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

Abstract

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.

FIG. 1.

Isolation, culture expansion, and verification of human bone marrow mesenchymal stem cells (MSCs). (A) In passage 0 (P0), freshly isolated human MSCs appeared as individual, fibroblast-like cells. (B) During culture expansion up to P3, they showed uniform growth characteristics and still maintained a fibroblast-like morphology. (C) In fluorescence-activated cell sorting analysis, P3 cells from three donors were positive for the typical MSC antigens CD44, CD73, CD90, CD105, and CD166 and negative for the hematopoietic cell markers CD14, CD34, and CD45.

FIG. 2.

Adipogenic differentiation of human MSCs. (A) MSCs were induced into the adipogenic lineage and at day 5, adipogenesis was assessed by oil red O staining of lipid droplets. (B) At day 5, unstimulated control MSCs (NC) did not form any lipid droplets. (C) At day 15, the number of oil red O stained lipid droplets was increased, (D) whereas the control cultures did not form any lipid droplets. (E) Real-time polymerase chain reaction revealed that the adipogenic marker genes peroxisome proliferator-activated receptor gamma (PPARG), and (F) fatty acid binding protein-4 (FABP4) were expressed significantly higher in induced cultures than in primary (day 0) or control (not stimulated) cultures (*P<0.05, **P<0.01, ***P<0.001). Bar: 200 μm. NC, negative control.

FIG. 3.

Matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF-MS) of endo-β-N-acetylglucosaminidase H (Endo H)-released N-glycans. Glycoproteins were isolated from undifferentiated and adipogenically differentiated MSCs (A) using membrane extraction. N-glycans of high-mannose- and hybrid-type were then enzymatically cleaved with Endo H, permethylated, and measured by MALDI-TOF-MS. (A) A representative mass spectrum of Endo H-released N-glycans of undifferentiated human MSCs. (B) The average relative amounts of all Endo H-released N-glycans of undifferentiated human MSCs (black) and adipogenically differentiated MSCs (A) at day 5 (dark gray) and day 15 (light gray) derived from three different preparations. Asterisks mark statistical significant differences between the two sample groups undifferentiated MSCs and 15 days adipogenically differentiated MSCs according to the Mann-Withney U test (*P≤0.05). The black square represents N-acetylglucosamine, gray circle mannose, white circle galactose, black circle glucose and black diamond N-acetylneuraminic acid. H, hexose; N, N-acetylhexosamine; S, N-acetylneuraminic acid.

FIG. 4.

MALDI-TOF-MS of peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F (PNGase F)-released complex-type N-glycans. Glycoproteins were isolated from undifferentiated and adipogenically differentiated MSCs. After isolation of high-mannose- and hybrid-type N-glycans by Endo H digestion, digested glycopeptides were subjected to PNGase F digestion to release complex-type N-glycans, permethylated and measured by MALDI-TOF-MS. A representative mass spectrum of PNGase F-released N-glycans of undifferentiated human MSCs is shown together with the 70 most abundant structures. Polyhexose contaminations are marked with asterisks and are negligible. Unidentified peaks were marked with complex asterisks. The black square represents N-acetylglucosamine, gray circle mannose, white circle galactose, black triangle fucose and black diamond N-acetylneuraminic acid.

FIG. 5.

Quantification of PNGase F-released N-glycans with MALDI-TOF-MS. (A) The average relative amounts of the 48 most abundant PNGase F-released N-glycans of undifferentiated human MSCs (black) and adipogenically differentiated MSCs (A) at day 5 (dark gray) and day 15 (light gray) derived from three different preparations, (B) grouped according to their N-glycan type, antennarity and fucosylation. Statistical significant difference between the two sample groups undifferentiated MSCs and 15 days adipogenically differentiated MSCs was assessed using Mann-Whithney U test (*P≤0.05). The black square represents N-acetylglucosamine, dark gray circle mannose, white circle galactose, black triangle fucose and black diamond N-acetylneuraminic acid. H, hexose; N, N-acetylhexosamine; F, deoxyhexose; S, N-acetylneuraminic acid.

FIG. 6.

An example of a MALDI-TOF/TOF mass spectrum of m/z 2605.3 of the composition S1H5N4F1 derived from human MSCs. Black square represents N-acetylglucosamine, dark gray circle mannose, white circle galactose, black triangle fucose and black diamond N-acetylneuraminic acid.