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

Summary

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Abstract

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.

Introduction

Numerous preclinical studies and clinical trials have confirmed the efficiency of cell-based cardiac regenerative therapies for failing hearts^1,2,3. 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 lineages^4,5, and allogenicity. In addition, tissue engineering technologies have made it possible to transfer millions of cells onto a damaged heart^5,6,7,8.

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 tissues^5,7. 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.

Protocol

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.

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 ...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
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
Reagents
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
Flowcytometry
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
EDTA	Any
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