Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating normal human somatic cells for developmental studies, toxicity testing, and cellular therapies. Cardiac myocytes are a particularly promising cell type that can be derived from human pluripotent stem cells since cardiac myocytes cannot easily be attained from primary sources or adult stem cells and are of tremendous importance in disease and pharmaceutical evaluation. While substantial progress has been made in generating contractile cardiac myocytes from human pluripotent stem cells, yields remain low and cell populations are heterogeneous, containing atrial, ventricular, and nodal cardiac myocytes, as a result of our lack of fundamental understanding of factors that govern cardiac myocyte development. Current differentiation protocols utilize chemical factors to stimulate cardiogenesis in pluripotent stem cells. In the initial project period we identified cell-cell contact as an important contributor to cardiac myocyte yield from human embryonic stem cells and discovered that an optimum colony size exists for generating cardiac myocytes. This optimum size correlates with reduced signaling through the Want/ -catenin pathway, which has both stimulatory and inhibitory effects on cardiac myocyte development in vivo. In the next project period we propose to elucidate the mechanism by which intercellular interactions affect yield and type specification of cardiac myocytes from pluripotent stem cells. Specifically, we will test the hypothesis that confinement of human pluripotent stem cells in microwells during differentiation affects the yield and type of cardiac myocytes generated by modulating the Want/ -catenin signaling pathway which is regulated by direct cell-cell contact and soluble factors. Our team's expertise in biomaterials development, stem cell biology, signaling pathway analysis, and cardiac myocyte physiology will permit us to construct culture systems that systematically vary colony morphology and presentation of biochemical signaling cues that regulate Want/ -catenin signaling, and quantitatively assess the effects on cardiac myocyte development.
Our specific aims to test the hypothesis of this proposal are: 1. quantify the effects of human pluripotent stem cell colony confinement during embryoid body formation and directed differentiation to cardiac myocytes on the yield and phenotype of the resulting cardiac myocytes. 2. Evaluate the mechanistic role of Want/ -catenin signaling in specifying cardiac myocyte differentiation in microwell-confined human pluripotent stem cells. 3. Assess the ability of microwells functionalized with Want signaling regulators to control human pluripotent stem cell differentiation to cardiac myocytes.

Public Health Relevance

Cardiac myocytes derived from human pluripotent stem cells offer a system to study development of the human heart in vitro, a tool to screen the effectiveness of drugs to treat cardiac diseases and to assess cardiac toxicity of other pharmacologic agents, and a potential cellular therapy to treat damaged and diseased hearts. Understanding how cell-cell contact regulates cardiac myocyte differentiation will improve our understanding of human heart development, permit rational design of methodologies to improve yield and functionality of pluripotent stem cell-derived cardiac myocytes, and facilitate translational applications of pluripotent stem cell- derived cardiac myocytes to research and clinical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB007534-06
Application #
8235081
Study Section
Biomaterials and Biointerfaces Study Section (BMBI)
Program Officer
Hunziker, Rosemarie
Project Start
2007-07-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
6
Fiscal Year
2012
Total Cost
$368,170
Indirect Cost
$118,170
Name
University of Wisconsin Madison
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Laperle, Alex; Masters, Kristyn S; Palecek, Sean P (2015) Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies. Biotechnol Prog 31:212-9
Hsiao, Cheston; Tomai, Matthew; Glynn, Jeremy et al. (2014) Effects of 3D microwell culture on initial fate specification in human embryonic stem cells. AIChE J 60:1225-1235
Musah, Samira; Wrighton, Paul J; Zaltsman, Yefim et al. (2014) Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification. Proc Natl Acad Sci U S A 111:13805-10
Lalit, Pratik A; Hei, Derek J; Raval, Amish N et al. (2014) Induced pluripotent stem cells for post-myocardial infarction repair: remarkable opportunities and challenges. Circ Res 114:1328-45
Hazeltine, Laurie B; Badur, Mehmet G; Lian, Xiaojun et al. (2014) Temporal impact of substrate mechanics on differentiation of human embryonic stem cells to cardiomyocytes. Acta Biomater 10:604-12
Lian, Xiaojun; Zhang, Jianhua; Zhu, Kexian et al. (2013) Insulin inhibits cardiac mesoderm, not mesendoderm, formation during cardiac differentiation of human pluripotent stem cells and modulation of canonical Wnt signaling can rescue this inhibition. Stem Cells 31:447-57
Lian, Xiaojun; Zhang, Jianhua; Azarin, Samira M et al. (2013) Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/?-catenin signaling under fully defined conditions. Nat Protoc 8:162-75
Lian, Xiaojun; Selekman, Joshua; Bao, Xiaoping et al. (2013) A small molecule inhibitor of SRC family kinases promotes simple epithelial differentiation of human pluripotent stem cells. PLoS One 8:e60016
Hazeltine, Laurie B; Selekman, Joshua A; Palecek, Sean P (2013) Engineering the human pluripotent stem cell microenvironment to direct cell fate. Biotechnol Adv 31:1002-19
Azarin, Samira M; Lian, Xiaojun; Larson, Elise A et al. (2012) Modulation of Wnt/?-catenin signaling in human embryonic stem cells using a 3-D microwell array. Biomaterials 33:2041-9

Showing the most recent 10 out of 20 publications