Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray

Abstract

Glycans are one of the major building blocks of extracellular vesicles (EVs). However, their roles and applications have not been completely explored. Here, we analyzed the glycome of EVs derived from human induced pluripotent stem cells (hiPSCs) using high-density lectin microarray. The glycan profiles of hiPSC-derived EVs were different from those of non-hiPSC-derived EVs. Moreover, rBC2LCN that shows specific binding to hiPSCs, showed strong specificity for hiPSC-derived EVs but not non-hiPSCs-derived EVs. Further, other hiPSC-specific probes, such as anti-TRA-1-60, anti-SSEA4, and anti-R-10G, exhibited specific, but weaker binding to hiPSC-derived EVs than rBC2LCN. We then developed a sandwich assay using rBC2LCN and a phosphatidylserine receptor, Tim4, to specifically detect hiPSC-derived EVs. The Tim4–rBC2LCN sandwich assay allowed for specific detection of hiPSC-derived EVs but not non-hiPSC-derived EVs, indicating that rBC2LCN could also be used for the specific detection of hiPSC-derived EVs. Together, our findings demonstrate that the characteristic glycan signature of hiPSCs are retained by EVs derived from them. The EV glycome could be novel targets for the identification and characterization of stem cells for use in regenerative medicine.

A Hybrid Approach Enabling Large-Scale Glycomic Analysis of Post-Golgi Vesicles Reveals a Transport Route for Polysaccharides

“…The large-scale aspect is only limited by the number of mAbs present in the collection and can be expanded with the availability of new Abs. In mammalian systems, the use of lectin arrays has been explored to identify surface glycoproteins in extracellular vesicles (Saito et al, 2018;Williams et al, 2018), but this work did not make use of a largescale polysaccharide Ab array. Given the high specificity of mAbs compared with lectins (Cummings and Etzler, 2009), our hybrid approach is very promising for detecting specific glycan populations in isolated vesicles.…”

“…Studies have underlined the role of the TGN and potentially unconventional vesicle trafficking, such as through exosomal compartments, in the plant pathogen response; however, these studies have mainly focused on the roles of proteins or RNA cargo under biotic stress (LaMontagne and Heese, 2017; Rutter and Innes, 2017;Yun and Kwon, 2017;Cai et al, 2018). Exosomal cargo has received a lot of attention with respect to the roles of protein and nucleic acid cargo in human disease (Bae et al, 2018); analysis of their glycomic cargo might provide further valuable insights (Saito et al, 2018;Williams et al, 2018). The current study lays the foundation for dissecting cellular pathways in both plants and animal systems by extending our proteomics perspective with a glycomic perspective.…”

Human Pluripotent Stem Cell-Derived Extracellular Vesicles: Characteristics and Applications

“…61,62 mRNAs in iPSC-EVs were found to contain reprogramming factors Oct3/4, Nanog, Klf4, and c-Myc. Glycome of EVs derived from hiPSCs was analyzed using high-density lectin microarray, 63 which found that the characteristic glycan signature of hiPSCs was captured in the derived EVs. Since glycosylation is a major posttranslational modification and glycan molecules attach to membrane proteins and lipids, glycans located on EV surface play an important role in EV function.…”

“…For example, podocalyxin is a glycoprotein ligand of rBC2LCN, an hiPSC-specific lectin. 63 EVs/exosomes secreted by hiPSCs reprogrammed from urine exfoliated renal epithelial cells were also evaluated as the RNA interference delivery system. 64 The secreted EVs/exosomes expressed CD63, TSG101, and ALIX, the typical exosomal markers.…”

Application of Lectin Microarrays for Biomarker Discovery

“…Moreover, significant affinity of rBC2LCN for a branched O ‐glycan with a H type 3 structure in human iPSCs suggest its potential as a pluripotency marker . Furthermore, rBC2LCN showed strong specificity for human iPSC‐derived extracellular vesicles (EVs) but not for non‐human iPSC‐derived EVs . Recombinant lectins from diverse lectin families were engineered to ensure broader coverage of glycan‐binding specificities.…”