Cell-based Heart Regeneration: Proteomic Characterization of Cell Surface Markers in Cardiomyocyte Differentiation

Differentiation of human embryonic stem cells (hESCs) is an invaluable model for the investigation of molecular events during embryonic development, and supplies functional differentiated cells for regenerative therapies. Cardiomyocyte (CM) differentiation of hESC often yields heterogeneous cell populations, making it difficult to perform systematic, quantitative analyses of the differentiation process. There is also a need for improving differentiation and isolation procedures that generate highly pure populations of CM. In the first part of this project, we are isolating surface proteins from hESC, and from differentiated CM, by biotinylating proteins exposed on intact, viable cell population. The cells are lysed under stringent conditions, and the biotinylated proteins were purified by streptavidin affinity purification. The isolated proteins will then be analysed by shotgun proteomics. This approach allows the characterisation of surface proteins that are differentially expressed in hESC and CM. Combination of these differential expression patterns can be used as “molecular barcodes” for the defining cell states and for purification of cells at different stages of differentiation. In the second part of the project, we will develop a novel approach based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) for the characterisation of the differentiation of hESC into cardiomyocytes (CM). Buffer washed cells are directly mixed with a matrix solution and subsequently deposited onto the stainless steel target for MALDI analysis. The resulting mass spectrometric (MS) profiles are highly reproducible and can be used to reflect cell phenotypes. We will test if these MS features can be used as signatures for hESC and differentiating CM, and develop this technique into a reliable, fast, label-free and high throughput method for barcoding hESC differentiation.