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High Content Screening for Modulators of Cardiac Differentiation in Human Pluripotent Stem Cells

  • Sean Spiering
  • Herman Davidovics
  • Paul J. Bushway
  • Mark Mercola
  • Erik Willems
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1263)

Abstract

Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.

Key words

Human pluripotent stem cells Small molecules Small RNAs High content screening Cardiac differentiation 

Notes

Acknowledgements

This work was supported by grants from the California Institute for Regenerative Medicine (CIRM) (RC00132) and NIH (HL113601, HL108176, and AG043917) to M.M. E.W. was supported by American Heart Association and CIRM training grants.

References

  1. 1.
    Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680PubMedCrossRefGoogle Scholar
  2. 2.
    Mercola M, Colas A, Willems E (2013) Induced pluripotent stem cells in cardiovascular drug discovery. Circ Res 112:534–548PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Bruneau BG (2008) The developmental genetics of congenital heart disease. Nature 451:943–948PubMedCrossRefGoogle Scholar
  4. 4.
    Burridge PW et al (2012) Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10:16–28PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Garbern JC, Lee RT (2013) Cardiac stem cell therapy and the promise of heart regeneration. Cell Stem Cell 12:689–698PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Willems E et al (2011) Small-molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell-derived mesoderm. Circ Res 109:360–364PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Willems E et al (2012) Small molecule-mediated TGF-β type II receptor degradation promotes cardiomyogenesis in embryonic stem cells. Cell Stem Cell 11:242–252PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Lanier M et al (2012) Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: a structure–activity relationship study based on inhibitors for the Wnt response. J Med Chem 55:697–708PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Schade D et al (2012) Synthesis and SAR of b-annulated 1,4-dihydropyridines define cardiomyogenic compounds as novel inhibitors of TGFβ signaling. J Med Chem 55:9946–9957PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Borowiak M et al (2009) Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell 4:348–358PubMedCrossRefGoogle Scholar
  11. 11.
    Chen S et al (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5:258–265PubMedCrossRefGoogle Scholar
  12. 12.
    Willems E, Bushway PJ, Mercola M (2009) Natural and synthetic regulators of embryonic stem cell cardiogenesis. Pediatr Cardiol 30:635–642PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Kita-Matsuo H et al (2009) Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. PLoS One 4:e5046PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Yang L et al (2008) Human cardiovascular progenitor cells develop from a KDR + embryonic-stem-cell-derived population. Nature 453:524–528PubMedCrossRefGoogle Scholar
  15. 15.
    Bushway PJ, Mercola M, Price JH (2008) A comparative analysis of standard microtiter plate reading versus imaging in cellular assays. Assay Drug Dev Technol 6:557–567PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Bushway P, Mercola M (2006) High-throughput screening for modulators of stem cell differentiation. Methods Enzymol 414:300–316PubMedCrossRefGoogle Scholar
  17. 17.
    Gustafson TA et al (1987) Thyroid hormone regulates expression of a transfected alpha-myosin heavy-chain fusion gene in fetal heart cells. Proc Natl Acad Sci U S A 84:3122–3126PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Dubois NC et al (2011) SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 29:1011–1018PubMedCrossRefGoogle Scholar
  19. 19.
    Cai W et al (2013) Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment. Genes Dev 27:2332–2344PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Li Z, Rana TM (2012) A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation. Nat Commun 3:1085PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Zhang J, Chung T, Oldenburg K (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sean Spiering
    • 1
  • Herman Davidovics
    • 1
  • Paul J. Bushway
    • 1
  • Mark Mercola
    • 1
    • 2
  • Erik Willems
    • 1
  1. 1.Muscle Development and Regeneration ProgramSanford-Burnham Medical Research InstituteLa JollaUSA
  2. 2.Department of BioengineeringUniversity of CaliforniaSan DiegoUSA

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