Induced pluripotent stem cells (iPSC), which have the capacity to become any cell type, have the potential to transform drug discovery, regenerative therapies, and personalized medicine for a broad range of human disease. Unfortunately, current methods yield mixed cell types with variable reproducibility and poor subsequent maturation, leaving many cells stalled in embryonic-like states. The goal of this project is to develop a novel framework based on live imaging and real-time manipulation that will determine the basic biology of stem cell differentiation into cardiac lineages. The knowledge gained will be used to develop practical tools to enhance control of cardiac-directed stem cell differentiation and maturation. These tools, including materials (cells), analytic tools (software) and an online database for standardizing differentiation protocols and outcomes, will be made widely available. The approach taken has the potential to impact the ability to control stem cell differentiation across a broad range of cell and tissue types. The project’s educational component involves multiple efforts designed to introduce students to the cross-disciplinary fields of developmental biology, genetics, systems/computational biology, and mechanobiology. Activities include involvement with existing programs (e.g. Girls in Science and Engineering, Detroit Area Pre-College Engineering Program), development of a new hands-on laboratory module centered on stem cell differentiation and a new course in stem cell biology, and providing rotation opportunities for medical students, residents and fellows.

This project’s goals are to develop a comprehensive understanding of cardiac lineage specification from the earliest stage in iPSC differentiation, to create robust methods to control differentiation from mesodermal precursors through several cardiac lineage bifurcations, and to direct maturation signals in specific cardiac lineages. The project builds on several innovative approaches developed in the investigators’ labs, including the ability to constrain where cells can adhere to a substrate with micro precision, to image BMP, Nodal, and Wnt signaling at the single cell level over multiple days of differentiation and to fabricate a micron-scale 2D (M2D) muscle bundle platform that generates >4000 muscle bundles per device and can be used as an efficient testbed for dissection of multiple variables that drive maturation. To accomplish the project’s goals, tools will be developed to enable real time adjustment of differentiation methods to control lineage specification and minimize unwanted heterogeneity during differentiation in diverse iPSC lines. The Research Plan is organized under two Aims. AIM 1 is to direct cardiomyocyte (CM) differentiation through single cell tracking and real-time manipulation. Mechanistic insight into cardiac differentiation will be obtained by tracking signaling at the single cell level, manipulating exogenous cues in response to these real time observations, and linking the cell environment to specific transcription factor activity using CRISPR activation/inhibition. Heterogeneous differentiation will be dissected as cells move down the lineage tree: from iPSC to mesoderm, from mesoderm to endoderm and first and second heart fields (FHF, SHF), and from FHF to the morphologic left ventricle and SHF to the atria and right ventricle/outflow tract. AIM 2 is to improve maturation of iPSC-CMs from FHF and SHF cardiac lineages by identifying the key nodes that drive CMs toward a mature and stable state. The M2D muscle bundle platform will be used to dissect combinatorial influences of exogenous and endogenous soluble cues with real-time monitoring. The relationships will be further interrogated with additional modulation of the mechanical and extracellular matrix. This aim will identify microenvironmental cues and underlying transcriptional networks that regulate CM maturation. In summary, this project’s emphasis on deciphering the underlying mechanisms of stem cell fate decisions and tissue-specific maturation is consistent with NSF’s “Rules of Life” Big Idea. The approaches established will yield critical insights into stem cell differentiation, define the key determinants of cardiac lineage specification and maturation, and enable real time monitoring and control of iPSC differentiation. Moreover, this project will address major hurdles that currently limit the use of stem cell technologies for a variety of applications.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

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Regents of the University of Michigan - Ann Arbor
Ann Arbor
United States
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