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Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

doi: 10.3791/61246 Published: June 9, 2020
Takeichiro Nakane1,2,6, Mosha Abulaiti1,2, Yuko Sasaki1, William J. Kowalski3, Bradley B. Keller4,5,7, Hidetoshi Masumoto1,2


The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.


Numerous preclinical studies and clinical trials have confirmed the efficiency of cell-based cardiac regenerative therapies for failing hearts1,2,3. Among various cell types, human induced pluripotent stem cells (hiPSCs) are promising cell sources by virtue of their proliferative ability, potential to generate various cardiovascular lineages4,5, and allogenicity. In addition, tissue engineering technologies have made it possible to transfer millions of cells onto a damaged heart5,6,7,8.

Previously, we reported the generation of three-dimensional (3D) linear engineered cardiac tissues (ECTs) from hiPSC-derived cardiovascular lineages using a commercially available culture system for 3D bioartificial tissues5,7. We found that the coexistence of vascular endothelial cells and mural cells with cardiomyocytes within the ECT facilitated structural and electrophysiological tissue maturation. Furthermore, we validated the therapeutic potential of implanted hiPSC-ECTs in an immune tolerant rat myocardial infarction model to improve cardiac function, regenerate myocardium, and enhance angiogenesis5. However, the linear ECTs constructed by this method were 1 mm by 10 mm cylinders and therefore not suitable for the implantation in preclinical studies with larger animals or clinical use.

Based on the successful use of tissue molds to generate porous engineered tissue formation using rat skeletal myoblasts and cardiomyocytes9, human ESC-derived cardiomyocytes10 and mouse iPSCs11, we developed a protocol to generate scalable hiPSC-derived larger implantable tissue using polydimethylsiloxane (PDMS) molds. We evaluated a range of mold geometries to determine the most effective mold characteristics. Mesh-shaped ECTs with multiple bundles and junctions exhibited excellent characteristics in cell viability, tissue function and scalability compared to plain-sheet or linear formats that lacked pores or junctions. We implanted the mesh-shaped ECT in a rat myocardial infarction model and confirmed its therapeutic effects similar to implanted cylindrical ECTs12. Here we describe the protocol to generate a hiPSC-derived mesh-shaped ECT.

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1. Maintenance of hiPSCs and cardiovascular differentiation

  1. Expand and maintain hiPSCs on thin-coat basement membrane matrix (growth factor reduced, 1:60 dilution) in conditioned medium extracted from mouse embryonic fibroblasts (MEF-CM) with human basic fibroblast growth factor (hbFGF)4.
    NOTE: We used a hiPSCs (4-factor (Oct3/4, Sox2, Klf4 and c-Myc) line: 201B6). Add hbFGF at the appropriate concentration for each cell line. Laminin-511 fragment can also be used for the coating of culture dish instead of basement membrane matrix. Commercially available medium designed for feeder-free culture of hiPSCs can be used as a substitute for MEF-CM.
  2. Use Versene (0.48 mM ethylenediaminetetraacetic acid (EDTA) solution) to detach and dissociate cells when cell confluency reaches 90‒100%.
    NOTE: Other commercially available products for cell dissociation can also be used.
  3. Plate cells at a density of 10,000 cells/mm2 on matrix-coated cell culture plates in MEF-CM with hbFGF and culture for two to three days.
  4. When the culture becomes fully confluent, cover the cells with matrix (1:60 dilution with MEF-CM) for one day.
  5. Replace the MEF-CM with RPMI 1640 + B27 medium. Add 100 ng/mL of Activin A and 100 ng/mL of Wnt3A to the medium for one day.
    NOTE: This is day 0 of differentiation. To upregulate canonical Wnt signaling, GSK3β inhibitors can also be used instead of Wnt3A.
  6. On day 1 of differentiation, change the medium to new RPMI 1640 + B27 with 10 ng/mL of BMP4 and 10 ng/mL of hbFGF. Culture cells for two (MC protocol) or four (CM+EC protocol) days without medium change5.
    NOTE: CM+EC protocol is optimized to simultaneously induce cardiomyocytes (CMs) and vascular endothelial cells (ECs). MC protocol is optimized to preferentially induce vascular mural cells (MCs).
  7. CM+EC protocol for the induction of CMs and ECs (Figure 1A).
    1. Replace the medium on day 5 of differentiation with RPMI 1640 + B27 supplemented with 50 ng/mL of VEGF165.
    2. Change the culture medium every 48 h until day 13‒15 of differentiation.
  8. MC protocol for the induction of vascular mural cells (Figure 1B)
    1. Replace the medium with RPMI1640 + 10% fetal bovine serum (FBS) at day 3 of differentiation.
    2. Change the culture medium every 48 h until day 13‒15 of differentiation.

2. Cell harvest and lineage analysis on differentiation day 13‒15

  1. Wash the cells with Ca2+ and Mg2+ free phosphate-buffered saline (PBS).
  2. Add cell dissociation solution (containing proteases, collagenases and DNAses) in the cell culture dish to cover the plate. Incubate the plate for 5 min at 37 °C.
  3. Collect and dissociate the cells with culture medium using a pipette after incubation.
  4. Allocate 1 x 106 cells for lineage analysis with flow cytometry. To eliminate dead cells, stain the cells with fixable viability dye.
  5. Stain the cells with membrane surface markers in PBS with 5% FBS. Use the following dilutions of antibody in fluorescence activated cell sorting (FACS) staining buffer: anti-PDGFRβ (1:100), anti-VE cadherin (1:100), anti-TRA-1-60 (1:20).
  6. For intracellular proteins, resuspend and fix the cells with 4% paraformaldehyde (PFA) in PBS.
  7. Stain the cells with anti-cardiac isoform of Troponin T (cTnT) in PBS with 5% FBS and 0.75% Saponin, then label the cTnT antibody with 488 mouse IgG1 (dilution 1:50).
  8. Resuspend the stained cells in PBS with 5% FBS and put them in FACS tubes with cell strainer.
  9. Analyze the cell composition of the stained cells from each differentiation protocol with flow cytometry to facilitate the generation of cell suspensions with defined lineage distributions.
    NOTE: While performing this procedure, the remaining cell suspension for ECTs is preserved in a 4 °C refrigerator.

3. Fabrication of PDMS tissue mold

  1. Cast a 0.5 mm thick and over 30 mm x 30 mm layer of polydimethylsiloxane (PDMS) by mixing the prepolymer and cross-linking solution at a ratio of 10:1 and then cure at 80 °C for 3 h.
  2. Cut the PDMS sheet and bond it with silicone adhesive to fabricate a 21 mm x 20.5 mm rectangular tray with 7 mm long, 0.5 mm wide, and 2.5 mm high rectangular posts at a staggered position. Horizontal post spacing between two lines of posts is 2.5 mm (Figure 2B).
  3. Autoclave the tray at 120 °C for 20 min.
  4. Coat the tray with 1% poloxamer 407 in PBS for 1 h. Rinse the poloxamer 407 and rinse the mold with PBS sufficiently prior to use.

4. ECT construction

  1. After the cell lineage analysis, combine the cells from CM+EC and MC protocols so that the final concentration of MCs is 10 to 20% in a total cell number of six million cells per construct5.
  2. Suspend mixed six million cells in ECT culture medium (alpha minimum essential medium supplemented with 10% FBS, 50 µM 2-mercaptoethanol and 100 U/mL Penicillin-Streptomycin).
  3. Preparation of matrix solution
    1. Mix 133 µL of acid-soluble rat tail collagen type I solution (2 mg/mL, pH 3) with 17 µL of 10x minimum essential medium (MEM). Then mix the solution with 17 µL of alkali buffer (0.2 M NaHCO3, 0.2 M HEPES, and 0.1 M NaOH).
      NOTE: Collagen must be kept on ice (4 °C). Check buffer color and if the medium does not become pinkish when all solutions are mixed, add additional alkali buffer to the medium. The mixing steps must be mix collagen I + 10x MEM, and then add alkali buffer. DO NOT change this order. Do NOT generate bubbles in the mix.
    2. Add 67 µL of basement membrane matrix to the neutralized collagen solution.
      NOTE: The mixed solution must be kept on ice (4 °C).
  4. Centrifuge the prepared cell suspension containing six million cells at 1,100 rpm (240 x g) for 5 min and resuspend the cells with 167 µL of high-glucose DMEM + 20% FBS + 1% penicillin-streptomycin (100x).
  5. Mix the cell suspension and the matrix solution. The total volume of cell/matrix mixture for one construct is 400 μL.
    NOTE: The cell/matrix mixture should be non-viscous and a pinkish color at this step. Keep it on ice as the gel will solidify at room temperature. The final concentration of collagen type I is 0.67 mg/mL.
  6. Pour the cell/matrix mixture evenly over the poloxamer 407 coated PDMS tissue mold, which is placed in a six-well culture plate12.
    NOTE: Pour the mixture carefully to avoid generating bubbles in order to prevent filling defects in the poured gel.
  7. Incubate the cell/matrix mixture in a standard CO2 incubator (37C, 5 % CO2) for 60 min.
  8. After the tissue is formed, soak the tissue mold with 4 mL of ECT culture medium.
    NOTE: Although the cell/matrix is crosslinked in 60 min, the construct is still very fragile. Add medium gently to avoid damaging it.
  9. Culture the tissue for 14 days with medium change every day.
  10. Prior to ECT implantation, remove the ECT from the loading posts gently using sterilized fine forceps.
    NOTE: The final ECT dimensions after removal from the mold are less than the original mold. A 2.1 cm x 2.05 cm mold generates a released ECT of approximately 1.5 cm x 1.5 cm. A larger 3.9 cm x 4.05 cm mold generates an ECT of approximately 3 cm x 3 cm. Unloaded ECT shows intrinsic spontaneous beating in warm culture medium. Although it initially maintains a mesh structure, an unloaded ECT shrinks and condenses over time. It is possible to hold the tissue softly with fine forceps and then use 7-0 silk suture to attach the ECT to the epicardium.

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Representative Results

Figure 1A,B shows the schematics of CM+EC and MC protocol. After inducing CMs and ECs from CM+EC protocol and MCs from MC protocol, the cells are mixed adjusting final MC concentrations to represent 10 to 20% of total cells. The 2 cm wide tissue mold is fabricated according to the design drawing from 0.5 mm thick PDMS sheet (Figure 2A,B). Six million of CM+EC+MC cells are combined with collagen I, and matrix and poured onto the tissue mold precoated with poloxamer 407 (Figure 2C). During preliminary experiments, we fabricated tissue molds with various patterns characterized by various post lengths and spacing and confirmed final geometries of ECTs (Figure 3). We selected the mold with 7 mm long posts with 2.5 mm wide intervals for this study.

Poloxamer 407 prevents cell adhesion to the mold and enabled the formation of characteristic mesh structure following rapid gel compaction in 14 days (Figure 4). This structure is maintained even after the ECT is released from its mold. The whole tissue is approximately 1.5 cm wide and 0.5 mm thick, and the width of each bundle in the mesh is approximately 0.5 mm in average.

It is possible to generate a 3 cm final width mesh ECT containing twenty-four million cells from a four times larger mold with the same staggered post design (Figure 5A,B). This larger mesh ECT can be removed from the mold easily and generates local active force equivalent to the smaller mesh ECT (Figure 5C).

Figure 1
Figure 1: Protocols to differentiate cardiovascular cells from human induced pluripotent stem cells. Schematic diagrams of protocols used to induce cardiomyocytes and vascular endothelial cells (A, CM+EC protocol) and to induce vascular mural cells (B, MC protocol).CM = cardiomyocyte; EC = endothelial cell; MC = vascular mural cell; iPSC = induced pluripotent stem cell; MG = basement membrane matrix; ActA = Activin A; Wnt3a, BMP4, Bone morphogenetic protein 4; bFGF = basic fibroblast growth factor; VEGF = vascular endothelial cell growth factor; FBS = fetal bovine serum. This figure is adapted from reference Masumoto et al.5. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Fabrication of PDMS tissue mold and ECT construction. (A) Representative image of an ECT mold fabricated from 0.5 mm thick PDMS sheets. (B) Design of the PDMS tissue mold with 7 mm long internal posts arranged in staggered position; front view (upper panel) and side view (lower panel). (C) The schematic protocol of ECT construction from hiPSC-derived cardiovascular cells and matrix gel. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Impact of tissue mold designs on final ECT geometries. (A) Definitions of post length (PL) and horizontal post spacing (HPS). (B) A tissue mold without rectangular posts (PL0) and formed a sheet ECT. (CE) Tissue molds with different PL/HPS and mesh ECTs. (F) A tissue mold with long parallel posts and formed a multiple linear ECT. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Formation of mesh ECT following gel compaction. Representative series of images of a mesh ECT maturation from day 0 to 14 are shown. Cell/matrix is poured in the PDMS tissue mold (Day 0_0 h), then culture medium is added one hour later (Day 0_1 h). On day 1, elliptical pores are observed around loading posts. The construct showed rapid gel compaction and matured into a mesh tissue thereafter. On day 14, the tissue is released from the mold. The unloaded ECT maintains the mesh geometry. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Representative images of a larger mesh ECT intended for large animal preclinical trials and clinical trials. (A) Design of the 4 cm x 4 cm PDMS tissue mold with 7 mm long posts and (B) an image of the mold. (C) Representative image of a 3 cm x 3 cm larger mesh ECT. Please click here to view a larger version of this figure.

Supplemental Figure 1: Pathological and electrophysiological evaluation of mesh ECTs. (A) Representative image for a bundle in a mesh ECT stained with Hoechst 33342 (blue) for live cells and Ethidium Homodimer III (red) for dead cells. Scale bar: 250 μm. (B) Representative image of a three-dimensional confocal image of a bundle in a mesh ECT stained with cardiac troponin T (green). Local cardiomyocyte orientations within the bundle are visualized as small lines, where line color indicates magnitude in the circumferential (green), radial (red), and axial (blue) directions. (C) The spherical histogram displays local cardiomyocyte orientations. The volume of each ray represents the relative count for each direction and the thick red line shows the mean CM orientation. (B) and (C) are adapted from reference13 with revision. (D) Representative image of contractile force measurement. A segment was cut off at the red dotted line in a 1.5 cm x 1.5 cm mesh ECT and attached to the muscle testing system using 10-0 nylon suture. The white arrowhead indicates force transducer and the yellow arrowhead indicates high-speed length controller. (E) Representative waveforms of active stress at different pacing frequencies from 1.5 Hz to 4 Hz. Please click here to download this figure.

Supplemental Video 1: Intrinsic beating of a junction in a mesh ECT on day 14. Please click here to download this video.

Supplemental Video 2: Intrinsic beating of a bundle in a mesh ECT on day 14. Please click here to download this video.

Supplemental Video 3: Intrinsic beating of a mesh ECT on day 14 after released from the tissue mold. Please click here to download this video.

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Following the completion of our investigation of a linear format, hiPSC derived ECT5, we adapted the protocol to mix hiPSC-derived CMs, ECs, and MCs to facilitate the in vitro expansion of vascular cells within ECTs and subsequent in vivo vascular coupling between ECTs and recipient myocardium.

To facilitate the generation of larger, implantable mesh ECT geometries we used thin PDMS sheets to design the 3D molds with loading posts arrayed at staggered positions. During preliminary experiments, we noted that ECTs adhered to the PDMS posts during gel compaction and so we modified our method to coat each mold with the surfactant poloxamer to prevent cell adhesion which is a critical step of the present protocol9. ECTs remain detached from the internal loading posts and the mold bottom during in vitro maturation, which facilitates gel compaction and ECT removal from the mold. Another critical step of the protocol is to evenly pour the cell-gel matrix mixture into the PDMS mold before the matrix solidifies. It is critical to accomplish the procedure within a short time. Bubbles in the cell-gel matrix mixture should also be avoided because it may cause structural vacuolation and functional disruption of the ECTs.

According to viability assays, approximately 97% of cells were alive within the day 14 constructs (Supplemental Figure 1A). Supplemental Figure 1B shows the whole mount confocal image stained with cTnT representing myofiber alignment parallel to the local bundle long axis (Supplemental Figure 1C)13. All constructs start intrinsic spontaneous beating in vitro within 72 h and continued beating throughout the duration of culture (Supplemental Videos 1‒3). We analyzed electromechanical properties of 1.5 cm x 1.5 cm ECTs using a custom isolated muscle testing system (Supplemental Figure 1D). ECTs displayed maximal pacing capture rates of 4 Hz and generated an average active stress of 0.55 mN/mm2 at 2 Hz, 5 V pacing protocol (n = 11, standard error of means = 0.063; Supplemental Figure 1E).

Although we selected 7 mm long internal loading posts with 5 mm intervals, it is possible to vary the final ECT geometry by arranging the length of loading posts and the interval between adjacent posts (Figure 3). Moreover, it is possible to expand the scale of 1.5 cm x 1.5 cm ECTs to larger formats such as 3 cm x 3 cm large mesh ECT. According to force measurement, 3 cm x 3 cm ECTs showed electromechanical properties similar to 1.5 cm x 1.5 cm ECTs12. The flexibility of the tissue shapes and the scalability with preserved tissue function is significances of the present protocol with respect to already existing methods.

One limitation of the present protocol is that the PDMS molds are hand-assembled from PDMS sheets. Although the construction of millimeter-unit molds would be feasible by hand-assembly, molds from photolithography or casting from master molds would be more suitable for expanded and stable ECT generation.

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The authors have no financial or scientific conflicts to disclose.


This work was financially supported by the Kosair Charities Pediatric Heart Research Program at the University of Louisville and the Organoid Project at the RIKEN Center for Biosystems Dynamics Research. HiPSCs used in our published protocols were provided by the Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.


Name Company Catalog Number Comments
Cell Culture Dishes 100x20 mm style Falcon/ Thomas scientific 9380C51
Multiwell Plates For Cell Culture 6well 50/CS Falcon / Thomas scientific 6902A01
Sylgard 184 Silicone Elastomer Kit Dow Corning 761036
Accumax Innovative Cell Technologies AM-105
BMP4, recombinant (10µg) R&D RSD-314-BP-010
Collagen, Type I solution from rat tail Sigma C3867
Growth factor-reduced Matrigel Corning 356231
Human VEGF (165) IS, premium grade Miltenyi 130-109-385
Pluronic F-127, 0.2 µm filtered (10% Solution in Water) Molecular Probes P-6866
Recombinant human bFGF WAKO 060-04543
Recombinant Human/Mouse/Rat ActivinA (50µg) R&D 338-AC-050
rh Wnt-3a (10µg) R&D 5036-WN
Versene solution Gibco 15040066
Culture medium and supplements
10x MEM Invitrogen 11430
2 Mercaptro Ethanol SIGMA M6250
B27 supplement minus insulin Gibco A1895601
DMEM, high glucose Gibco 11965084
Fetal Bovine Serum (500ml) Any
Fetal Bovine Serum (500ml) Any
L-Glutamine Gibco 25030081
NaHCO3 Any
PBS 1x Gibco 10010-031
Penicillin-Streptomycin (5000 U/mL) Gibco 15070-063
RPMI1640 medium Gibco 21870092
αMEM Invitrogen 11900024
anti-TRA-1-60, FITC, Clone: TRA-1-60, BD Biosciences BD / Fisher 560380
anti-Troponin T, Cardiac Isoform Ab-1, Clone: 13-11, Thermo Scientific Lab Vision Fisher MS-295-P0
BD FACS Clean Solution BD 340345
BD FACSFlow Sheath Fluid BD 342003
BD FACSRinse Solution BD 340346
Falcon Tube with Cell Strainer Cap (Case of 500) Corning 352235
Fetal Bovine Serum (500ml) Any
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation Molecular Probes L34957
PDGFRb; anti-CD140b, R-PE, Clone: 28D4, BD Biosciences BD / Fisher 558821
Saponin Sigma-Aldrich 47306-50G-F
VEcad-FITC; anti-CD144, FITC, Clone: 55-7H1, BD Biosciences BD / Fisher 560411
Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit Molecular Probes Z25002



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Cite this Article

Nakane, T., Abulaiti, M., Sasaki, Y., Kowalski, W. J., Keller, B. B., Masumoto, H. Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair. J. Vis. Exp. (160), e61246, doi:10.3791/61246 (2020).More

Nakane, T., Abulaiti, M., Sasaki, Y., Kowalski, W. J., Keller, B. B., Masumoto, H. Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair. J. Vis. Exp. (160), e61246, doi:10.3791/61246 (2020).

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