The ability to isolate well-characterized human induced pluripotent stem cell derived cardiomyocytes (iCM) from mixed cell populations is a fundamental and major unfulfilled goal of cardiac regenerative medicine, disease modeling and drug discovery efforts. The overall goal of this proposal is to develop a clinically- applicable method for high-throughput isolation of purified ventricular iCM, one of the most sought after cell types for translational applications. The isolation strategy is based on immunophenotyping, wherein functional potential, identity, and isolation of clinically-relevant cells are achieved via panels of antibodies that recognize exposed surface markers. Our preliminary studies have identified more than 40 informative cell surface markers for this purpose and we have developed novel monoclonal antibodies to one novel cell surface protein not previously described in the heart. We have already determined that several of these markers are restricted to specific chambers in the heart, indicating they will be highly informative for sorting subtype-specific iCM in vitro. The work will be carried out in two Aims designed to test the hypothesis that antibodies to cell surface proteins can be used to isolate ventricular iCM (Aim 1) and to determine their functional role in ventricular cardiomyogenesis (Aim 2). This work will contribute a new, non-transgene based high-throughput method for isolating live ventricular cardiomyocytes, which is not possible by current methodologies. The significance of this proposal lies with the functional outcomes enabled by the technological developments, as when an informative marker panel for sorting stage and subtype specific cells is developed, this will enable the reproducible isolation of ventricular iCM suitable for drug-discovery, therapeutic, and disease modeling studies.

Public Health Relevance

This proposal will develop a strategy to use novel cell surface markers to enable the isolation of ventricular cardiomyocytes derived from human induced pluripotent stem cells. The ability to isolate well-characterized cardiomyocytes derived from pluripotent stem cells will facilitate drug-discovery, therapeutic, and disease modeling studies to treat cardiac disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL126785-04
Application #
9656888
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Lee, Albert
Project Start
2016-04-01
Project End
2019-08-12
Budget Start
2019-03-01
Budget End
2019-08-12
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Fujinaka, Chelsea M; Waas, Matthew; Gundry, Rebekah L (2018) Mass Spectrometry-Based Identification of Extracellular Domains of Cell Surface N-Glycoproteins: Defining the Accessible Surfaceome for Immunophenotyping Stem Cells and Their Derivatives. Methods Mol Biol 1722:57-78
Moehring, Francie; Waas, Matthew; Keppel, Theodore R et al. (2018) Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin. J Proteome Res 17:2635-2648
Haverland, Nicole A; Waas, Matthew; Ntai, Ioanna et al. (2017) Cell Surface Proteomics of N-Linked Glycoproteins for Typing of Human Lymphocytes. Proteomics 17:
Mallanna, Sunil K; Waas, Matthew; Duncan, Stephen A et al. (2017) N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm. Proteomics 17:
Kropp, Erin M; Broniowska, Katarzyna A; Waas, Matthew et al. (2017) Cardiomyocyte Differentiation Promotes Cell Survival During Nicotinamide Phosphoribosyltransferase Inhibition Through Increased Maintenance of Cellular Energy Stores. Stem Cells Transl Med 6:1191-1201
Boheler, Kenneth R; Gundry, Rebekah L (2017) Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets. Stem Cells Transl Med 6:131-138
Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk et al. (2016) Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes. Stem Cell Reports 7:543-556
Boheler, Kenneth R; Gundry, Rebekah L (2016) Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets. Stem Cells Transl Med :