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.
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Method Article
Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair
In This Article
Summary
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 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,
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Protocol
1. Maintenance of hiPSCs and cardiovascular differentiation
- 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 subst....
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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 .......
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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 .......
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Disclosures
The authors have no financial or scientific conflicts to disclose.
Acknowledgements
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.
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Materials
| 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 |
References
- Sanganalmath, S. K., Bolli, R. Cell therapy for heart failure: A comprehensive overview of experimental and clinical studies, current challenges, and future directions. Circulation Research. 113, 810-834 (2013).
- Fisher, S. A., Doree, C., Mathur, A., Martin-Rendon, E.
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