Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model

Reagents for hiPSC culture and differentiation

hiPSC culture

Induced pluripotent stem cells Kolfc2 (WTSIi018-B) were maintained on six-well plates coated with Geltrex (1:100; for details of reagents and suppliers see Table 1) according to the manufacturer’s protocol. Cells were cultured in mTeSR Plus media at 37 °C and 5 % CO₂ and passaged at 70-80 % confluency. Cells were passaged by removing the media in the well, washing once with PBS and adding TrypLE Express Enzyme (1X), phenol red for 3 minutes at 37°C. The TrypLE Express was removed and the cells were gently washed off the surface of the well with 1 mL of warm mTeSR Plus medium containing 10 μM Rock Inhibitor (Y-27632). Cells were passaged according to a seeding density of 20,000 cells per cm² and cultured in 10 μM Rock Inhibitor (Y-27632) mTeSR Plus media for the first 24 hours before switching to mTeSR Plus. The Rho-kinase inhibitor (Rock Inhibitor), Y-27632 was added to prevent dissociation-induced apoptosis. It should be removed 24 hours after the passaging of the cells to maintain iPSC pluripotency.

hiPSC-CM differentiation

The method for differentiation of hiPSC-CM was broadly adapted from the protocol outlined in Smith et al. (2018) (Figure 1). hiPSCs were seeded onto six-well plates coated with Geltrex and cultured in 2 mL per well of mTeSR Plus. The medium was changed on the cells every 48 hours until the cells reached 60 % confluency. The medium was then changed for 24 hours with StemPro-34 SFM (1X) supplemented with 2 mM L-Glutamine, 1 ng/mL Recombinant Human BMP-4 Protein and 1:100 Geltrex. The cells were subsequently changed with StemPro medium supplemented with 10 ng/mL BMP-4, 8 ng/mL Activin A and 2 mM L-Glutamine and incubated for 48 hours (day 0). The medium was changed with RPMI 1640 Medium with B-27 Supplement, minus insulin, 10 μM XAV 939 and 10 μM KY 02111 for 48 hours (day 2). The cells were subsequently changed with RPMI 1640 Medium with B-27 Supplement (50X), 10 μM XAV 939 and 10 μM KY02111 for 48 hours (day 4). The medium on the cells was changed with RPMI 1640 Medium with B-27 Supplement (50X) every other day. Atrial differentiation was achieved through the addition of 1 μM retinoic acid to the media on days 4 and 6. At 12 days after the initiation of differentiation, glucose starvation was performed to purify the population of cells for cardiomyocytes. This was achieved by changing the medium of the cells with RPMI 1640 Medium supplemented 1:50 with no glucose with B-27 Supplement (50X) for 48 hours. Cells were dissociated at day 15 for incorporation into EHT models. This media was removed from the well(s), the well was washed once with 1 mL of PBS and the cells were incubated in 2 mL of TrypLE Select 10× for 30 minutes at 37 °C. The cells were then washed from the surface of the plate with pre-warmed DMEM-F12.

Figure 1. Differentiation of hiPSC-CM.

Schematic representation of the hiPSC-CM differentiation protocol. Mesodermal specification is achieved through the activation of the Wnt signalling pathway using the recombinant proteins: Bone morphogenic protein- 4 (BMP-4) and Activin A. Subsequent inactivation of the Wnt signalling pathway by small molecules XAV 939 and KY02111 enables the differentiation of cardiac progenitor cells into induced pluripotent stem cell derived cardiomyocytes. Differentiation can be driven towards atrial cardiomyocyte specification through the addition of retinoic acid on days 4 and 6 only. The medium on the cells was changed every 48 hours after day 6 with RPMI 1640 with B27 + Insulin. Selection is achieved from days 12-14 by culturing the cells in RPMI 1640 No Glucose with B27 + insulin for 48 hours. Created with Biorender.com.

hiPSC-CF differentiation

The method for the differentiation of quiescent induced pluripotent stem cell-derived cardiac fibroblasts was broadly adapted from the protocol outlined in Zhang et al. (2019) (Figure 2). Induced pluripotent stem cells (Kolfc2/WTSIi018-B) were seeded at 30,000 cells/cm² in a T25 flask pre-coated with 1:100 Geltrex for 2 hours at 37 °C, in 10 μM Rock Inhibitor (Y-27632) mTeSR Plus medium. The cells were cultured for 48 hours prior to the medium being changed with RPMI 1640 supplemented with B27 minus insulin and 6 μM CHIR-99021 for 48 hours. The medium was then changed with RPMI 1640 supplemented with B27 minus insulin for 24 hours. The cells were subsequently treated with a RPMI 1640 with B27 minus insulin and 5 μM IWR1 for 48 hours. The cells were dissociated by washing with 3 mL PBS and then treating with 5 mL accutase enzyme for 3 minutes at 37 °C. The cells were removed from the flask, diluted with 5 mL pre-warmed RPMI 1640 and spun down at 200 g for 3 minutes. The cells were resuspended in Advanced DMEM/F-12 supplemented with 5 μM CHIR-99021 and 2 μM Retinoic Acid and seeded at 20,000 cells/cm² in a Geltrex coated T25 flask. The cells were incubated in this medium for 3 days and then subsequently changed to Advanced DMEM/F-12 for a further 4 days. The cells were passaged and plated into a Geltrex coated T25 flask at a density of 20,000 cells/cm² in Fibroblast Growth Medium 3 supplemented with 10 ng/mL FGF2 and 10 μM of the TGF-β1 inhibitor SB431542. The cells were cultured in this media for 6 days, with media changes taking place every 48 hours. Following the 6 days, the cells can be assessed for purity using immunocytochemistry. It is recommended that the cells are expanded, passaged at 70-80 % confluency for 2 passages and cryopreserved for use in future experiments in serum free freezing medium. Cells were passaged by washing once with PBS, and then incubating in TrypLE™ Express for 3 minutes at 37 °C. Cells can be dislodged from the surface of the flask by gently tapping the sides of the container. Following the detachment of the cells, a 2 × volume of DMEM/F12 was added to dilute the TrypLE. The TrypLE/cell mix was centrifuged at 300 g for 3 minutes. The cells were resuspended in Fibroblast Growth Medium 3 supplemented with 10 ng/mL FGF2 and 10 μM SB431542 and seeded at a density of 10,000 cells/cm².

Figure 2. Differentiation of hiPSC-CF.

Schematic representation of the hiPSC-CF differentiation protocol. Mesodermal specification is achieved through the activation of the Wnt signalling pathway using CHIR-99021. Subsequent inactivation of the Wnt signalling pathway by the small molecule IWR1 allows the generation of cardiac progenitor cells. Culture in CHIR-99021 and retinoic acid drives the cells towards the derivation of cardiac fibroblasts. Cardiac fibroblasts are maintained in medium supplemented with FGF2 and the TGF-β1 inhibitor SB 431542 to prevent activation into myofibroblasts. Created with Biorender.com.

hiPSC-derived EHTs

Protocol for EHT construction

Here we describe a step-by-step protocol for the construction of four EHTs (one strip) containing iPSC-CF. The reagents required are listed in Table 2.

Preparation of reagents for EHT construction

Dissociation of hiPSC-CM

Dissociation of hiPSC-CF (performed during Step 2 of hiPSC-CM dissociation)

Combining cells and centrifugation

Making agarose moulds (performed during 10-minute centrifugation)

Preparing the EHT mix (See Table 3)

Casting EHTs

Removal and culture of EHTs

RT-qPCR

hiPSC-CF were cultured in T25 flasks for 72 hours in Fibroblast Growth Media 3 supplemented with 10 ng/mL FGF2 + 10 μM SB 431542 or 10 ng/mL FGF2 + 10 ng/mL TGF-β1 (R&D Systems, 240-B-002/CF). RNA extraction was performed using the Direct-zol RNA Miniprep Kit (Zymo Research, R2050) according to the manufacturer’s protocol. cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, 4368814) according to the manufacturer’s protocol. RT-qPCR was performed using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, 4444557) following the manufacturer’s protocol and on a QuantStudio 5 Real-Time PCR System, 384-well (ThermoFisher Scientific, A28140). RT-qPCR was performed using TaqMan Gene Expression Assay (FAM) (ThermoFisher Scientific, 4331182) for ACTA2 (Hs00426835_g1) and COL1A1 (Hs00164004_m1). All reactions were multiplexed and normalised to the housekeeping gene GAPDH using Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe, primer limited) (ThermoFisher Scientific, 4326317E). 10 ng of cDNA were used per reaction. Transcript levels were measured from three samples of iPSC-CF, with each sample being obtained from cells at different passages. Cycle threshold values were obtained from three technical replicates of each sample. Relative gene expression was calculated using the comparative cycle threshold method (Schmittgen and Livak, 2008).

Immunofluorescence

Induced pluripotent stem cell derived cardiac fibroblasts

hiPSC-CF were split into wells of a Geltrex-coated 24 well plate (10,000/well) and cultured for 72 hours in Fibroblast Growth Media 3 supplemented with 10 ng/mL FGF2 + 10 μM SB 431542 or 10 ng/mL FGF2 + 10 ng/mL TGF-β1. Cells were fixed for 14 minutes at room temperature using 4 % paraformaldehyde Solution (ThermoFisher Scientific, J19943.K2). Fixed cells were blocked for 1 hour in blocking buffer consisting of PBS, Fetal bovine serum (5 %), Bovine Serum Albumin (1 %) (Merck Millipore, A9418-10G) and Triton X-100 (0.5 %) (Merck Millipore, X100-100ML). Primary antibodies were added at dilutions listed in Table 4 in blocking buffer for 2 hours at room temperature. This was followed by 3 washes in blocking buffer and the addition of secondary antibodies (Table 4) for 1 hour in the dark at room temperature. DAPI (ThermoFisher Scientific, D1306) was added with the secondaries at 0.1 μg/mL to visualise nuclei. Samples were washed 3 times in blocking buffer and then once with PBS. Samples were imaged using a EVOS M5000 Imaging System (ThermoFisher Scientific, AMF5000).

Engineered heart tissues

EHTs constructed with and without the addition of hiPSC-CF were removed from posts after 15 days in culture (30 days post initiation of differentiation), washed three times in PBS and fixed overnight at 4 °C in 4 % paraformaldehyde solution. A scalpel was used to longitudinally section EHTs. Sectioned pieces of EHTs were blocked overnight in blocking buffer. Primary antibodies were added in blocking buffer, at the dilutions listed in Table 4, overnight at 4°C. Samples were washed three times in blocking buffer before the addition of secondary antibodies for 4 hours in the dark at room temperature. DAPI was added with the secondaries at 0.1 μg/mL to visualise nuclei. Tissue sections were placed onto coverslips (VWR, 631-0170) and mounted onto cover slides using Hydromount Histology Mounting Media (Scientific Laboratory Supplies, NAT1324). Images were acquired using a Zeiss LSM780 Confocal laser scanning microscope equipped with a C-Apochromat 63x/1.20 W objective.

Analysis of contractile function

EHTs constructed with and without hiPSC-CF were cultured for 15 days in EHT media (30 days after initiation of differentiation) prior to recording and analysis. 10 second videos of each EHT was recorded at 1080p/30 fps using an inverted camera with a macroscopic lens (3 EHTs with iPSC-CF, 3 EHTs without iPSC-CF). Videos were converted into tif files and analysed using the ImageJ Plugin TrackMate for displacement over time. All EHTs with contractile function were included in the analysis. Contractile function was defined as at least two macroscopically visible contractions within 15 seconds. All EHTs constructed with iPSC-CF demonstrated contractile function. Only three of the nine EHTs constructed (one of three batches, three EHTs per batch) without the addition of iPSC-CF showed contractile function. The analysis of contractile function was performed blinded to the presence or absence of hiPSC-CF. Outputs were semi-automated and included contraction duration, time to peak, relaxation time, total contraction amplitude and beat rate. Contraction amplitude/area (tensile strength) was calculated by dividing the total contraction amplitudes by the cross-sectional area of the EHTs. For converting displacement into force, elastic bending of the pillars was assumed with a measured Young’s modulus of 3.0 MPa, yielding a conversion factor of 0.26 mN/mm.

Generation of quiescent hiPSC-CF

Induced pluripotent stem cells were differentiated into cardiac fibroblasts using the protocol outlined in materials and methods. Following differentiation, the activation status and plasticity of the cells was assessed using immunofluorescence. The cells were cultured in the presence and absence of 10 ng/mL TGF-β1 for 72 hours. The cells were then fixed and stained for vimentin, αSMA and collagen 1 with the results shown in Figure 3A. Vimentin is a cardiac fibroblast marker irrespective of activation status whilst αSMA and collagen are predominantly expressed in activated or myofibroblasts. The hiPSC-CF cultured in the absence of TGF-β1 were positive for vimentin but negative for αSMA, indicating a quiescent cardiac fibroblast phenotype. The cells cultured without the TGF-β1 inhibitor, SB 431542, and in the presence of 10 ng/mL TGF-β1 were positive for vimentin and αSMA and showed greater expression of collagen. The gene expression of ACTA2 (the transcript which encodes αSMA) and COL1A1 (a transcript which encodes collagen) was assessed using RT-qPCR as markers for cardiac fibroblast activation. Expression analysis was performed on three biological samples, with three technical repeats performed for each sample. No data were excluded from the analysis. The results are shown in Figure 3B. The hiPSC-CF showed a three-fold increase in the mean expression of COL1A1 following TGF-β1 treatment (-TGF-β1: M = 1, SD = 0.21, +TGF-β1 M = 2.9, SD = 0.47). The mean expression of ACTA2 in the hiPSC-CF increased approximately five-fold following TGF-β1 treatment (TGF-β1: M = 1, SD = 0.13, +TGF-β1 M = 4.9, SD = 1.59).

Figure 3. Activation state of hiPSC-CF.

hiPSC-CF were cultured in the standard media (FGM3 + 10 ng/ml FGF2 + 10 μM SB 431542, ‘-TGF-β1’) and with 10 ng/mL TGF-β1 instead of the SB 431542 (‘+TGF-β1’) for 72 hours to assess activation status. A: The cells were fixed and stained for vimentin (top red), Collagen (bottom red) αSMA (green) and DAPI (blue, indicating nuclei). Scale bar = 150 microns. B: RNA was extracted, and RT-qPCR was performed on the cDNA for ACTA2 and COL1A1.

N = 3 (three biological samples with 3 technical replicates per sample are plotted).

Macroscopic EHT structure

A workflow delineating the generation of EHTs from hiPSCs is displayed in Figure 4. EHTs containing hiPSC-CM and hiPSC-CF started twitching approximately 48 hours after construction, with maximal beating capacity being reached after approximately 14 days in culture. Those generated in the absence of hiPSC-CF started twitching approximately 96 hours after construction and showed diminished contractile function throughout the duration of culture. EHTs constructed with and without hiPSC-CF demonstrated marked differences in shape and structure after 72 hours in culture as shown in Figure 5. EHTs constructed with hiPSC-CF showed tightening around the silicon posts whilst hiPSC-CM-only EHTs remained more block-like throughout time in culture.

Figure 4. Generation of engineered heart tissues (EHTs).

An outline of the workflow to generate of EHTs with and without the addition of hiPSC-CF. The EHT shown on the top right was generated without the addition of hiPSC-CF. The EHT shown on the bottom right was constructed with the addition of hiPSC-CF. Created with Biorender.com.

Figure 5. Morphology of EHTs 72 hours after construction.

EHTs were constructed with and without the addition of hiPSC-CF according to the protocol outlined above. A and B show side by side comparisons. C - Close-up of an EHT with hiPSC-CF and hiPSC-CM demonstrated compaction after 72 hours, which was not observed in EHTs generated from hiPSC-CMs alone (D). Scale bar = 4 mm.

Contractile analysis of EHTs

At day 30 after the initiation of differentiation (15 days in EHT form), 1080p/30 fps videos of EHTs with and without hiPSC-CF were analysed using the Open-source ImageJ plugin TrackMate (Figure 6). All EHTs which demonstrated contractility were included in the analysis (Section 3.8). EHTs with and without hiPSC-CF demonstrated similar contraction durations and relaxation times of approximately 400 and 240 ms respectively. The mean time to peak time of the EHTs containing hiPSC-CF was similar to that of their hiPSC-CM-only counterparts at approximately 170 ms. The average contraction amplitude of the EHTs containing hiPSC-CF was approximately 30 % greater than that of the EHTs consisting of hiPSC-CM only. The EHT containing the hiPSC-CFs demonstrated an approximate five-fold increase in force, normalised to the cross-sectional area. EHTs constructed with hiPSC-CFs showed a marked decrease in spontaneous beat rate.

Figure 6. Contractile analysis of EHTs.

EHTs constructed with and without the addition of hiPSC-CF were analysed to assess contractile activity. Frames of the videos of the EHTs were analysed using the ImageJ Plugin TrackMate for displacement over time, the box represents the analysis window where movement of the pillar was tracked (A). Contractile profiles were generated (a representative profile of an EHT with and without iPSC-CF is shown) (B) and used to ascertain Contractile Duration, Relaxation Time, Time to Peak, Contraction Amplitude, Contraction Amplitude/Area and Spontaneous Beat Rate (C). N = 3 EHTs. Scale bar = 4 mm.

Composition of hiPSC-derived EHT

EHTs consisting of hiPSC-CM and hiPSC-CM/hiPSC-CF were cultured for 15 days after construction (30 days after initiation of hiPSC-CM differentiation). The tissue was fixed and sectioned using a scalpel and then stained for DAPI, titin and vimentin to assess composition and alignment (Figures 7, 8). Vimentin is a key marker of cardiac fibroblasts whilst titin is an integral component of the sarcomeres of cardiomyocytes. The hiPSC-CM demonstrated longitudinal alignment in EHTs constructed with and without hiPSC-CF (Figure 7). Cardiac fibroblasts were present in EHTs constructed with hiPSC-CF and were absent in the hiPSC-CM only tissues (Figure 8).

Figure 7. Alignment of engineered heart tissues.

EHTs were constructed with and without iPSC-CFs. Tissues were fixed, sectioned and stained for titin (TTN) and DAPI. Scale bar = 20 microns.

Figure 8. Composition of engineered heart tissues.

EHTs were constructed with and without iPSC-CFs. Tissues were fixed, sectioned and stained for titin (TTN), vimentin and DAPI. Scale bar = 20 microns.