, D5546) plus 4% replicate and mimic cardiac–neural responses seen in other oleic acid–BSA (Sigma, O3008), 10 µg ml−1 insulin (Sigma, mammalian systems remains to be established. I9278), 2 mM glutamine, 10% horse serum) was applied prior to Here we adapted current protocols of cardiac myocytes experiments to enhance myocyte maturation when required and sympathetic neuronal differentiation from hiPSC and (figure 1a). conducted physiological phenotyping of these cells to confirm cell type and cell function. Simulation of a human disease was tested by exposure of hiPSC-cardiac myocytes (c) hiPSC-SN differentiation (hiPSC-CM) to pressor agents to induce hypertrophy. We Sympathetic neurons were differentiated from hiPSC according also developed hiPSC-sympathetic neurons (hiPSC-SN) in to the protocol proposed by Winbo et al. [24]. Briefly, after mono-culture and co-culture with hiPSC-CM, and assessed three passages, hiPSC were seeded on Matrigel-coated 12-well the utility of these cultures to respond to physiological stimuli. plates and cultured in mTeSR until 80% confluent. Then a series of chemical compounds were added sequentially (500 nM LDN193189 (Sigma, SML0559) days 0–3, 10 µM SB431542 (Sigma, S4317) days 0–4, 3 µM CHIR99021 + 10 µM DAPT 2. Methods (Sigma, D5942) + 0.2 µM PD173074 (Sigma, P2499) days 2–7, 60 ng ml−1 Shh C25II (Gibco, PMC8034) + 1 µM PMP (Sigma, (a) hiPSC culture SML0868) days 3–12, 10 ng ml−1 BMP4 (R&D Systems, 314-BP- The two healthy hiPSC lines (SFC854-03-02 and iPS-OX1-19) used 010) days 10–14) to the culture medium to modify pathways for cardiac myocyte differentiation in this study have been pub- involved in the induction of neuromesodermal progenitors and lished previously [27,28]. Lines were derived from dermal neural crest cells. Culture medium mTeSR was gradually replaced fibroblasts from healthy donors through StemBANCC (SFC854- by N2 medium (Neurobasal Plus medium (Gibco, A3582901) sup- 03-02) or the Oxford Parkinson’s Disease Centre (iPS-OX1-19): par- plemented with 2 mM L-glutamine, B-27 Plus (Gibco, A3582801), ticipants were recruited to this study having given signed N-2 supplements (Gibco, 11520536), 0.2 mM ascorbic acid (Sigma, informed consent, which included derivation of hiPSC lines from A8960), 0.2 nM dbcAMP (Sigma, D0260), 10 ng ml−1 NGF skin biopsies (Ethics Committee that specifically approved this (Bio-Techne, 256-GF-100), 10 ng ml−1 BDNF (R&D Systems, 248- part of the study: for control donors, National Health Service, BDB-010), 10 ng ml−1 GDNF (R&D Systems, 212-GD-010)) to Health Research Authority, NRES Committee South Central, promote sympathetic neuron development from day 4 (days 4–5: Berkshire, UK, REC 10/H0505/71); all experiments were per- 75% mTeSR + 25% N2 medium; days 6–7: 50% mTeSR + 50% N2 formed in accordance with UK guidelines and regulations and as medium; days 8–9: 25% mTeSR + 75% N2 medium; days 10–12: set out by the Research Ethics Committee (REC). Three cell lines, N2 medium). On day 12, differentiated cells were detached with iPS-OX1-19, UKKi007-A and ReBl-PATRYR2-WT, were used for sym- Accutase (Gibco, A1110501), re-seeded on 24-well plates at a den- pathetic neuron induction. UKKi007-A was provided by the sity of 106 cells per well and cultured in neuronal medium for 40 European Collection of Authenticated Cell Cultures, and ethically days for maturation, after which characterization and co-culture approved by the Ethics Committee of the Ruhr-University with iPSC-CM was carried out (figure 1b). (a) cardiac myocyte induction (b) sympathetic neuron induction 3 royalsocietypublishing.org/journal/rstb hiPSC seeding iPSC-SN differentiation iPSC-SN maturation hiPSC seeding hiPSC-CM differentiation + purification day –1 day 0 2 3 4 7 9 10 day 12 14 day 40+ day –1 day 0 day 3 day 11 day 15+ replate LDN medium mTeSR RPMI+B27(-insulin) RPMI(-Glu)+B27 RPMI+B27 SB m3i chemicals CHIR99021 Wnt-C59 sodium lactate SP BMP4 isolate hiPSC-CMs maturation medium neuronal medium to single cells mTeSR day 15+ day 20+ (c) hiPSC-CM (e) hiPSC-CM Q1 Q2 cTnT α-actinin α-actinin/cTnT/Hoechst 105 10.2 80.0 104 MLC2v-PE Phil. Trans. R. Soc. B 378: 20220173 103 0 Q4 Q3 –103 7.25 2.55 10 mm 10 mm 10 mm –103 0 103 104 105 TnT-APC (d) hiPSC-SN (f) hiPSC-SN TH PRPH PRPH/TH/Hoechst TH (–) TH (+) 100 15.7 84.3 80 count 60 40 20 20 mm 20 mm 20 mm 0 –103 0 103 104 105 TH Figure 1. Induction of cardiac myocytes and sympathetic neurons from human induced pluripotent stem cells. (a,b) Schematic summary of cardiac myocyte (a) and sympathetic neuron (b) induction protocols. (c,d) Flow cytometric results from day 20 cardiac myocytes (c) and day 50 sympathetic neurons (d). In (c) the right upper quadrant (Q2) shows positive population for ventricular myosin light chain 2 (MLC2v) and cardiac troponin (cTnT) in hiPSC-CM (from SFC854-03-02) of 80%. (d) Blue curve represents blank control and red curve represents hiPSC-SN (from UKKi007-A) stained with anti-tyrosine hydroxylase (anti-TH). Compared with negative control, 84.3% of hiPSC-SN expressed TH. (e,f) Struc- tural characterization of day 20 cardiac myocytes (e) and day 42 sympathetic neurons ( f ) by immunofluorescence staining. The alignment of cTnT and α-actinin shows sarcomere structure within cardiac myocytes (e). Anti-TH-stained neuron cell body and axon; peripherin (PRPH) staining outlines the axon extension and formation of varicosities ( f ). (d) Neurocardiac co-culture times. After each 20 min stirring, supernatants containing cardiac Contracting hiPSC-CM over day 20 and hiPSC-SN over day 40 myocytes were collected and centrifuged, and cell pellets were with abundant axon extensions were digested on the same day resuspended. After digestion, all cell suspensions were combined and reseeded together in Matrigel-coated culture plates for neu- to spin at 1250 r.p.m. (175g), 5 min, 4°C. Pellets containing cardi- rocardiac co-culture establishment. TrypLE enzyme was used for omyocytes were resuspended in culture medium (DMEM high hiPSC-CM dissociation by incubating for 7–10 min in a 37°C CO2 glucose (Invitrogen, 42430025) plus 17.5% M199 (Invitrogen, incubator. hiPSC-SN were dissociated by 20–40 min incubation 31150022), 10% horse serum, 5% newborn calf serum, 2 mM glu- in 1 mg ml−1 collagenase A (Roche, 10103586001) in Hanks’ tamine, 0.1% penicillin/streptomycin) and seeded on cell culture balanced salt solution. Detached cells were centrifuged and dishes for 1 h to get rid of fibroblasts and endothelial cells. Unat- resuspended in 1 : 1 RB+ plus N2 medium, and the seeding den- tached myocytes were collected from supernatant and plated on sity of myocytes to neurons was 1 : 1. The medium was changed appropriate cell culture dishes for further experiments. every other day. (f ) Immunofluorescence staining Induced cardiomyocytes and sympathetic neurons from hiPSC (e) Isolated rat ventricular myocytes were dissociated and seeded on FluoroDish tissue culture Animal use complied with the University of Oxford Local Ethical dishes at desirable density for subsequent confocal imaging. Guidelines and was in accordance with the Guide for the care and Two to five days after seeding, cells were fixed with 4% formal- use of laboratory animals [29] published by the US National Insti- dehyde, permeabilized and blocked in Tris-buffered saline (TBS) tutes of Health ( publication no. 85-23, revised 2011) and the plus 0.5% Triton and 6% donkey serum. hiPSC-CM were then Animals (Scientific Procedures) Act 1986 (United Kingdom). incubated in TBS with primary antibodies, anti-α-actinin Experiments were performed under British Home Office (Sigma, A7811) and anti-cardiac troponin T (anti-cTnT, Abcam, Project License P707EB251 (D.J.P.). Ventricles were removed ab45932), overnight at 4°C. For hiPSC-SN, anti-tyrosine from ca 1–3-day-old Wistar neonatal rats in accordance with hydroxylase (anti-TH, Sigma, T2928), anti-peripherin (anti- approved Schedule 1 of the Act, killing by dislocation of the PRPH, Sigma, AB1530) and anti-synapsin (anti-Syn, Abcam, neck, and digested by stirring for 20 min in collagenase (Roche, ab254349) were used. Then secondary antibodies conjugated to 10103586001) and pancreatin (Sigma, P1750) buffer at 37°C five Alexa Fluor (488 and 594) were applied at room temperature for 2 h. Cell nuclei were stained by Hoechst (Invitrogen, H3570) supplied in the proBNP kit) and stop solution sequentially. Absor- 4 for 10 min. Images were taken by Leica confocal microscope bance at 450 nm was measured by spectrophotometer (Thermo (Leica TCS SP5) and analysed with LAS X software. Multiskan GO). royalsocietypublishing.org/journal/rstb (g) Flow cytometry (k) Förster resonance energy transfer (FRET) imaging Differentiated cells were collected in single cell suspension Cardiomyocytes were transfected with adenoviral vectors and centrifuged. Pellets were resuspended and incubated with encoding the Förster resonance energy transfer (FRET) cAMP Live/Dead stain (ThermoFisher, 65-0865-14), then fixed with sensor Epac-SH187 by incubation overnight at 37°C, 5% CO2 prior 4% formaldehyde, permeabilized and blocked in blocking to the FRET measurements. Cytosolic cAMP response to different buffer, followed by incubation with anti-cTnT (MACS 130-120- stimuli in the transfected cells was recorded as cyan fluorescent 403) and anti-ventricular myosin light chain-2 (MLC2v, MACS protein (CFP)/yellow fluorescent protein (YFP) emission intensity 130-119-581) antibodies for hiPSC-CM, and anti-TH (Abcam, change. Cells were bathed in Tyrode solution (135 mM NaCl ab209921) for hiPSC-SN, at required concentrations. Dead cell (Sigma, S7653), 4.5 mM KCl (Sigma, P9541), 20 mM HEPES sample, unstained sample and compensation beads were pre- (Sigma, H3375), 11 mM glucose (Sigma, G7021), 1 mM MgCl2 pared as control groups. Cells were then resuspended in FACS Phil. Trans. R. Soc. B 378: 20220173 (SERVA, 39772.02), 2 mM CaCl2 (BioVision, B1010), titrated with buffer ( phosphate-buffered saline (without calcium, magnesium, NaOH to pH 7.4) to reach a stable baseline before isoprenaline Corning, 21-040-CV) + 2% fetal bovine serum (Gibco, 16140071) (Sigma, I6504), IBMX (non-selective phosphodiesterase inhibitor; + 0.02% NaN3 (Sigma, 71289)) for running in a flow machine (BD 3-isobutyl-1-methylxanthine, Sigma, I5879) and forskolin (FSK, Fortessa X20). Results were analysed by FlowJo software. adenylyl cyclase agonist; Cayman Chemical, 11018) were applied. Images were taken using a Nikon Eclipse Ti2 inverted microscope with a 40× oil immersion objective, connected to a Cairn Research (h) Cell size and nuclear size measurement dual OptoLED light source, and a Prime BSI photometric camera. Day 20 hiPSC-CM were re-plated on FluoroDish tissue culture Excitation wavelength was set at 430 nm, and emission was dishes, and treated with AngII (MedChem Express, HY- measured at 480 and 535 nm for CFP and YFP, respectively. 13948A) 100 nM, 1 μM for 48 h to induce cardiac hypertrophy Background fluorescence was subtracted from the recorded [30]. Then, control and hypertrophic myocytes were fixed and emission intensities, and FRET ratio was calculated as CFP over stained for cTnT and α-actinin. Images taken were analysed by YFP and normalized to the average FRET ratio at baseline for ImageJ software. Nuclear area was automatically selected using comparison. the ImageJ wand (tracing) tool after thresholding each Hoechst- stained image layer in ImageJ to identify pixels that shape the nuclear area. The selected area was entered into ROI Manager, (l) Calcium imaging where pixel size could be automatically measured. Pixel size Intracellular calcium concentration ([Ca2+]i) was measured in was converted to physical size in micrometres according to hiPSC-induced sympathetic neurons using Fura-2 AM live cell cal- image scale. cium indicator. Cells were loaded with 5 µM Fura-2 AM (Invitrogen, F1221) for 30 min at 37°C, then were washed three times prior to imaging. Throughout imaging, cells were bathed in (i) Reverse transcription-quantitative polymerase chain Tyrode solution at 36.5°C, supplemented with AngII (100 or reaction (RT-qPCR) 200 nM) at 2 min and 50 mM KCl at 5 min. Images were captured Total RNA from control and hypertrophic hiPSC-CM was at 340 and 380 nm excitation with a QIClick digital CCD extracted using the RNeasy mini kit (Qiagen, 74104) according camera (QImaging) connected to an OptoLED fluorescence ima- to the manufacturer’s instructions. Reverse transcription of ging system housed on a Nikon Eclipse Ti2 inverted microscope extracted RNA was performed using the iScript cDNA synthesis with a 40× oil immersion objective. Calcium concentration was kit (Bio-Rad, 1708891). Subsequent reverse transcription-quanti- measured as ratio of emission triggered by 340 nm over 380 nm tative polymerase chain reaction (RT-qPCR) was conducted with and expressed as fold change from the normalized baseline. 20 µl reaction mixture composed of 2 µl cDNA plus 7 µl RNase- free water, 1 µl TaqMan Gene Expression Assay (20X) (NPPB Hs00173590_m1; NPPA Hs00383230_g1; MYH6 Hs01101425_m1; (m) Statistical analysis MYH7 Hs01110632_m1) and 10 µl TaqMan Universal PCR All data were processed and analysed using Prism 9.0 software. Master Mix (Applied Biosystems, 4304437). Amplification was Differences between groups were assessed by t-test (two groups) conducted in QuantStudio 5 with thermal cycling parameters as and ANOVA (more than two groups); non-parametric t-test and follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C ANOVA were used for data that were not normally distributed. and 1 min at 60°C. Amplification curves were generated auto- Results are expressed as mean ± s.e. Significance is indicated as matically by the real-time PCR system. Relative quantification *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001, n.s., not significant. compared target gene expression between control and hyper- trophic groups using the comparative ΔCT method. GAPDH were used as internal control. 3. Results ( j) Pro-B-type natriuretic peptide (proBNP) secretion (a) hiPSC differentiation to cardiac myocytes and The proBNP ( precursor of active BNP that is secreted from cardiac sympathetic neurons myocytes, an indicator of ventricular injury) concentration in The induction procedures of cardiac myocytes and sympath- cardiomyocyte culture media was measured using the Human etic neurons from hiPSC are illustrated in figure 1a,b. proBNP ELISA kit (Invitrogen, EHPRONPPB) according to the manufacturer’s instructions. Cell culture media were Cardiomyocyte differentiation was adapted from a well-estab- collected from hypertrophic and control groups. Collected lished small-molecule-modulated differentiating approach samples were transferred to the supplied proBNP antibody- [8,31,32]. There have been several published methods reporting coated 96-well plates in duplicate, followed by adding of biotin, sympathetic neuronal differentiation from hiPSC [24,33–35]. streptavidin-HRP, TMB substrate (3,3’,5,5’-tetramethylbenzidine, Here we adapted the method reported by Winbo et al. [24]. Flow cytometry was used to validate induction efficiency Elevated cAMP level during beta-adrenergic stimulation is 5 of hiPSC-CM and hiPSC-SN. Day 20 hiPSC-CM were stained commonly associated with hypertrophied myocytes [36,37]. hiPSC-CM loaded with the cAMP FRET-sensor Epac-SH187 royalsocietypublishing.org/journal/rstb by anti-cTnT and anti-MLC2v. According to positive and negative signals set by compensation beads, and compared were sequentially stimulated by 0.3 nM isoprenaline, 100 µM with non-stained control, 80% of hiPSC-CM induced from IBMX and 10 µM FSK. Cytosol cAMP generation during SFC854-03-02 hiPSC expressed both cardiac-specific proteins beta-receptor activation was significantly higher in AngII (figure 1c). Day 42 hiPSC-SN (derived from UKKi007-A) (1 µM)-treated iPSC-CM (1.377 ± 0.021, n = 28 versus 1.607 ± was stained by anti-TH, with 84.3% of hiPSC-SN showing 0.051, n = 39, p < 0.001). The non-selective phosphodiesterase positive for TH expression compared with non-stained con- inhibitor IBMX further increased cytosolic cAMP levels. To trol sample (figure 1d ). demonstrate the dynamic operating range of the sensor, FSK Cell morphology was further characterized by immuno- (10 µM) was added to saturate cytosolic cAMP at the end of fluorescence staining. Anti-cTnT and anti-α-actinin were experiments. Both cAMP elevations were more prominent in used for hiPSC-CM staining. cTnT is part of the troponin hypertrophic myocytes (figure 2d,e; electronic supplementary complex that covers the actin thin filament, spanning along material, data S3). Similarly, the FRET response of hiPSC-CM Phil. Trans. R. Soc. B 378: 20220173 the longitudinal sarcomere, and α-actinin crosslinks the thin closely resembled the cAMP response of hypertrophied neo- filaments and serves as a binding point for thick filaments natal rat ventricular cardiomyocytes when stimulated with to the Z-disc. Staining of cTnT and α-actinin in hiPSC-CM isoprenaline, indicating a conserved phenotypic response colocalized and showed the structure of adjoining sarcomeres across model systems (figure 2d,e; electronic supplementary (figure 1e), establishing evidence to support the cells being material, data S3). cardiac myocytes. Anti-TH and anti-PRPH were used for hiPSC-SN staining. As a filamentous protein responsible for neurite elongation, PRPH labelled the axons of hiPSC-SN, (c) Functional connection between hiPSC-SN and hiPSC- and cell bodies were dominated by TH, confirming their CM in co-culture nature as sympathetic neurons (figure 1f ). To test whether myocytes and neurons differentiated from the same cell line could be a reliable in vitro model for the study of neurocardiac disease, we plated mature hiPSC-CM (b) Pathological hypertrophy modelling using hiPSC-CM and hiPSC-SN in co-culture to establish neurocardiac connec- To develop myocyte hypertrophy, healthy hiPSC-CM tivity (figure 3a). Re-plated hiPSC-SN extended axons toward (derived from SFC854-03-02 hiPSC) were exposed to AngII hiPSC-CM and formed varicosities in close relation to (100 nM and 1 µM) for 48 h. Myofibril structure of hyper- myocytes, which indicated the formation of neurocardiac trophic cardiac myocytes was distorted, as represented by junctions. Staining with cTnT and TH showed the co-exist- discrete immunofluorescence staining of α-actinin and cTnT ence and connection of cardiac myocytes and sympathetic (figure 2a). Myofilament rearrangement is an indicator of neurons in co-culture. Anti-synapsin staining showed the stress-induced pathological hypertrophy. To assess cell enlar- formation of varicosities around cardiac myocytes. Cardiac gement, cell size and nuclear size were measured by myocytes joined adjacent myocytes, forming clusters. immunostaining. Imaging fields for control and AngII-trea- Sympathetic neurons dispersed around cardiac myocytes ted myocytes were randomly selected, all cells and nuclei clusters and axons stretched out to form connections with with clear and complete borders in the view were used for myocytes and other neurons (figure 3b) in a similar manner surface area measurements to avoid selection bias. Cell sur- to early reports [35]. face area significantly increased from 606.3 ± 53.02 µm2 Prior to evaluation of sympathetic regulation of cardiac (control) to 1096 ± 79.89 µm2 (AngII 100 nM) and 964.7 ± myocytes in hiPSC-derived co-culture, functional tests were 161.0 µm2 (AngII 1 µM), p < 0.01. Nuclei were significantly first performed in hiPSC-SN monocultures. [Ca2+]i was enlarged, from 98.46 ± 5.879 µm2 (control) to 135.6 ± measured in hiPSC-SN derived from the healthy cell line 5.63 µm2 (AngII 100 nM) and 135.9 ± 11.0 µm2 (AngII 1 µM), ReBl-PATRYR2-WT using the calcium-sensitive fluorescent p < 0.01. This is consistent with activated biosynthesis in ratiometric dye Fura-2 AM. hiPSC-SN were bathed in the nucleus and enhanced protein synthesis in the cytosol Tyrode solution to reach a steady basal Ca2+ level, then (figure 2b; electronic supplementary material, data S1). Fetal stimulated by AngII (100 or 200 nM) followed by 50 mM gene reactivation is well documented in hypertrophic cardiac KCl. [Ca2+]i showed significant elevation from baseline level myocytes in the process of cardiac remodelling. Therefore, during the application of AngII (figure 3c; electronic sup- we performed qPCR to ascertain the RNA expression of BNP plementary material, data S4; p < 0.05) given the well- as one of these fetal genes in hiPSC-CM. BNP transcription in established presence of AT1 receptors on the neurons. 1 µM AngII-treated myocytes was significantly higher ( p < Depolarization of neurons with 50 mM KCl also induced 0.01) than in the untreated control group. Co-incubation an abrupt [Ca2+]i increase from 1.09 ± 0.01 to 1.38 ± 0.05, with the AngII receptor blocker losartan (20 µM) reversed the p < 0.0001 (in 100 nM AngII), and 1.03 ± 0.01 to 1.44 ± 0.04, increase in BNP expression (figure 2c; electronic supplemen- p < 0.0001 (in 200 nM AngII) (figure 3c; electronic supplemen- tary material, data S2). The proBNP secretion level measured tary material, data S4). These results are consistent with the by ELISA also showed a dose-dependent increase in cell cul- behaviour of neurons in response to receptor and voltage ture medium of hypertrophic hiPSC-CM (figure 2c; electronic activation in the regulation of [Ca2+]i. supplementary material, data S2). Reactivation of BNP Subsequently, we recorded cAMP FRET changes in expression was confirmed by iPS-OX1-19 hiPSC differentiated hiPSC-CM in monoculture and co-culture with hiPSC-SN in cardiomyocytes, along with another important cardiac response to two concentrations of FSK to verify the functional hypertrophic marker MYH7 (figure 2c; electronic supplemen- neurocardiac connection (figure 3d; electronic supplementary tary material, data S2). material, data S5 ). Original traces showed increased cAMP (a) human myocyte model (b) cell size measurement 6 AngII royalsocietypublishing.org/journal/rstb 48 h * * healthy hypertrophy? hiPSC-CM # ** 2500 250 D-actinin/cTnT/Hoechst D-actinin/cTnT/Hoechst nuclear surface area (µm2) cell surface area (µm2) 2000 200 1500 150 1000 100 500 50 0 0 5 Pm 5 Pm ng M M l A 0 nM M 10 l ro o ng ontr 0n 1µ 1µ nt AngII 1 PM co 10 control II II c ng II II ng A Phil. Trans. R. Soc. B 378: 20220173 A A * (c) * # 1.0 n.s. BNP ANP MYH6 MYH7 25 proBNP secretion (OD450 nm) 150 4 relative BNP gene expression 40 60 ** 0.8 * relative gene expression relative gene expression relative gene expression relative gene expression 20 30 3 0.6 100 40 15 0.4 20 2 10 50 20 5 0.2 10 1 0 0 0 0 0 0 control AngII control AngII control AngII control AngII M M rta I 1 M PM + l I l ro o 20 M A ontr 0n lo An 1 P 1P nt n P co 10 II c I ng ng sa gI II ng A A (d) 4 4 hiPSC-CM ** FRET ratio change (R/R0) 3.5 *** control FSK (10 µM) 3 3 cAMP CFP/YFP ratio (R/R0) 3.0 AngII 1 µM 2.5 IBMX (100 µM) 2 2 2.0 Iso (0.3 nM) 1.5 1 1 1.0 0 0 0.5 control AngII control AngII 0 2 min 1 µM 1 µM Iso peak IBMX plateau (e) Wistar neonatal CM 4 4 # * FRET ratio change (R/R0) 3.5 control FSK (10 µM) 3 3 cAMP CFP/YFP ratio (R/R0) 3.0 AngII 1 µM 2.5 IBMX (100 µM) 2 2 Iso (0.3 nM) 2.0 1.5 1 1 1.0 0 0 0.5 control AngII control AngII 0 2 min 1 µM 1 µM Iso peak IBMX plateau Figure 2. Hypertrophic phenotype induced by angiotensin II (AngII) in hiPSC-CM (from one differentiation batch of hiPSC SFC854-03-02) versus Wistar neonatal cardio- myocytes. (a) The cartoon shows the induction of cardiac hypertrophy by exposing healthy hiPSC-CM to AngII for 48 h. Staining of α-actinin and cTnT shows disruption of cardiac structural protein by 1 µM AngII. Sarcomeres were fragmented and not organized into parallel myofilament bundles. (b) Treatment with AngII resulted in significant increase of cell size (left) and nuclear size (right). (c) Brain natriuretic peptide (BNP) mRNA expression significantly increased in 1 µM AngII-treated myocytes. Pretreatment of myocytes with AngII receptor blocker losartan antagonized BNP enhancement. Quantitative ELISA in cell culture supernatant revealed a tendency for increased proBNP secretion in hypertrophic groups. RT-qPCR repeated with iPS-OX1-19 hiPSC-CM confirmed significant increase of BNP and MYH7 (for myosin heavy chain 7), and an increased tendency for ANP and MYH6. (d,e) Grouped data showing dynamic increases in cAMP in response to isoprenaline (Iso), IBMX and forskolin (FSK) in hiPSC-CM (d) and Wistar neonatal cardiomyocyte (e) models. One micromolar Ang II-treated group caused beta-adrenergic hyper-responsiveness and increased maximal cAMP level in both models. generation in hiPSC-CM in response to FSK. Cytosolic cAMP significantly higher in myocytes co-cultured with hiPSC-SN at baseline showed no significant difference between hiPSC- (n = 9) than monocultured myocytes alone (n = 19) (FSK CM in monoculture and co-culture. However, when stimu- 0.1 µM: 24.73 ± 3.49% versus 9.30 ± 0.53%, p < 0.0001; FSK lated by FSK (0.1 and 0.3 µM), the increase of cAMP was 0.3 µM: 38.87 ± 1.71% versus 21.33 ± 1.26%, p < 0.0001). The (a) (b) 7 cTnT/TH/Hoechst cTnT/Syn/Hoechst in vitro royalsocietypublishing.org/journal/rstb differentiation co-culture healthy CM healthy iPSC healthy SN 10 Pm 10 Pm hiPSC-CM/SN co-culture (c) hiPSC-SN AngII-induced [Ca2+] rise KCl-induced [Ca2+] rise 2.0 AngII 100 nM # # Phil. Trans. R. Soc. B 378: 20220173 AngII 200 nM KCl 4 # 2.5 # [Ca2+] ratio (R/R0) 1.5 AngII [Ca2+] ratio (R/R0) 2.0 [Ca2+] ratio (R/R0) 3 1.5 1.0 2 1.0 1 0.5 0.5 0 2 4 6 0 0 time (min) baseline peak baseline peak (d) (e) monoculture co-culture FSK 10 µM + 0.4 60 60 250 IBMX 100 PM # FRET change (% of max.) cAMP CFP/YFP ratio # cAMP FRET change (%) 200 0.3 40 40 150 0.2 100 FSK 0.3 PM 20 20 FSK 0.1 PM 0.1 50 0 0 0 0 5 10 15 20 25 30 basal FSK 0.1 PM FSK 0.3 PM –50 time (min) Figure 3. Functional phenotype of hiPSC-SN monoculture, hiPSC-CM monoculture and neurocardiac co-culture. (a) Cartoon showing cardiac myocytes and sym- pathetic neurons differentiated from a healthy hiPSC line and co-cultured together. (b) hiPSC-derived neurocardiac co-culture fixed and stained with cTnT (CM) and TH (SN) (left), cTnT (CM) and Syn (SN) (right). Overlay shows sympathetic neurons dispersed around cardiac myocyte clusters and extended axons towards myocytes, and dot-staining of anti-synapsin shows varicosities. (c) Representative normalized intracellular calcium ([Ca2+]i) trace of healthy hiPSC-SN monoculture (ReBl-PATRYR2-WT). [Ca2+]i level increased significantly at the application of 100 or 200 nM angiotensin II (AngII) and 50 mM KCl. (d) Original Förster resonance energy transfer (FRET) traces showing dynamics of cytosolic cAMP changes in response to adenylyl cyclase activator forskolin (FSK, 0.1 and 0.3 µM) in healthy 20-day iPSC-CM. Saturation of the sensor was achieved by using FSK (10 µM) and IBMX (100 µM). (e) Comparison of myocyte cytosolic cAMP changes between iPSC-CM monoculture and co-culture with iPSC-SN. Baseline cAMP level showed no significant difference between monoculture and co-culture, while co-culture with SN activated myocytes’ response to FSK with significantly higher increase of cytosolic cAMP. ((d,e) from cell line iPS-OX1-19.) greater cAMP response of the myocytes in the co-culture monoculture hiPSC-SN and hiPSC-CM, we established hiPSC (compared with myocyte monoculture) is probably a combi- neurocardiac co-cultures and demonstrated anatomical and nation of both FSK-activated myocyte cAMP, and the likely functional connectivity. FSK-activated cAMP in neurons enhancing transmission to further increase cAMP in myocytes via beta-receptor acti- vation of the second messenger pathway. (a) hiPSC cardiac myocyte differentiation and characterization In the past decades, many protocols have been established for the differentiation of cardiac myocytes from hiPSC [8,9,31,38]. 4. Discussion In this study, we adapted a widely used defined RPMI/B27 Here, we present a differentiation protocol for the induction of medium monolayer small-molecule-guided differentiation sympathetic neurons and for generation of cardiac myocytes protocol [10]. The Wnt signalling pathway has been shown from two human iPSC cell lines. Both myocytes and neurons to have a biphasic effect in directing cardiogenesis. Early expressed cell-type-specific structural proteins and membrane exposure to the GSK3 inhibitor CHIR99021 promotes the receptors that underpin specific physiological function in induction of mesoderm progenitor cells. Moreover, sub- response to stimuli. Derived myocytes were also capable sequent inhibition of canonical Wnt signalling by Wnt-C59 of replicating pathological hypertrophy when challenged results in further differentiation to cardiac fate [8]. Indeed, with AngII. Following calcium and cAMP phenotyping of the concentration of these small molecules influences the induction proficiency across different cell lines [8]. Here we neurotransmission [44]. Using live cell calcium imaging we 8 used 6 µM CHIR99021 for 48 h (days 0–2) and 2.5 µM observed that [Ca2+]i could be enhanced in response to AngII royalsocietypublishing.org/journal/rstb Wnt-C59 for 48 h (days 3–5), yielding ca 80% cTnT and and high extracellular potassium. AngII acts through AT1 MLC2v double-positive cardiac myocytes from two hiPSC receptors, which activate phospholipase C, which cleaves the lines. In addition, we found that hiPSC confluency when phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) commencing cardiomyocyte differentiation is critical in opti- into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate mizing cell yield. A high confluency of 90% in a 12-well plate (IP3). IP3 then diffuses in the cytosol and binds to IP3 receptors was optimal in generating cardiomyocytes with spontaneous on the endoplasmic reticulum, which in turn releases stored contraction from days 7–9 following induction. We also Ca2+ into the cytosol [45]. High potassium-induced depolariz- observed that derived myocytes were best purified by shift- ation activates plasma membrane voltage-gated calcium ing the nutritional supply from glucose to lactate using channels, which allows extracellular Ca2+ influx to activate RPMI-glucose medium supplemented with 5 mM sodium downstream signalling pathways involved in exocytosis of clas- lactate from day 11 to day 14. This helped minimize non-car- sical transmitters [46]. In particular, Winbo et al. [26] diac cell proliferation over cardiac myocyte maturation since demonstrated the utility of this neuronal monoculture system Phil. Trans. R. Soc. B 378: 20220173 other cell types required glucose for differentiation and are when sympathetic neurons derived from an LQT1 lineage intolerant to lactate metabolism, as reported by Gaspar had increased excitability and transmission, suggesting that a et al. [39]. Therefore, we conducted experiments at around component of the disease phenotype may also reside in the day 20 to avoid over-expansion of other cell types. For neuron [26]. When taken together with those of Winbo et al., longer cell maintenance, medium could be changed to low- our observations are consistent with the notion that our glucose maturation medium supplemented with fatty acids neuronal hiPSC are sympathetic in nature and can respond to to minimize non-cardiac cell enhancement [40]. classical physiological stimuli. Pressure and volume overload and inherited mutations are common causes of pathological cardiac hypertrophy. For in vitro modelling, isoprenaline, adrenaline or noradrenaline (c) Neural-cardiac signalling in co-culture mimics neurohormonal activation during pressure and stress Several groups have successfully coupled healthy iPSC sym- overload. Agonists for AngII type I receptors and endothelin- pathetic neurons and cardiac myocytes and shown the power 1 receptors are all effective inducers for cardiac hypertrophy of the neuron to drive cardiac excitability [21,23,24]. We have [41]. To characterize pathophysiological function of hiPSC- further demonstrated the utility of the hiPSC neurocardiac co- CM, these cells hypertrophied following incubation with culture system. First, both neurons and myocytes were able to 100 nM or 1 µM AngII for 48 h. We observed significant survive and thrive in co-culture. Secondly, immunostaining increases in cell size and nuclear enlargement with sarcomere confirmed the formation of neuronal varicosities adjacent to disarray. In addition, expression of the fetal gene BNP, which myocyte clusters, which is the anatomical basis for functional is an established marker of cardiac hypertrophy, was also elev- connectivity. Physiologically, the increased cytosolic cAMP ated. When stimulated by isoprenaline, cAMP levels were response to adenylyl cyclase activation was found to be significantly enhanced in hypertrophic hiPSC-CM, which indi- enhanced in myocytes co-cultured with neurons compared cates remodelling of beta-receptor-coupled cAMP signalling. with myocyte monocultures. The augmented enzyme func- This adrenergic hyper-responsiveness was also replicated tion could be a result of myocyte maturation when co- in neonatal Wistar rat ventricular myocytes, thus supporting culturing with neurons [25,47], where recent studies suggest the idea that the signalling pathway is conserved across secreted neurotrophic factors enhance the synaptic efficacy mammalian systems. of the co-culture [42,48,49]. Our co-culture responded in a dynamic fashion to activation of cAMP with FSK, resulting in a larger FRET signal in co-cultured myocytes compared (b) hiPSC sympathetic neurons with myocytes in monoculture. Here, FSK application to Coupling of sympathetic neurons to cardiac myocytes is an the co-culture would give rise to increased neurotrans- emerging field to study the role of neuronal-myocyte signal- mission, which would act on cardiac myocytes as an extra ling systems in a dish [22,42], although there are only a few stimulus to raise myocyte cAMP. studies that have investigated this coupling using human In conclusion, we have replicated various anatomical and iPSC cell models [21,23,24]. We differentiated sympathetic physiological characteristics of human cells by hiPSC-CM neurons from hiPSC based on the published technique from and hiPSC-SN. Whether these cell types have the Winbo et al. [24]. Since no standardized sympathetic neuronal full transcriptomic atlas of actual human cells remains to induction protocol is available to date, our data serve as be established. Nevertheless, the hiPSC monoculture and another validation for this feeder-free, chemical-directed co-culture preparations behave in a manner that mimics key induction process. We found that hiPSC-SN showed typical physiological responses seen in other mammalian cell round cell bodies with extensive axonal outgrowth types, and therefore may provide a framework for target (figures 1f and 3b). Staining of TH and PRPH confirmed discovery and a mechanistic understanding of human these neuron-like cells as catecholaminergic in nature, under- pathophysiological states. scoring their being sympathetic neurons. Their functional electrophysiological properties, including resting membrane Ethics. The two healthy hiPSC lines (SFC854-03-02 and iPS-OX1- potential and action potential kinetics, have been described 19) that were used for cardiac myocyte differentiation in this study have been published previously. Lines were derived from dermal previously [24]. fibroblasts from healthy donors through StemBANCC (SFC854-03- We further phenotyped these cells and tested their 02) or the Oxford Parkinson’s Disease Centre (iPS-OX1-19): partici- [Ca2+]i in response to cell stimulation, since Ca2+ is a crucial pants were recruited to this study having given signed informed messenger that underpins synaptic plasticity [43] and consent, which included derivation of hiPSC lines from skin biopsies (Ethics Committee that specifically approved this part of the study: Authors’ contributions. N.L.: data curation, formal analysis, writing— 9 for control donors, National Health Service, Health Research Auth- original draft; M.E.: methodology, writing—review and editing; ority, NRES Committee South Central, Berkshire, UK, REC 10/ K.L.: data curation; C.D.: resources, writing—review and editing; royalsocietypublishing.org/journal/rstb H0505/71); all experiments were performed in accordance with UK J.B.: data curation; O.C.N.: data curation; D.L.: data curation, project guidelines and regulations and as set out by the Research Ethics administration, supervision, writing—review and editing; D.J.P.: Committee (REC). Three cell lines, iPS-OX1-19, UKKi007-A and funding acquisition, project administration, resources, writing— ReBl-PATRYR2-WT, were used for sympathetic neuron induction. review and editing. UKKi007-A was provided by the European Collection of Authenti- All authors gave final approval for publication and agreed to be cated Cell Cultures, ethically approved by Ethics Committee of the held accountable for the work performed herein. Ruhr-University Bochum, Bad Oeynhausen, Germany, registration Conflict of interest declaration. We declare we have no competing interests. no. 41/2008. ReBl-PATRYR2-WT isolation and use of patient fibroblasts Funding. N.L. was supported by the China Scholarship Council, were approved by the Nottingham Research Ethics Committee and the Chinese Academy of Medical Sciences and Peking Union (License 09/H0408/74) and sample collections are registered with Medical College (CAMS/PUMC) Innovation Fund for Medical the UK Clinical Research Network under project 8164. Science (CIFMS). We acknowledge the British Heart Foundation Data accessibility. The datasets generated during and/or analysed during (grant no. 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