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Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells

Abstract

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.

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Figure 1: Schematic of the procedure and process of PDMS stencil fabrication.
Figure 2: Resulting patterns and seeded cells after oxygen plasma etching.
Figure 3: 3D reconstructed images of cardiac microchambers.
Figure 4: Contractile motion tracking of beating cardiac microchambers grown from 400-μm and 600-μm (ref. 7) circle patterns.

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Acknowledgements

This work was supported by the Nappi Family Foundation Research Scholar Project and NIH-NIBIB R21EB021003, and in part by NIH-NCATS UH3TR000487. Z.M. acknowledges support from American Heart Association (AHA) postdoctoral fellowship 16POST27750031. P.H. acknowledges support from a National Science Foundation Integrative Graduate Education and Research Traineeship (NSF IGERT) and DMR-DGE-1068780.

Author information

Affiliations

Authors

Contributions

P.H., K.E.H., and Z.M. conceived the protocol development and finalization. P.H. and Z.M. performed biological experiments and analyzed data. J.W. contributed to the initial protocol development. B.R.C. provided the hiPSC line. P.H. wrote the manuscript with discussions and suggested improvements from all authors. Z.M. and K.E.H. funded the study, and B.R.C., K.E.H., and Z.M. supervised the project development and management.

Corresponding author

Correspondence to Zhen Ma.

Ethics declarations

Competing interests

B.R.C. is a founder of Tenaya Therapeutics, a company focused on finding treatments for heart failure, including the use of CRISPR interference to interrogate genetic cardiomyopathies. B.R.C. holds equity in Tenaya, and Tenaya provides research support for heart failure-related research to B.R.C. K.E.H. has a financial relationship with Organos Inc., and both he and the company may benefit from commercialization of the results of this research. The other authors declare no competing financial interests.

Supplementary information

400-μm cardiac microchamber.

Video showing the beating cardiac microchamber engineered from 400-μm circle patterns, with motion vectors calculated from optical flow analysis of the motion-tracking software. (MP4 3511 kb)

600-μm cardiac microchamber.

Video showing the beating cardiac microchamber engineered from 600-μm circle patterns, with motion vectors calculated from optical flow analysis of the motion-tracking software. Reproduced with permission from ref. 7, Springer Nature. (MOV 5333 kb)

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Hoang, P., Wang, J., Conklin, B. et al. Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells. Nat Protoc 13, 723–737 (2018). https://doi.org/10.1038/nprot.2018.006

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