Suvi Natunen

Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM‐MSCs) and umbilical cord blood (UCB‐MSCs). The BM‐MSCs and UCB‐MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB‐MSCs had higher expression levels of α4 integrin (CD49d, VLA‐4), α6 integrin (CD49f, VLA‐6), and the hepatocyte growth factor receptor (c‐Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. STEM CELLS2013;31:317–326

The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope

The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and α2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.

Are globoseries glycosphingolipids SSEA-3 and -4 markers for stem cells derived from human umbilical cord blood?

Umbilical cord blood (UCB) is an efficient and valuable source of hematopoietic stem cells (HSCs) for transplantation. In addition to HSCs it harbours low amounts of mesenchymal stem cells (MSCs). No single marker to identify cord blood-derived stem cells, or to indicate their multipotent phenotype, has been characterized so far. SSEA-3 and -4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells, where SSEA-3 rapidly disappears when the cells start to differentiate. Lately SSEA-3 and -4 have also been observed in MSCs. As there is an ongoing discussion and variation of stem-cell markers between laboratories, we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB. We have performed complementary analysis using gene expression analysis, mass spectrometry and immunochemical methods, including both flow cytometry and immunofluoresence microscopy. SSEA-4, but not SSEA-3, was expressed on MSCs but absent from HSCs. Our findings indicate that SSEA-3 and/or -4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood, as their expression may be altered by cell-culture conditions.

Glycosylation pattern of anti-platelet IgG is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia

Fetal or neonatal alloimmune thrombocytopenia (FNAIT) is a potentially life‐threatening disease where fetal platelets are destroyed by maternal anti‐platelet IgG alloantibodies. The clinical outcome varies from asymptomatic, to petechiae or intracranial haemorrhage, but no marker has shown reliable correlation with severity, making screening for FNAIT impractical and highly inefficient. We recently found IgG Fc‐glycosylation towards platelet and red blood cell antigens to be skewed towards decreased fucosylation, increased galactosylation and sialylation. The lowered core‐fucosylation increases the affinity of the pathogenic antibodies to FcγRIIIa and FcγRIIIb, and hence platelet destruction. Here we analysed the N‐linked glycans of human platelet antigen (HPA)‐1a specific IgG1 with mass spectrometry in large series of FNAIT cases (n = 166) including longitudinal samples (n = 26). Besides a significant decrease in Fc‐fucosylation after the first pregnancy (P = 0·0124), Fc‐glycosylation levels remained stable during and after pregnancy and in subsequent pregnancies. Multiple logistic regression analysis identified anti‐HPA‐1a –fucosylation (P = 0·006) combined with galactosylation (P = 0·021) and antibody level (P = 0·038) correlated with bleeding severity, making these parameters a feasible marker in screening for severe cases of FNAIT.

Nanoscale reversed-phase liquid chromatography–mass spectrometry of permethylated N-glycans

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.

Novel data analysis tool for semiquantitative LC-MS-MS2 profiling of N-glycans

Despite recent technical advances in glycan analysis, the rapidly growing field of glycomics still lacks methods that are high throughput and robust, and yet allow detailed and reliable identification of different glycans. LC-MS-MS2 methods have a large potential for glycan analysis as they enable separation and identification of different glycans, including structural isomers. The major drawback is the complexity of the data with different charge states and adduct combinations. In practice, manual data analysis, still largely used for MALDI-TOF data, is no more achievable for LC-MS-MS2 data. To solve the problem, we developed a glycan analysis software GlycanID for the analysis of LC-MS-MS2 data to identify and profile glycan compositions in combination with existing proteomic software. IgG was used as an example of an individual glycoprotein and extracted cell surface proteins of human fibroblasts as a more complex sample to demonstrate the power of the novel data analysis approach. N-glycans were isolated from the samples and analyzed as permethylated sugar alditols by LC-MS-MS2, permitting semiquantitative glycan profiling. The data analysis consisted of five steps: 1) extraction of LC-MS features and MS2 spectra, 2) mapping potential glycans based on feature distribution, 3) matching the feature masses with a glycan composition database and de novo generated compositions, 4) scoring MS2 spectra with theoretical glycan fragments, and 5) composing the glycan profile for the identified glycan compositions. The resulting N-glycan profile of IgG revealed 28 glycan compositions and was in good correlation with the published IgG profile. More than 50 glycan compositions were reliably identified from the cell surface N-glycan profile of human fibroblasts. Use of the GlycanID software made relatively rapid analysis of complex glycan LC-MS-MS2 data feasible. The results demonstrate that the complexity of glycan LC-MS-MS2 data can be used as an asset to increase the reliability of the identifications.

The i Blood Group Antigen as a Marker for Umbilical Cord Blood-Derived Mesenchymal Stem Cells

Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by β3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.

Metabolic glycoengineering of mesenchymal stromal cells with N-propanoylmannosamine

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 N-glycan 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.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been shown to exert immunomodulatory properties that have great potential in therapies for various inflammatory and autoimmune disorders. However, intravenous delivery of these cells is followed by massive cell entrapment in the lungs and insufficient homing to target tissues or organs. In targeting to tissues, MSCs and other therapeutic cells employ similar mechanisms as leucocytes, including a cascade of rolling and adhesion steps mediated by selectins, integrins and their ligands. However, the mechanisms of MSCs homing are not well understood. We discovered that P‐selectin (CD62P) binds to umbilical cord blood (UCB)‐derived MSCs independently of the previously known sialyl Lewis x (sLex)‐containing ligands such as P‐selectin glycoprotein ligand‐1 (PSGL‐1, CD162). By biochemical assays, we identified galectin‐1 as a novel ligand for P‐selectin. Galectin‐1 has previously been shown to be a key mediator of the immunosuppressive effects of human MSCs. We conclude that this novel interaction is likely to play a major role in the immunomodulatory targeting of human UCB‐derived MSCs.