Metabolic glycoengineering of mesenchymal stromal cells with N-propanoylmannosamine

Abstract

There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the Nglycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri-and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.

Human Umbilical Cord Blood‐Derived Mesenchymal Stromal Cells Display a Novel Interaction between P‐Selectin and Galectin‐1

“…The experiments were carried out with cells from two to four individual UCB‐MSCs lines, derived from different donors, using cells in passages between 3 and 7. Even though impaired or absent adipogenic differentiation of cord blood MSCs has been indicated in some reports , we have not experienced problems with the adipogenic differentiation of UCB‐MSCs . For the experiments, the cells were quickly detached from cell culture plates by trypsin (TrypLE; Gibco, Life Technologies Ltd., Paisley, UK) or by pronase (Roche Applied Science, Mannheim, Germany) as described .…”

Metabolic Glycoengineering with N‐Acyl Side Chain Modified Mannosamines

“…After growth of MSC in the presence of ManNProp,more than half of their natural Neu5Ac was replaced by Neu5-Prop. [382] Theg lycan structures in the treated cells were altered following incorporation of Neu5Prop.Inparticular,an increase in fucosylated glycan species compared to untreated cells was observed. Interestingly,m ost of the fucosylated glycans were of the Lewis X or blood group Hepitopes,but not sLe X (sLe X ).…”

Cell Surface Sialylated N-Glycan Alterations during Development

“…MSCs are considered very valuable cell sources for stem cell-based therapy because of the probability of teratoma formation in ESCs and iPSCS (73,74). The ability to adhere to plastic surfaces is one of the main characteristics of MSCs and it is also useful for glycoengineering studies (71).…”