Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Cardiovascular diseases are the leading cause of mortality worldwide¹ and cardiomyopathy represents the third cause of cardiac-related deaths².Cardiomyopathy is a collective group of diseases that affect cardiac muscles. The recent developments of induced pluripotent stem (iPS) cells and the directed-differentiation of iPS cells toward cardiomyocytes (PSC-CMs) have opened the door for studying cardiomyocytes with patient genome as an in vitro model of cardiomyopathy. These cells can be used to understand the pathophysiology of cardiac diseases, to elucidate their molecular mechanisms, and to test different therapeutic candidates³. There is a tremendous amount of interest, thus, patient-derived iPS cells have been generated (e.g., hypertrophic cardiomyopathy [HCM]^4,5, arrhythmogenic right ventricular cardiomyopathy [ARVC]⁶, dilated cardiomyopathy [DCM]⁷, and mitochondrial-related cardiomyopathies^8,9). Because one of the characteristics of cardiomyopathy is the dysfunction and disruption of sarcomeres, a valid tool that uniformly measures sarcomere function is needed.

Sarcomere shortening is the most widely used technique to assess sarcomere function and the contractility of adult cardiomyocytes derived from animal models and humans. To perform sarcomere shortening, well-developed sarcomeres that are visible under phase-contrast are required. However, PSC-CMs cultured in vitro display underdeveloped and disorganized sarcomeres and, therefore, are unable to be used to properly measure sarcomere shortening¹⁰. This difficulty to properly assess the contractility of PSC-CMs hinders their usage as a platform to assess cardiac functions in vitro. To assess PSC-CMs contractility indirectly, atomic force microscopy, micro-post arrays, traction force microscopy, and impedance measurements have been used to measure the effects of the motion exerted by these cells on their surroundings^11,12,13. More sophisticated and less invasive video-microscopy recordings of actual cellular motion (e.g., SI8000 from SONY) can be used to alternatively assess their contractility, however, this method does not directly measure sarcomere motion or force generation kinetics¹⁴.

To directly measure sarcomere motion in PSC-CMs, new approaches, such as fluorescent-tagging to sarcomere protein, are emerging. For example, Lifeact is used to label filamentous actin (F-actin) to measure sarcomere motion^15,16. Genetically modified iPS cells are another option for tagging sarcomere proteins (e.g., α-actinin [ACTN2] and Myomesin-2 [MYOM2]) by fluorescent protein^17,18,19.

In this paper, we describe how to perform time-lapse imaging for measuring sarcomere shortening using Myom2-TagRFP (mouse embryonic stem [ES] cells) and ACTN2-mCherry (human iPS cells). We also show that a patterned culture facilitates sarcomere alignment. In addition, we describe an alternative method of sarcomere labeling, using adeno-associated viruses (AAVs), which can be widely applied to patient-derived iPS cells.

1. Differentiation of mouse pluripotent stem cells

1. Maintenance of mouse ES cells
   1. Maintenance medium: Mix 50 mL of fetal bovine serum (FBS), 5 mL of L-alanine-L-glutamine, 5 mL of non-essential amino acid (NEAA), 5 mL of 100 mM Sodium Pyruvate, and 909 μl of 55 mM 2-Mercaptoethanol with 450 mL of Glasgow Minimum Essential Medium (GMEM). Supplement Leukemia inhibitory factor (LIF), CHIR-99021, and PD0325901 at a final concentration of 1000 U/mL, 1 μM, and 3 μM, respectively. Sterilize the medium through a 0.22 μm filter.
   2. FBS medium: Mix 55 mL of FBS, 5.5 mL of L-alanine-L-glutamine, 5.5 mL of Sodium Pyruvate, and 5.5 mL of NEAA to 500 mL of Dulbecco's Modified Eagle Medium (DMEM) high glucose. Filtrate the medium through a 0.22 μm filter to sterilize.
   3. Culture SMM18 mouse ES cells in which TagRFP was knocked into the Myom2 locus on a gelatinized 6 cm dish in maintenance medium as previously described¹⁸. Passage every 2-3 days.
2. Preparation of serum-free differentiation (SFD) medium
   1. Basal SFD: Mix 250 mL of Ham's F-12, 750 mL of Iscove's Modified Dulbecco's Medium (IMDM), 10 mL of B27 supplement minus Vitamin A, 5 mL of N2 supplement, 10 mL of L-alanine-L-glutamine, 5 mL of 10% bovine serum albumin in phosphate-buffered saline (PBS), and 10 mL of penicillin and streptomycin (10,000 U/mL). Filter through 0.22 μm strainer to sterilize.
   2. Dissolve ascorbic acid at 5 mg/mL in distilled water and filter through 0.22 μm strainer to sterilize.
   3. Dilute 13 µL of 1-Thioglycerol to 1 mL of IMDM. Herein, refer to this diluted 1-Thioglycerol as MTG.
   4. Add 10 µL of ascorbic acid (5 mg/mL) and 3 µL of MTG to 1 mL of basal SFD on the day of use. Herein, refer to this mixture as complete SFD.
3. Day 0, embryoid body (EB) formation for differentiation
   1. Harvest SMM18 mouse ES cells with a recombinant trypsin-like protease (rTrypsin) and count the cells.
   2. Centrifuge 5 x 10⁵ cells at 300 x g for 3 min in 4 °C, resuspend in 10 mL of complete SFD, and seed into a 10-cm Petri dish. Culture the cells at 37 °C and 5% CO₂ for 50 h.
4. Differentiation day 2
   1. Add Activin A, human vascular endothelial growth factor (hVEGF), and bone morphogenetic protein 4 (BMP4) to complete SFD at a final concentration of 5 ng/mL, 5 ng/mL, and 1.9 ng/mL, respectively.
      ​NOTE: BMP4 concentrations may differ depending on the lot of BMP4. Test several concentrations in a small-scale trial prior to using a new lot and determine the best concentration for cardiac differentiation.
   2. Transfer EBs from a Petri dish into a 15 mL tube and centrifuge at 50-100 x g for 3 min at 4 °C.
   3. Meanwhile, add the medium prepared in step 1.4.1 to the Petri dish to protect the remaining EBs being dry.
   4. Aspirate the supernatant from the 15 mL tube, resuspend the EBs with the medium in the Petri dish, and transfer the EB solution back to the dish. Then, cultivate the EBs at 37 °C and 5% CO₂ for 46 h.
5. Differentiation day 4
   1. Gelatinize a 10 cm tissue culture-treated dish with 5 to 10 mL of 0.1% gelatin for at least 5 min. Aspirate gelatin right before seeding cells.
   2. Prepare medium: Mix basic fibroblast growth factor (bFGF), FGF10, and hVEGF to complete SFD at 5 ng/mL, 25 ng/mL, and 5 ng/mL final concentrations, respectively. For a 10 cm dish, prepare 10 mL.
   3. Transfer cells from the Petri dish to a 15-mL tube. Add 5 mL of PBS to the Petri dish, wash several times, and transfer to the 15-mL tube to collect the remaining cells. Centrifuge at 50-100 x g, 4 °C, 3 min.
   4. Aspirate supernatant, add 1 mL of rTrypsin, and incubate at 37 °C for 3 min.
   5. Vortex briefly to dissociate EBs, add 9 mL of 10% FBS medium, vortex again, and count the cells.
   6. Centrifuge 1.5 x 10⁷ cells at 300 x g, 4 °C for 3 min, resuspend with the media prepared in step 1.4.2, and seed into the gelatinized dish. Incubate at 37 °C and 5% CO₂ for 2 days.
      NOTE: By day 7 or 8, extensive beating of PSC-CMs can be observed.
6. Drug selection at differentiation days 7 and 9: Refeed the media with puromycin (2 μg/mL at the final concentration) to eliminate non-cardiomyocytes at day 7 and 9 of differentiation.
   ​NOTE: Parental line of SMM18 is syNP4 mouse ES cells, harboring NCX1 promoter-driven puromycin-resistant gene²⁰.
7. Day 10, replate for future experiments
   1. Coat a glass-bottom culture plate or a 35 mm imaging dish containing a polymer coverslip with 0.1% Gelatin. To enhance maturation, coat the dishes with laminin-511 E8 fragment (LN511-E8) at 1 μg/cm² for 30-60 min at room temperature¹⁸. To culture PSC-CMs in specific patterns of interest, please refer to steps 4 and 5 for preparing polydimethylsiloxane (PDMS) stamps.
   2. To harvest SMM18 PSC-CMs, wash the dish twice with PBS, apply 1 mL of rTrypsin, and incubate 3 min at 37 °C.
   3. Collect cells in 9 mL of 10% FBS medium, suspend, and count the cells. Plate the cells at 50,000-100,000 cells in one well of a 24-well plate or 250,000-500,000 cells in a 35 mm imaging dish.
   4. Centrifuge a sufficient number of cells (300 x g, 3 min) and resuspend the cells with complete SFD supplemented with FBS (final concentration at 10%).
   5. Incubate overnight and change the culture medium to complete SFD with puromycin.
   6. From day 14, change the culture medium two to three times a week with complete SFD until day 21-28, when Myom2-RFP becomes prominent. For AAV-based transduction of fluorescent-tagged sarcomere proteins, please refer to Step 3.

2. Differentiation of human pluripotent stem cells

1. Preparation of differentiation media
   1. RPMI+B27-Ins: mix 500 mL of RPMI 1640 medium, 10 mL of B27 minus insulin, and 5.25 mL of L-alanine-L-glutamine.
   2. RPMI+B27+Ins: mix 500 mL of RPMI, 10 mL of B27 supplement, and 5.25 mL of L-alanine-L-glutamine.
2. Maintenance of human iPS cells
   1. Passage human iPS cells twice a week with AK02N on LN511-E8 following previously published method with some modifications²¹.
   2. Harvest cells with a 3 min treatment of rTrypsin and collect into 10% FBS medium. Count cells and centrifuge at 300 x g for 3 min at 4 °C. Seed 75,000-125,000 cells in one well of 6 well plate with 2 mL of AK02N supplemented with LN511-E8 and Y27632 at the final concentration of 0.5 μg/mL (0.1 µg/cm²) and 10 μM, respectively.
   3. Incubate at 37 °C and 5% CO₂ and replace the medium the following day with 2 mL of AK02N without any supplement. Change media every two to three days and passage every three to four days.
3. Day -4: replate prior to differentiation
   1. Coat a 6 well plate with 0.5 μg/cm² of LN511-E8 diluted in PBS. Then, incubate for at least 30 min at 37 °C and 5% CO₂ or 1 h at room temperature. Aspirate coating solution right before seeding cells.
   2. Harvest human iPS cells with rTrypsin and count cells as in Step 2.2.2.
   3. Centrifuge 1.25 x 10⁵ cells for a well of a 6-well plate at 300 x g for 3 min at 4 °C and resuspend in 2 mL of AK02N supplemented with LN511-E8 (final concentration 0.5 μg/mL or 0.1 μg/cm²) and Y27632 (final concentration 10 μM) per well.
   4. Aspirate coating solution, seed resuspended cells into the coated plate, and incubate at 37 °C and 5% CO₂.
4. Days -3 and -1: replace medium with 2 mL of AK02N.
5. Day 0: Replace medium with 2 mL of RPMI+B27-Ins supplemented with CHIR99021 (final concentration 6 μM) per well to start differentiation.
6. Day 2: Replace medium with 2 mL of RPMI+B27-Ins with WntC59 (final concentration 2 μM) per well.
7. Day 4: Replace medium with 2 mL of RPMI+B27-Ins per well.
8. Day 7 and day 9: replace medium with 2 mL of RPMI+B27+Ins with puromycin (final concentration 10 μg/mL) per well to selectively culture PSC-CMs.
   ​NOTE: ACTN2-mCherry line, used in this study, has a cassette of internal ribosomal entry site (IRES), puromycin-resistant gene inserted to the 3′-untranslated region (UTR) of TNNT2 locus, and mCherry replacing the stop codon of ACTN2. To purify cardiomyocyte without knock-in, please refer to Steps 3 and 4.
9. Day 10: replate for future experiments
   1. Coat a 35 mm imaging dish with a polymer coverslip with 0.5-1 μg/cm² of LN511-E8 diluted in 0.1% Gelatin. Incubate 2-4 h at room temperature for long-term viability. To culture PSC-CMs in desired patterns, please refer to steps 4 and 5 for preparing PDMS stamps.
   2. To harvest human PSC-CMs, wash the dish twice with PBS, apply 1 mL of rTrypsin per well, and incubate 3 min at 37 °C.
   3. Collect cells in 4 mL of 10% FBS medium, suspend, and count the cells. Plate 250,000-500,000 cells per 35 mm imaging dish.
   4. Centrifuge a sufficient number of cells at 300 x g for 3 min at 4 °C, resuspend with RPMI+B27+Ins with puromycin (10 μg/mL), and plate on the coated 35 mm imaging dish.
   5. Incubate overnight. The next morning, replace the culture medium with RPMI+B27+Ins with puromycin (10 μg/mL).
   6. From day 14, change culture medium 2-3 times a week with RPMI+B27+Ins until day 21-28 for imaging. For AAV-based transduction of fluorescent-tagged sarcomere proteins, please refer to Step 3.

3. Fluorescent labeling of sarcomeres using adeno-associated viruses

1. Preparation before AAV production
   1. Maintain HEK293T cells in DMEM supplemented with FBS (final concentration 10%) on a 10 cm tissue culture plate. Passage cells three times a week.
   2. Prepare polyethylenimine (PEI) at 1 mg/mL. Mix 50 mg of polyethylenimine MAX 40000 and 40 mL of ultrapure water. Adjust pH to 7.0 using 1 N NaOH. Then, make the final volume to 50 mL with ultrapure water and filter through a 0.22 μm strainer.
   3. Prepare a shuttle vector with a sarcomere labeling gene (e.g., TCAP or PDLIM3 fused with a green fluorescent protein [GFP]), driven by a cardiomyocyte-specific promoter, such as cardiac troponin T (cTNT)²².
      ​NOTE: For this instance, we used monomeric enhanced GFP with mutations of V163A, S202T, L221V²³.
2. Day 0, passage HEK cells
   1. When cells reach confluency, passage 2.0 x 10⁷ HEK293T cells to a 15 cm tissue culture plate with 20 mL of DMEM with 10% FBS.
3. Day 1, transfection
   1. Mix 13.5 μg of the shuttle vector, 26 μg of pHelper (a vector coding E2A, E4, and VA of adenovirus), 16.5 μg of pRC6 (a vector coding AAV2 Rep and AAV6 Cap genes), and 1 mL of DMEM without sodium pyruvate (DMEM-Pyr).
   2. Mix 224 μL of PEI (1 mg/mL, prepared in step 3.1.2) and 776 μL of DMEM-Pyr.
   3. Mix and incubate the plasmid solution and the PEI solution at room temperature for 30 min.
   4. Add the plasmid/PEI solution to the HEK293T cells prepared in step 3.2.
4. Day 2, medium change
   1. At 24 h after transfection, change medium to DMEM-Pyr. Culture cells until harvesting AAV on day 7. AAV will be released into the culture media.
5. Day 7, AAV collection, concentration, and buffer substitution using minimal purification method²⁴
   1. Incubate a centrifugal ultrafiltration unit (100k molecular weight cut-off [MWCO]) with 5 mL of 1% BSA in PBS at room temperature for 15 min. Then, centrifuge the ultrafiltration unit at 500 x g for 2 min and aspirate both filtered and remaining solutions.
   2. Transfer medium from the 15 cm dish that produced AAV to a new 50mL conical tube and centrifuge (500 x g, 5 min). Filter the supernatant through a 0.45 μm syringe strainer to remove cell debris and apply suspension to the ultrafiltration unit.
   3. Centrifuge at 2000 x g for 90 min or until concentrating the culture supernatant 0.5 to 1 mL.
   4. Aspirate the filtrate and apply 15 mL of PBS to the ultrafiltration unit.
   5. Repeat centrifugation until the concentrate becomes 0.5-1 mL.
   6. Repeat 3.5.4 and 3.5.5.
   7. Transfer concentrated AAV to a new 1.5 mL tube and store at 4 °C or -20 °C.
      ​NOTE: AAV can be used in P1 facilities but follow local rules and regulations. AAV can be produced by conventional methods as well.
6. Calculation of AAV titer
   1. Mix 5 μL of AAV, 195 μL of DMEM-Pyr, and 10 U of benzonase and incubate at 37 °C for 1 h.
   2. Add 200 μL of proteinase K buffer (0.02 M Tris HCl and 1% SDS) and 5 μL of proteinase K (20 mg/mL) and incubate at 37 °C for 1 h.
   3. Carefully prepare 400 μL of a 25:24:1 Phenol/chloroform/isoamyl alcohol solution, vortex for 1 min, and centrifuge at 20,000 x g for 1 min.
   4. Transfer 200 μL of the aqueous phase to a new 1.5 mL tube, which will yield approximately half of the original AAV genomes.
   5. Add 1 μL of Glycogen (20 mg/mL) to 20 μL of 3 M CH₃COONa (pH 5.2) and vortex. Add 250 μL of 2-Propanol to 100 μL of 100% ethanol and vortex again.
   6. Incubate at -80 °C for 15 min. Then centrifuge at 20,000 x g for 30 min at 4 °C.
   7. Aspirate supernatant and add 70% ethanol to the tube. Then, centrifuge at 20,000 x g, 4 °C for 5 min.
   8. Aspirate supernatant and air dry until the pellet becomes clear.
   9. Add 200 μL of Tris-Ethylenediaminetetraacetic acid (TE; pH 8.0) to resolve the AAV genomes. Then, dilute the sample 100-fold with TE.
   10. Prepare a standard with pAAV-CMV-Vector at 6.5 ng/μL with TE to obtain 10⁹ vector genomes (vg)/ μL. Then, make a series of 10-fold dilution from 10⁴ to 10⁸ with TE.
   11. Mix 1 μL of sample DNA (or the standards), 0.4 μL of primers (5 μM), 3.6 μL of distilled water, and 5 μL of SYBR Green master mix. Primers, located on ITR, are 5′-GGAACCCCTAGTGATGGAGTT-3′ and 5′-CGGCCTCAGTGAGCGA-3′.
   12. Perform real-time PCR with the following condition: Initial denature at 95 °C for 60 s, 40 cycles of denaturing at 95 °C for 15 s, and annealing and extension at 60 °C for 30 s, followed by melting curve.
   13. Based on the standards and Ct values, a real-time PCR machine provides the copy number of vector genome in 1 μL of a sample. Calculate original AAV titer using the following equation: a copy number provided by real-time PCR (vg/μL) x 8 x 10³ x 2, wherein 8 x 10³ as a dilution factor during AAV genome isolation, and 2 as the difference factor of AAV (single strand) and plasmid (double strand).
7. Transduction to PSC-CMs
   1. Count PSC-CMs in an extra well or extra dish.
   2. Dilute AAVs (1 x 10⁴ to 1 x 10⁶ vg/cell) to make up 50 μL with PBS. Apply AAVs at the multiplicity of infection (MOI) of 1 x 10⁴ to 1 x 10⁶ vg/cell to PSC-CMs and culture PSC-CMs for 3 days with AAV in the corresponding differentiation media for mouse and human PSC-CMs, then change media to culture medium without AAV.
   3. Use PSC-CMs for live-cell imaging after 7 days or more post-transduction.

4. [Optional] AAV-based purification of PSC-CMs

1. Preparation of AAV
   1. Prepare AAV as described in Step 3 using a shuttle vector expressing blasticidin-resistant gene under the control of cTNT promoter.
2. Transduction to differentiating iPS cells
   1. Differentiate human iPS cells for 4 days following the protocol described in Step 2 and count the number of cells in an extra well.
   2. After changing medium at day 4, apply AAVs at the MOI of 1 x 10⁵ vg/cell to differentiating PSCs in RPMI+B27-Ins media.
   3. At day 7, refresh medium with RPMI+B27+Ins and add 2.5-10 μg/mL of blasticidin.
   4. At day 10, PSC-CMs are ready to replate.

5. Preparation of PDMS stamps

1. Design the device pattern of 200 μm strips along with 10-25 μm grooves in between the strips using a computer-aided design (CAD) drawing software.
2. Draw the pattern of devices onto a chromium photomask coated with AZP1350 using UV light of a maskless lithography tool.
3. Develop the pattern on the photomask in a series of positive photoresist developer (e.g., chromium etchant) and rinse with DI water.
4. Dehydrate a silicon wafer on a hot plate at 120 °C for 15 min.
5. Allow the wafer to cool to room temperature and spin-coat a negative photoresist SU-8 3010 to make a height of 10-20 μm with 1500 rpm for 30 s using a spin-coater.
6. Soft bake the wafer in two steps on a hot plate according to the manufacturer's protocol.
7. After the wafer cools to room temperature, load the wafer onto the mask aligner.
8. Use a mask aligner to align the mask on the wafer and expose the wafer to UV light.
9. Conduct the post-exposure bake to the wafer in two steps on a hot plate according to the manufacturer's protocol.
10. Develop the wafer in a series of SU-8 developer and 2-Propanol, then dry the wafer with a nitrogen stream.
11. Transfer the wafer into a Petri dish of a suitable size.
12. Mix PDMS elastomer and its curing agent in a ratio 10:1 w/w and pour the mixture into the Petri dish.
13. Degas the PDMS in a desiccator until all air bubbles disappear, then cure PDMS on hot plate at 80 °C for 2 h.
14. Peel the cured PDMS off from the master mold using a tweezer, then cut out the portion with the design to be a PDMS stamp.
    ​NOTE: Shape can be square, however, an octagonal shape transfers the pattern better at the edge.

6. Patterned culture of pluripotent stem cell-derived cardiomyocytes

1. Remove dust from the surface of PDMS stamps using mending tape.
2. Submerge the stamps into 70% ethanol to sterilize. Then, blow ethanol off the surface of the stamps using an air duster.
3. Apply 5-10 μL of 0.5 wt% 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer/ethanol on the surface of PDMS stamps.
   NOTE: Uneven distribution of MPC polymer may cause a disrupted pattern.
4. Incubate 10-30 min until MPC polymer is completely dried.
5. Place the stamps in contact with coverslips of a glass-bottom culture plate or a 35 mm imaging dish with a polymer coverslip and put a weight (e.g., a AAA battery) on the stamp for 10 min.
6. Remove the weight and stamps. Then, confirm that the pattern has been transferred under microscope.
   NOTE: Stamped plates/dishes can be stored up to 1 week at room temperature.
7. Wash the stamped wells/dishes with PBS two times.
8. Dilute LN511-E8 with PBS at 2-4 μg/mL and coat the dish with LN511-E8 at 0.5-1 μg/cm². For human PSC-CMs, dilute LN511-E8 with 0.1% gelatin solution instead of PBS. Then, incubate for at least 1 h (optimally, more than 4 h).
9. Plate cells as described in previous sections.

7. Time-lapse imaging of sarcomeres under fluorescent microscope

1. Turn on and connect the microscope, associated computer, and also all of the required peripherals.
2. To perform time-lapse imaging, capture time-lapse images with the highest magnification (100X objective lens with oil emersion).
3. Select live-imaging conditions. To obtain good representative data, try to adjust to the highest framerate (minimum of 20 ms or 50 frames per second is recommended). Set the shutter open and apply a necessary binning (4 X 4) and a crop of the acquisition area to achieve the shortest intervals between images during the time-lapse imaging.
   NOTE: The setting may vary depending on the configurations of microscopes. The camera needs to be high-sensitivity and is able to transfer the data to the connected PC fast enough. To this end, we used ORCA flash with Camera-link. We have tested a spinning confocal microscopy and have acquired images at 400 frames per second.
   1. [Optional] If the beating rate of the cells is low, evoke the cells by electrical field stimulation.
4. Run the time-lapse record
   1. Ensure that the imaged fields remain in focus during recording the time-lapse image.
   2. Save the time-lapse images into an appropriate folder.

8. Analysis of time-lapse imaging using SarcOptiM, an ImageJ/Fiji plugin

1. Load a series of time-lapse images into ImageJ. For Olympus VSI format, open files through OlympusViewer Plugin.
2. Adjust brightness and contrast of the image to observe the sarcomere pattern clearly (Image | Adjust | Brightness/Contrast).
3. Open SarcOptiM by clicking More tools menu (>>) and selecting SarcOptiM.
4. Calibrate the program by pressing Ctrl + Shift + P and 1 μm button on the toolbar and following the instructions of the dialog boxes.
5. Draw a line across the region of the sarcomere that will be used to measure the sarcomere shortening.
6. Start sarcomere shortening analysis by pressing SingleCell (AVI) on the toolbar. Representative data is shown in Figure 1 and Figure 2.

Measuring sarcomere shortening using knock-in PSC-CMs reporter lines. Sarcomere-labeled PSC-CMs were used to measure sarcomere shortening. The lines express Myom2-RFP and ACTN2-mCherry from endogenous loci. TagRFP was inserted to Myom2, coding M-proteins that localize to the M-line, while mCherry was knocked-in to ACTN2, coding α-Actinin, which localizes to the Z-line^18,25. Time-lapse images were obtained and used to determine sarcomere shortening as presented in Figures 1 and 2 and Movie 1-3.

To overcome the disorganized sarcomere of PSC-CMs, specific PDMS stamps were used to culture PSC-CMs in the stripe pattern. This patterned culture promoted an elongated cell shape and a more organized sarcomere pattern compared to cells cultured in the non-pattern area (Figures 2B and 2C). With this advantage, the patterned culture promoted better contraction of the cells and provided a smooth sarcomere length profile as shown in Movies 2 and 3 and Figure 2D.

Fluorescent tagging of Z-line protein using AAV vectors. To visualize the Z-line of PSC-CMs without generating knock-in iPS cells, fluorescent-tagged Z-line proteins were expressed using AAV transduction. Two of small Z-line proteins, Telethonin (TCAP) and Actin-associated LIM protein (PDLIM3) with GFP, were tagged and packaged using the AAV6 capsid (Figure 3A). Once PSC-CMs were differentiated and purified, AAVs were transduced to PSC-CMs (Figure 3B). The transduced PSC-CMs expressed sarcomeric GFP signals along the PSC-CMs as early as three days post-transduction (Figures 3C and 3D). Typically, the transduction of AAV at an MOI of 10⁵ vg/cell is sufficient to visualize fluorescent-tagged sarcomere proteins and a higher titer may cause non-specific localization of GFP to cytoplasm though it increases overall GFP intensity.

Purification of PSC-CMs using AAV vectors. Current methods rely on the drug selection cassette that is already on the genome of PSC-CMs, either transgenic or knock-in line. However, it is labor-intensive to produce such a line from patient-derived iPS cells. As AAV vectors have been demonstrated to drive the expression of fluorescent-labeled Z-line proteins without the need for knock-in, we sought to establish the purification method without knock-in as well (Figure 4). To this end, a new AAV vector, which encodes blasticidin-resistant gene under the control of cTNT promoter, was constructed (Figure 4A). The AAV (MOI of 10⁵ vg/cell) was transduced to differentiating human iPS cells at day 4. Then cells were treated with 2.5-10 μg/mL of blasticidin (need to titrate for each cell line) between days 7 and 9 (Figure 4B). At day 14, the purity of PSC-CMs was more than 90% (Figure 4C).

Figure 1: Sarcomere shortening of the mouse PSC-CMs derived from the Myom2-TagRFP cell line. A. The timeline for mouse PSC-CM differentiation. B. Representative images for sarcomere shortening in different time points with measuring regions as indicated by yellow bars. Scale bar = 10 μm. C. Sarcomere length profile during contraction of the cardiomyocytes that was stimulated with electricity at 1 Hz. The framerate was 50 frames per second. The pixel size was 0.26 μm. Please click here to view a larger version of this figure.

Figure 2: Representative data showing sarcomere shortening of the human PSC-CMs derived from the ACTN2-mCherry cell line in non-patterned and patterned culture. A. The timeline for human PSC-CM differentiation. B. and C. Cardiomyocytes cultured in non-patterned cultures show disorganized sarcomere patterns (B) while patterned cultures promote a good alignment of the sarcomere (C). Measuring regions presented by yellow bars. D. Corresponding sarcomere length profiles obtained during cell contraction induced by electrical stimulation at 0.5 Hz and a 100 frames per second frame rate. Pixel size = 0.26 μm, scale bar = 10 µm. Please click here to view a larger version of this figure.

Figure 3: Mouse PSC-CMs after AAV transduction for 3 days. A. Schematic vector map of AAV for sarcomere labeling. A sarcomere protein (gene of interest, GOI) is linked to GFP with a Gly-Gly-Gly-Ser linker (L) and expressed under the control of cardiac troponin T (cTNT) promoter. B. The timeline for mouse PSC-CM differentiation and AAV transduction. C. and D. Representative images showing clear sarcomere localization and the corresponding sarcomere length profile of TCAP-GFP (C) and PDLIM3-GFP (D) after 3 days of transduction into PSC-CMs generated from the Myom2-TagRFP cell line. Scale bar = 10 μm. Please click here to view a larger version of this figure.

Figure 4: Blasticidin Purification of human PSC-CMs without knock-in. 
A. Schematic vector map of AAV, in which a blasticidin-resistance gene cassette (BSR) is inserted downstream to cTNT promoter. B. The timeline of human PSC-CMs differentiation, transduction, and blasticidin selection. C. Representative data showing percentage of cTNT + cells in human PSC-CMs (transduced 10⁵ vg/cell AAV6 on day 4 then treated with 2.5 µg/mL blasticidin on days 7 and 9). Please click here to view a larger version of this figure.

Movie 1: Fluorescent time-lapse video of mouse PSC-CMs generated from the Myom2-TagRFP cell line. RFP signals showed a sarcomere pattern after PSC-CM culture for 28 days. The cells showed beating synchronously when stimulated with electricity at 1 Hz. The time-lapse images were acquired every 20 ms with a 100X lens. Scale bar = 5 μm. Please click here to download this movie.

Movie 2: Fluorescent time-lapse video of the human PSC-CMs with ACTN2-mCherry cultured on a non-patterned culture dish. The PSC-CMs expressing ACTN2-mCherry on a non-patterned culture dish not only showed disorganization of sarcomere but also presented a waving contraction, for which it is difficult to determine sarcomere shortening. The cells were stimulated with electricity at 0.5 Hz and images acquired every 10 ms with a 100X lens. Scale bar = 10 μm. Please click here to download this movie.

Movie 3: Fluorescent time-lapse video of the human PSC-CMs with ACTN2-mCherry cultured on a patterned culture dish. The patterned culture promoted alignment of the sarcomere and forced the cells to a rod shape. This method allowed the sarcomere shortening in PSC-CMs to be determined more easily. The video was obtained by stimulating the cells with electricity at 0.5 Hz. The framerate was 100 frames per second. Scale bar = 10 μm. Please click here to download this movie.

Supplemental CAD files. CAD files for creating stamps with strips of 200 μm width and grooves of 10 μm (Stamp_200x10.dxf), 25 μm (Stamp_200x25.dxf), and 50 μm (Stamp_200x50.dxf). Please click here to download this file.

PSC-CMs have great potential to be utilized as an in vitro platform to model heart disease and to test the effects of drugs. Nevertheless, an accurate, unified method to assess PSC-CMs functions must first be established. Most of functional tests work with PSC-CMs, e.g., electrophysiology, calcium transient, and metabolism²⁶, and one of the first patient-derived PSC-CM studies was about long-QT syndrome²⁷. However, contractility, one of the most important functions of a cardiomyocyte, is still difficult to assess. With adult cardiomyocytes, sarcomere shortening is widely used. In contrast, due to the underdeveloped and disorganized sarcomere of PSC-CMs, the standard method for sarcomere shortening does not work with these cells. Therefore, we have presented an alternative method for examining sarcomere shortening in PSC-CMs using fluorescent-tagged sarcomere proteins. As demonstrated, proteins localized to the M-line (MYOM2) or Z-line (ACTN2, TCAP, and PDLIM3) fused with fluorescent proteins can be used for this approach. We have also shown that fluorescent-tagged proteins can be expressed from endogenous loci or by AAVs. AAVs provide more flexibility for expressing fluorescent-tagged proteins than do endogenous loci, as AAVs can be applied to any type of patient-derived PSC-CMs. In contrast, expressing proteins from endogenous loci may have a lesser effect on sarcomere function, as the expression level of the genes is tightly regulated and can also be used for monitoring the maturation of PSC-CMs¹⁸.

Even though Myom2-TagRFP, ACTN2-mCherry, and Lifeact were all used to examine the sarcomere shortening^16,18,19, it is still unclear if these proteins interfere with sarcomere function. Recently, Lifeact was reported to disturb actin organization and cellular morphology²⁸. It is also important to note that fusion patterns (i.e., the GFP fusion site at N-term or C-term of target protein) also affect the sarcomere function²⁹. Therefore, before being used widely, it is important to thoroughly assess whether fluorescent-tagged sarcomere proteins interfere with sarcomere function and whether protein-protein interactions occur in sarcomeres in vitro, in vivo, and/or in adult cardiomyocytes. This repertoire of fluorescent-tagged sarcomere proteins provides a good starting point for future protein-engineering options (i.e., shortening the sarcomere proteins to only localization signals). Selecting proteins to tag is another key to success. We have tagged another Z-line protein with GFP, however, this protein displayed only a cytoplasmic distribution rather than localizing to the sarcomere. For live-imaging, protein stability may also play a role, as, for example, if a tagged protein is unstable, the signal level will be lower. The photostability of the fluorescent protein is also important, as unstable protein signals will be easily quenched during imaging.

To examine the contractility of PSC-CMs using methods other than described, indirect measurements of force generated by PSC-CMs (e.g., micro-post arrays, traction force microscopy) or motion (e.g., high-resolution motion detection using SI8000)^11,12,13,14 can be used. Our method can be combined with these methods or with dye-based action potential/calcium transient measurements. The combinatorial approach may provide further information on how a disease causes dysfunction in patient-derived PSC-CMs.

One of the challenges in sarcomere shortening in PSC-CMs is to find a good sarcomere that moves linearly, otherwise, the cells may easily come off the line for sarcomere detection of SarcOptiM and cause unstable sarcomere shortening results. Here, we demonstrate that using a patterned culture generated with PDMS stamps may provide a more stable and linear sarcomere movement. A patterned culture is also known to support the maturation of PSC-CMs¹⁶, which is important for sarcomere function.