Approach for Profiling of Glycosphingolipid Glycosylation
by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced
Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent
Stem Cells and Derived Cardiomyocytes
posted on 2019-05-06, 00:00authored byCharlotte Rossdam, Sarah A. Konze, Astrid Oberbeck, Erdmann Rapp, Rita Gerardy-Schahn, Mark von Itzstein, Falk F. R. Buettner
Application
of human induced pluripotent stem cell-derived cardiomyocytes
(hiPSC-CMs) as tissue transplants in regenerative medicine depends
on cell-surface marker-based characterization and/or purification.
Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed
biomolecules that have been neglected as potential surface markers
for hiPSC-CMs due to significant analytical challenges. Here, we describe
the development of a novel and high-throughput-compatible workflow
for the analysis of GSL-derived glycans based on ceramide glycanase
digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and
multiplexed capillary gel electrophoresis coupled to laser-induced
fluorescence detection (xCGE-LIF). GSL glycans were detected with
highly reproducible migration times after repeated analysis by xCGE-LIF.
We built up a migration time database comprising 38 different glycan
species, and we showed exemplarily that as few as 10 pg of fucosyl
lactotetra was detectable. GSL glycan profiling could be performed
with 105 human induced pluripotent stem cells, and we quantitatively
dissected global alterations of GSL glycosylation of human induced
pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF.
In our study, we observed a general switch from complex GSLs with
lacto- and globo-series core structures comprising the well-known
human pluripotent stem cell marker stage-specific embryonic antigen
3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and
GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being
highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.