NHLBI Progenitor Cell Biology Consortium Ancillary Project Collaborative

Abstract:

The University of Washington-University of Pennsylvania PCBC focuses on the role of Notch and Wnt signaling in controlling cardiovascular and blood progenitors. One of our long term goals is to identify pathways to control differentiation and proliferation of human cardiomyocytes for clinical-scale heart repair studies. Recent advances, in part funded through the PCBC, have demonstrated that commitment of human embryonic stem cell (hESC)-derived progenitors to a cardiac fate involves the interplay of Wnt and Notch signaling, and further, that Notch signals provide the strongest known stimulus for proliferation of definitive human cardiomyocytes. These signals are sufficiently robust that we now have the capacity to control cardiomyocyte differentiation and proliferation for engineering human heart tissue at a scale to repair the heart of a large animal. We propose here a 3-way collaboration between two established members of the PCBC (Keller and Murry) and bring in Dr. Wolfram Zimmermann from the University of Göttingen, Germany, a world leader in cardiac tissue engineering. Our goal is to manipulate the Wnt and Notch pathways in hESC derivatives to generate sufficient human cardiomyocytes to generate human engineered heart tissue (EHT) constructs large enough to test in large animal models of infarction. We propose the following Specific Aims:

Aim 1 (Keller). The role of Wnt signaling during mesoderm induction and cardiovascular lineage specification.

Work from the Keller and Murry groups has shown that activation of Wnt and Notch signaling stimulates the differentiation of cardiovascular progenitors and leads to an increase differentiation of cardiomyocytes. Using small molecules to modulate Wnt signaling, we will evaluate multiple hESC lines and hiPSC lines and optimize the conditions that will promote the generation of greater than 70% cardiomyocytes from all cell lines tested. The ability to increase the consistency of cardiac differentiation would represent an important step forward, as it would enable the routine production of cells for the generation of EHT as detailed in Aim 3.

Aim 2 (Murry). Expand the proliferation of definitive human cardiomyocytes by promoting Notchsignaling.

Unlike their rodent counterparts, hESC-derived cardiomyocytes remain proliferative for several weeks after they begin beating. This provides the basis for expansion of cardiomyocytes in vitro and, eventually, in vivo after transplantation. As part of an ongoing screen to discover mitogens for human cardiomyocytes, we recently identified a Notch ligand as the strongest stimulus yet described. To exploit this, cardiomyocytes will be expanded by expressing a drug-inducible version of the Notch intracellular domain. We will use this technique to scale production of cardiomyocytes to levels needed for testing in large animals (~108 cardiomyocytes per EHT construct).

Aim 3 (Zimmermann). Generation of large scale engineered human heart tissue constructs by modulation of Wnt and Notch signaling.

We will first generate EHTs with hESC-cardiomyocytes derived using the optimized methods developed in aim 1 and we will determine if use of the drug-inducible Notch signaling system will promote proliferation of hESC-derived cardiomyocytes in 3D EHTs. We will then use computer simulation to generate a model for large-scale tissue assembly based on a modular concept developed by the Zimmermann lab, which makes use of the propensity of single EHTs to fuse into basically any geometry as a functional syncytium. Finally, based on this modeling and incorporating lessons from Aims 1 and 2, we will generate large scale constructs of human myocardium (~5x5x0.5 cm, containing 108 cardiomyocytes) suitable for testing in large animal models. Techniques for production of large-scale constructs and their electromechanical conditioning will be optimized.