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. 2018 Apr;13(4):723-737.
doi: 10.1038/nprot.2018.006. Epub 2018 Mar 15.

Generation of Spatial-Patterned Early-Developing Cardiac Organoids Using Human Pluripotent Stem Cells

Free PMC article

Generation of Spatial-Patterned Early-Developing Cardiac Organoids Using Human Pluripotent Stem Cells

Plansky Hoang et al. Nat Protoc. .
Free PMC article


The creation of human induced pluripotent stem cells (hiPSCs) has provided an unprecedented opportunity to study tissue morphogenesis and organ development through 'organogenesis-in-a-dish'. Current approaches to cardiac organoid engineering rely on either direct cardiac differentiation from embryoid bodies (EBs) or generation of aligned cardiac tissues from predifferentiated cardiomyocytes from monolayer hiPSCs. To experimentally model early cardiac organogenesis in vitro, our protocol combines biomaterials-based cell patterning with stem cell organoid engineering. 3D cardiac microchambers are created from 2D hiPSC colonies; these microchambers approximate an early-development heart with distinct spatial organization and self-assembly. With proper training in photolithography microfabrication, maintenance of human pluripotent stem cells, and cardiac differentiation, a graduate student with guidance will likely be able to carry out this experimental protocol, which requires ∼3 weeks. We envisage that this in vitro model of human early heart development could serve as an embryotoxicity screening assay in drug discovery, regulation, and prescription for healthy fetal development. We anticipate that, when applied to hiPSC lines derived from patients with inherited diseases, this protocol can be used to study the disease mechanisms of cardiac malformations at an early stage of embryogenesis.

Conflict of interest statement

All the authors declare no competing financial interests.


Figure 1.
Figure 1.. Schematic of procedure and process of PDMS stencil fabrication.
(a) Schematic outlining key steps of the entire procedure. (b) SU8 master with a small amount of PDMS prepolymer. (c) Completed assembly of the patterned SU8 master-PDMS-transparency-glass slide construct. (d) Thin film of PDMS removed from the assembly after curing and then placed on top of the optically clear PEG grafted surface (e).
Figure 2.
Figure 2.. Resulting patterns and seeded cells after oxygen plasma etching.
SEM images of (a) PDMS stencil with clear-through holes (Steps 8–12) and (b) etched PEG surface (Steps 13–20) with circular patterns. (c-e) hiPSCs patterned into an array of circles, triangles and squares, respectively. (f-h) Patterned hiPSCs still maintain pluripotency as indicated by OCT4 expression. (i) Timeline of the differentiation procedure and cell morphologies at key stages of differentiation. Scale bars: 200 μm (a, b, f-i); 400 μm (c-e).
Figure 3.
Figure 3.. 3D reconstructed images of cardiac microchambers.
Confocal microscopy images showing contracting cardiomyocytes at the center (red) and myofibroblasts (green) along the perimeter characterized by (a) myosin heavy chain and smooth muscle actin, (b) cardiac troponin T and SM22, and (c) sarcomeric α-actinin and calponin. (d) Two-photon microscopy image taken at a single plane of the cardiac microchamber showing a void chamber surrounded by cells. All scale bars 100 μm.
Figure 4.
Figure 4.. Contractile motion tracking of beating cardiac microchambers grown from 400 μm and 600 μm circle patterns.
(a, e) Videos of beating cardiac microchambers are analyzed to calculate motion vectors. (b, f) Motion vectors are plotted as contraction directions. (c, g) Motion vectors are used to compute localized mean contraction velocities as heatmaps. (d, h) Contraction motion waveforms are then generated to characterize beating physiology of the cardiac microchambers. Scale bar: 200 μm.

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