Characterization and Application of Bioengineered Heart Muscle as a New Tool to Study Human Heart Development and Disease
Farah Raad
Doctoral thesis
Date of Examination:
2016-06-13
Date of issue:
2016-09-20
Advisor:
Zimmermann, Wolfram-Hubertus Prof. Dr.
Referee:
Zimmermann, Wolfram-Hubertus Prof. Dr.
Referee:
Tietze, Lutz Prof. Dr. Dr. h.c.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-002B-7C05-A
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Abstract
English
Proof-of-concept for the generation of a bioengineered heart muscle (BHM) directly from human pluripotent stem cells (hPSCs) as a model to simulate human heart muscle development was introduced recently by our lab. BHMs undergo stage-specific directed differentiation and self-assembly guided by the sequential addition of growth factors and small molecules to support (1) mesoderm induction (3 days), (2) cardiac specification (10 days) and (3) cardiac maturation (up to 50 days studied). By culture day 22, initial findings revealed homogeneously contracting BHMs with robust inotropic responses to increasing extracellular calcium and β–adrenergic stimulation. In this thesis, a detailed characterization on the molecular, cellular and functional level revealed that BHMs (1) do indeed traverse through defined in-utero like developmental stages with characteristic transcriptome profiles, (2) are composed of mainly mesodermal cells (cardiomyocytes and fibroblast-like cells), (3) display continuous functional maturation over time with enhanced contractile performance on the cellular level and (4) develop in late cultures (day 60) a functional neural crest component with resemblance to the cardiac sympathetic nervous system. Assessments of drug responses revealed the utility of BHM in disease modeling and drug screening. Modulation of one of the pivotal signaling pathways implicated in early cardiac induction, such as the BMP pathway during BHM development revealed that low concentrations of BMP4 are needed for the optimal differentiation of hPSCs to both cardiomyocytes and fibroblast-like cells. In a first effort to reduce all protein stimuli from the BHM culture format, screening for BMP4 replacements was performed and identified two hit molecules (4’-hydroxychalcone and 4-fluoro-4’-methoxychalcone). Both successfully induced cardiac differentiation in monolayer culture when added as BMP4 replacements, but not in the BHM culture format. Collectively, this is the first detailed characterization of a novel cardiac organoid model (BHM), generated by a single step tissue engineering approach directly from hPSCs, with organotypic contractile functionality. Applications in drug screening and disease modeling are demonstrated. Further improvements may be achieved by the replacement of all protein culture medium supplements by bioactive small molecules, such as chalcones to replace BMP4.
Keywords: Human Pluripotent Stem Cells, Cardiac Differentiation, Bioengineered Heart Muscle, Chalcones
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Molekulare Medizin [149]