Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry.

Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.

N-glycosylation profile of undifferentiated and adipogenically differentiated human bone marrow mesenchymal stem cells: towards a next generation of stem cell markers.

Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.

A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures.

Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-ß3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.