Identification of Cell Surface Proteins for Antibody-Based Selection of Human Embryonic Stem Cell-Derived Cardiomyocytes

Abstract

The absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell types from mixed cell populations to genetic approaches. Here, we describe the use of mass spectrometry (MS)-based proteomics on cell membrane proteins isolated from hESCs that were differentiated into cardiomyocytes to identify candidate proteins for this particular lineage. Quantitative MS distinguished cardiomyocyte-specific plasma membrane proteins that were highly enriched or detected only in cardiomyocytes derived from hESCs and human fetal hearts compared with a heterogeneous pool of hESC-derived differentiated cells. For several candidates, cardiomyocyte-specific expression and cell surface localization were verified by conventional antibody-based methodologies. Using an antibody against elastin microfibril interfacer 2 (EMILIN2), we demonstrate that cardiomyocytes can be sorted from live cell populations. Besides showing that MS-based membrane proteomics is a powerful tool to identify candidate proteins that allow purification of specific cell lineages from heterogeneous populations, this approach generated a plasma membrane proteome profile suggesting signaling pathways that control cell behavior.

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

“…As the success of each of these applications will ultimately require the ability to efficiently and reproducibly select and track pluripotent cell derivatives at the appropriate developmental stage and subtype, new non-transgene based strategies for cell identification and selection will likely be required 55 . In this respect, proteomic technologies are predicted to a play major role in defining new cell surface markers to facilitate the identification and selection of cells [56][57][58][59][60] , similar to what is routine in the hematopoietic hierarchy [61][62][63][64] . However, proteomic technologies currently provide a snapshot averaged across the population of cells sampled.…”

Proteomic Analysis of Human Pluripotent Stem Cell–Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

“…10,12 Microarray-based experiments have been previously performed to characterize the expression profile of hESC-CMs, purified for ventricular lineage or not, for identifying signaling pathways implicated in their differentiation. [13][14][15][16][17] All profiling attempts to date almost exclusively relied on transcriptomic data [13][14][15][16][17] or cell surface protein assessments, 18 but the global proteome of hESCVCMs has not been examined. Furthermore, most hESC-VCM studies focus on pathways important for cardiac differentiation, rather than mechanisms implicated in their developmental maturation.…”

Anti-Proliferative Effects of Human Anti-FZD7 Single Chain Antibodies on Colorectal Cancer Cells

“…These Over-expression of FZD7 in colorectal cancer implies that this receptor may be involved in the carcinogenesis of this cancer and might be critical for the growth of these cells (7). Studies have demonstrated that FZD7 has a vital role in preserving self-renewal capacity of embryonic stem cells and it can be used as a novel embryonic stem cellspecific surface antigen (38). This study evaluated the anti-proliferative potential of anti-FZD7 scFv antibodies.…”