(Glyco)proteomic alterations during cardiomyogenic differentiation of human pluripotent stem cells

• Applicant: Professor Dr. Falk Büttner
• Subject Area: Developmental Biology ; Biochemistry
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394324341

Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide promising tools for in vitro applications such as drug testing or disease modeling and bear the potential to be used in regenerative therapies. Cell replacement e.g. upon disease-induced loss of functional heart muscle tissue in man will essentially depend on pure and well-defined populations of cardiomyocytes. On this background it is of great interest to find conditions that allow the isolation, maintenance, and controlled propagation of cells at specific stages during cardiomyogenic differentiation. Moreover, technologies that would enable the unequivocal identification and isolation of cardiomyocyte subtypes (e.g. atrial-, ventricular-, or nodal-like cardiomyocytes) are of utmost importance in terms of therapeutic application. These aims can be achieved by optimization of culture protocols together with the development of strategies for purification of the desired cell types from mixed populations.Cardiomyogenic differentiation strongly depends on factors of the cell-environment interface including secreted proteins that exert their autocrine or paracrine function by acting on membrane receptors. Aiming at a better understanding of this interplay, we will differentiate hPSCs in vitro into cardiomyocytes and assess the secretome as well as the surfaceome at different stages during cardiomyogenesis by quantitative proteomics (SILAC, LFQ). Secretomics is commonly hampered by an excess of serum proteins, mainly albumin, in cell culture supernatants. To circumvent these limitations, we established serum- and albumin-free conditions for cardiomyogenic differentiation. For assessment of the surfaceome, surface exposed glycoproteins will be captured by means of avidin-biotin interaction upon linking a newly synthesized cleavable biotinylation reagent covalently to glycan moieties of surface glycoproteins. The assessment of secretome and surfaceome are expected to reveal stage-specific signaling pathways that can be exploited to improve the differentiation process.We further intend to identify surface exposed structures on cardiomyocytes that are stage- or subtype-specific, and which can be used for cell type characterization and/or purification. Beyond membrane proteins, N-glycans represent ideal candidates for this purpose. N-glycans of certain differentiation stages or of different cardiomyocyte subtypes will be released from membrane proteins by PNGase F digestion and analyzed by capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). Identified marker structures (proteins or glycans) will subsequently be tested for their potential to either enrich the cell population of interest or for deprivation of unwanted cells.

• DFG Programme: Research Grants