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

Nature Protocols volume 13, pages 723–737 (2018)Cite this article

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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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References

  1. Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125–1247125 (2014).

    Article  Google Scholar 

  2. Yin, X. et al. Engineering stem cell organoids. Cell Stem Cell 18, 25–38 (2016).

    Article  CAS  Google Scholar 

  3. Willyard, C. The boom in mini stomachs, brains, breasts, kidneys and more. Nature 523, 520–522 (2015).

    Article  CAS  Google Scholar 

  4. Simian, M. & Bissell, M.J. Organoids: a historical perspective of thinking in three dimensions. J. Cell Biol. 216, 31–40 (2017).

    Article  CAS  Google Scholar 

  5. Dutta, D., Heo, I. & Clevers, H. Disease modeling in stem cell-derived 3D organoid systems. Trends Mol. Med. 23, 393–410 (2017).

    Article  CAS  Google Scholar 

  6. Ranga, A. et al. Neural tube morphogenesis in synthetic 3D microenvironments. Proc. Natl. Acad. Sci. USA 114, E3163 (2017).

    Article  Google Scholar 

  7. Ma, Z. et al. Self-organizing human cardiac microchambers mediated by geometric confinement. Nat. Commun. 6, 7413 (2015).

    Article  CAS  Google Scholar 

  8. Mohr, J.C. et al. The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. Biomaterials 31, 1885–1893 (2010).

    Article  CAS  Google Scholar 

  9. Warmflash, A., Sorre, B., Etoc, F., Siggia, E.D. & Brivanlou, A.H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847–54 (2014).

    Article  CAS  Google Scholar 

  10. Deglincerti, A. et al. Self-organization of human embryonic stem cells on micropatterns. Nat. Protoc. 11, 2223–2232 (2016).

    Article  CAS  Google Scholar 

  11. Fatehullah, A., Tan, S.H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–54 (2016).

    Article  Google Scholar 

  12. Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

    Article  CAS  Google Scholar 

  13. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    Article  CAS  Google Scholar 

  14. Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).

    Article  CAS  Google Scholar 

  15. Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 536, 238–238 (2016).

    Article  CAS  Google Scholar 

  16. Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    Article  CAS  Google Scholar 

  17. Li, Y., Xu, C. & Ma, T. In vitro organogenesis from pluripotent stem cells. Organogenesis 10, 159–163 (2014).

    Article  Google Scholar 

  18. Breckwoldt, K. et al. Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat. Protoc. 12, 1177–1197 (2017).

    Article  CAS  Google Scholar 

  19. Guo, X.M. et al. Creation of engineered cardiac tissue in vitro from mouse embryonic stem cells. Circulation 113, 2229–2237 (2006).

    Article  Google Scholar 

  20. Shkumatov, A., Baek, K. & Kong, H. Matrix rigidity-modulated cardiovascular organoid formation from embryoid bodies. PLoS One 9, 1–10 (2014).

    Article  Google Scholar 

  21. Sun, X. & Nunes, S.S. Biowire platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Methods 101, 21–6 (2016).

    Article  CAS  Google Scholar 

  22. Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616–623 (2014).

    Article  CAS  Google Scholar 

  23. Mathur, A. et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Sci. Rep. 5, 8883 (2015).

    Article  CAS  Google Scholar 

  24. Ma, Z. et al. Three-dimensional filamentous human cardiac tissue model. Biomaterials 35, 1367–1377 (2014).

    Article  CAS  Google Scholar 

  25. Hinson, J.T. et al. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science 349, 982–986 (2015).

    Article  CAS  Google Scholar 

  26. Pettinato, G., Wen, X. & Zhang, N. Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays. Sci. Rep. 4, 7402 (2014).

    Article  CAS  Google Scholar 

  27. Nunes, S.S. et al. Biowire: a platform for maturation of human pluripotent stem cell–derived cardiomyocytes. Nat. Methods 10, 781–787 (2013).

    Article  CAS  Google Scholar 

  28. Bergström, G., Christoffersson, J., Schwanke, K., Zweigerdt, R. & Mandenius, C.-F. Stem cell derived in vivo-like human cardiac bodies in a microfluidic device for toxicity testing by beating frequency imaging. Lab Chip 15, 3242–3249 (2015).

    Article  Google Scholar 

  29. Matsudaira, K. et al. MEMS piezoresistive cantilever for the direct measurement of cardiomyocyte contractile force. J. Micromech. Microeng. 27, 10 (2017).

    Article  Google Scholar 

  30. Huebsch, N. et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Sci. Rep. 6, 24726 (2016).

    Article  CAS  Google Scholar 

  31. Pavesi, A. et al. Controlled electromechanical cell stimulation on-a-chip. Sci. Rep. 5, 11800 (2015).

    Article  Google Scholar 

  32. Simmons, C.S., Petzold, B.C. & Pruitt, B.L. Microsystems for biomimetic stimulation of cardiac cells. Lab Chip 12, 3235–3248 (2012).

    Article  CAS  Google Scholar 

  33. Caiazzo, M. et al. Defined three-dimensional microenvironments boost induction of pluripotency. Nat. Mater. 15, 344–352 (2016).

    Article  CAS  Google Scholar 

  34. Jeon, O. & Alsberg, E. Regulation of stem cell fate in a three-dimensional micropatterned dual-crosslinked hydrogel system. Adv. Funct. Mater. 23, 4765–4775 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. McBeath, R., Pirone, D.M., Nelson, C.M., Bhadriraju, K. & Chen, C.S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 6, 483–495 (2004).

    Article  CAS  Google Scholar 

  36. Musunuru, K., Domian, I.J. & Chien, K.R. Stem cell models of cardiac development and disease. Annu. Rev. Cell Dev. Biol. 26, 667–687 (2010).

    Article  CAS  Google Scholar 

  37. Bruneau, B.G. The developmental genetics of congenital heart disease. Nature 451, 943–948 (2008).

    Article  CAS  Google Scholar 

  38. Bressan, M. et al. Reciprocal myocardial-endocardial interactions pattern the delay in atrioventricular junction conduction. Development 141, 4149–4157 (2014).

    Article  CAS  Google Scholar 

  39. Luxán, G. et al. Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy 19, 193–201 (2013).

  40. Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. USA 109, E1848–E1857 (2012).

    Article  CAS  Google Scholar 

  41. Huebsch, N. et al. Automated video-based analysis of contractility and calcium flux in human-induced pluripotent stem cell-derived cardiomyocytes cultured over different spatial scales. Tissue Eng. Part C Methods 21, 467–479 (2015).

    Article  CAS  Google Scholar 

  42. Tourovskaia, A. et al. Micropatterns of chemisorbed cell adhesion-repellent films using oxygen plasma etching and elastomeric masks. Langmuir 19, 4754–4764 (2003).

    Article  CAS  Google Scholar 

  43. Kattman, S.J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011).

    Article  CAS  Google Scholar 

  44. Burridge, P.W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014).

    Article  CAS  Google Scholar 

  45. Pei, F. et al. Chemical-defined and albumin-free generation of human atrial and ventricular myocytes from human pluripotent stem cells. Stem Cell Res. 19, 94–103 (2017).

    Article  CAS  Google Scholar 

  46. Maddah, M. et al. A non-invasive platform for functional characterization of stem-cell-derived cardiomyocytes with applications in cardiotoxicity testing. Stem Cell Rep. 4, 621–631 (2015).

    Article  CAS  Google Scholar 

  47. Lee, E.K., Kurokawa, Y.K., Tu, R., George, S.C. & Khine, M. Machine learning plus optical flow: a simple and sensitive method to detect cardioactive drugs. Sci. Rep. 5, 11817 (2015).

    Article  Google Scholar 

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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

Authors and Affiliations

  1. Department of Biomedical & Chemical Engineering, Syracuse University, Syracuse, New York, USA

    Plansky Hoang & Zhen Ma

  2. Syracuse Biomaterials Institute, Syracuse University, Syracuse, New York, USA

    Plansky Hoang & Zhen Ma

  3. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

    Jason Wang

  4. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    Bruce R Conklin

  5. Department of Medicine, University of California, San Francisco, San Francisco, California, USA

    Bruce R Conklin

  6. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    Bruce R Conklin

  7. Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA

    Kevin E Healy

  8. Department of Materials Science & Engineering, University of California, Berkeley, Berkeley, California, USA

    Kevin E Healy

Authors
  1. Plansky Hoang
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  3. Bruce R Conklin
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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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