Cell Surface Capture & The Cell Surface Protein Atlas
A central technology in our laboratory is the Cell Surface Capture Technology - a mass spectrometry approach that enables capture and identification of extracellular domains of plasma membrane and extracellular matrix N-glycoproteins from live cells. Our laboratory and collaborators have pioneered the use of this technology on multiple cell types, including cancer, blood, liver, heart and stem cells. The Cell Surface Capture Technology serves as the starting point in many of our studies, revealing proteins of interest we then target in either antibody-based or drug-based studies. Generally speaking, this workflow supports the development of novel panels of markers for live cell identification and sorting of specific cell types of interest and the identification of proteins involved in specific signaling/functional events important for cardiomyogenesis.
Working together with our collaborator Dr. Bernd Wollscheid, we have applied the CSC-Technology to more than 100 cell types. This database (stored internally, published & unpublished data) is a powerful resource for rapidly determining whether proteins identified on a particular cell type are broadly expressed among a variety of cell types or are more restricted to a few or a single cell type.
Human pluripotent stem cells are a viable source for in vitro generation of cardiomyocytes useful for the study of human development, heart disease modeling, and regenerative medicine. However, the inability to reliably identify chamber-specific cardiomyocytes of a specific maturation stage in a high-throughput fashion has been a barrier to utilizing such cells for repair of the damaged heart, discovering new drugs that avoid cardiotoxic effects, and improving strategies to accurately model congenital heart disease. Our long-term goals include the development of cell surface marker "barcodes" that enable identification and isolation of stage- and subtype-specific cardiomyocytes which can then be tested for their suitability for a wide variety of research and clinical applications.
Of the 5.7 million Americans with heart failure, ~10% will fail to respond to medical therapy and progressively worsen to develop advanced heart failure, for which the only definitive therapy is cardiac transplantation. As the supply of suitable donor hearts is limited to ~2000 per year in the US, the care of advanced heart failure patients requires therapeutic alternatives. For some patients, mechanical circulatory support in the form of a ventricular assist device (VAD) - an implantable pump - can provide short-term or long-term support and in 1-2% of VAD recipients, the heart improves to the point where the pump can be removed, termed myocardial recovery. Currently, it is not possible to predict which heart failure patients will respond to medical therapy alone, which will benefit from VAD support, and which have no potential for recovery. For VAD recipients, quantifying myocardial recovery to inform if, and when, to explant the device requires invasive testing.
Working closely with the uniquely skilled clinical team led by our collaborator Dr. Claudius Mahr at the University of Washington, our long term goals include the development of new, less-invasive strategies to quantitatively measure cardiac recovery and assist physicians in deciding if and when VAD explantation may be considered. Specifically, we are interested in cell surface markers for measuring metabolically-healthy cardiomyocytes and cardiac fibrosis, including markers that can be exploited for use in real-time patient imaging for tracking myocardial mass and structure. Moreover, we are actively pursuing the use of cell-type specific surface proteins as informative circulating biomarkers that can be detected independently of cell death (i.e. necrosis/apoptosis not required for their release) using non-invasive methods.
In 2014, we reported the first evidence that inhibitors of NAMPT (Nicotinamide phosphoribosyltransferase) can be used to selectively eliminate pluripotent stem cells. This has value for preparation of stem cell derivatives for transplantation without risk of tumor formation as well as the production of more homogeneous cultures of desired cells to minimize confounding factors for in vitro functional analyses. We are currently working to understand why pluripotent cells are susceptible to NAMPT inhibition, while differentiated progeny from all three germ lineages, including cardiomyocytes, hepatocytes, fibroblasts, and epithelial cells, are resistant.