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,

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 coatin.......

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

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