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Defining the role of non-myocytes for cardiomyocyte differentiation and function in an genetically engineered heart muscle model

Fachliche Zuordnung Pharmakologie
Förderung Förderung von 2008 bis 2014
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 69424651
 
Erstellungsjahr 2014

Zusammenfassung der Projektergebnisse

The aim of the study was to develop cell-specific reporter models (first in the mouse and then in the human) to study the role of myocardial cell-cell interplay for heart muscle formation in vitro. In preliminary work we had already established transgenic embryonic stem cells (ESCs) lines from the mouse, expressing a neomycin resistance (NeoR), green fluorescing protein (GFP), and nuclear localized beta-galactosidase (nLacZ) specifically in cardiomyocytes (aMHC-NIGIL). Building on this work we proposed to use alternative promoter elements to generate ESCs with distinguishable labels in endothelial cells, smooth muscle cells, fibroblasts, and macrophages. Accordingly, we cloned several DNA vectors containing red (NIRIL), yellow (NIYIL), and orange (NIOIL) fluorescing proteins for stable integration in murine ESCs. However, we realized that cell purification and amplification in the genetically modified mouse ESC models is limited, because of suboptimal differentiation and selection protocols. After extensive work to address these limitations, we decided to not aim at specifically selecting cell types for tissue engineering, but focus first on genetically naive fibroblasts isolated from different sources and purified by passaging in culture. With this approach, we could demonstrate that fibroblasts and cardiomyocytes are essential components for the construction of Engineered Heart Muscle (EHM). This was demonstrated for several mouse and human pluripotent stem cell types, including embryonic, induced pluripotent and parthenogenetic stem cells. Finally, in the human model, we developed a transgenic reporter model with RFP and GFP labeled cardiomyocytes and fibroblast-like stroma cells, respectively. This will enable us to gain fundamental insight into processes governing heart muscle assembly and the specific contnbution of fibroblasts in this process. The development of defined conditions for human EHM assembly also serves as the basis for our efforts to clinically translate tissue engineered heart repair, first with embryonic stem cells (1st generation of therapeutic EHM), followed by induced pluripotent stem cells (2nd generation of therapeutic EHM), and parthenogenetic stem cells (3rd generation of therapeutic EHM).

Projektbezogene Publikationen (Auswahl)

 
 

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