Exosome surface glycans reflect osteogenic differentiation of mesenchymal stem cells: Profiling by an evanescent field fluorescence-assisted lectin array system

Extracellular vesicles (EVs) carry information between cells in the form of biomolecules. Such molecules have been found to serve as biomarkers. Glycans attached to surface molecules on EVs are involved in their cellular uptake. In this study, we examined glycan profiles of small EVs which are generally termed exosomes before and after osteogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) by an evanescent field fluorescence-assisted (EFF)-lectin array system to discover glycan biomarkers for osteogenic differentiation. We found few differences between exosomes before and after osteogenic differentiation of MSCs in terms of fundamental characteristics such as size, morphology, and exosomal marker proteins. However, specific lectins bound strongly to exosomes from differentiated cells. Exosomes from osteogenically differentiated MSCs bound strongly to fucose- and mannose-binding lectins, especially at a high concentration of exosomes. In summary, we found that several lectins bound to exosomes from differentiated MSCs more strongly than to those from undifferentiated cells using an EFF-lectin array system, indicating that monitoring exosomal surface glycans may identify predictive indexes of osteogenic differentiation.

proteins, extracellular matrix proteins, and proteoglycans 13 . These findings suggest that glycans attached to surface molecules on exosomes are involved in cellular uptake of exosomes. We found that exosomes from human adipose-derived mesenchymal stem cells (MSCs) strongly interact with sialic acid-binding lectins, and sialic acids on exosomes are involved in cellular uptake of exosomes in vitro and in vivo 14 .
To elucidate further functions of exosomal surface glycans in biological events, we examined glycan profiles resulting from MSC differentiation, particularly during osteogenic differentiation. It is well known that MSCs can differentiate into osteoblasts, adipocytes, chondrocytes, neurons, and myocytes, of which the first three cell types have been particularly well studied 15 . Bone remodelling is the process through which bones are continuously regenerated by maintaining the balance of bone resorption and formation to maintain homeostasis 16 . Unbalanced bone remodelling can cause various bone disorders including osteoporosis, Paget disease, and heterotopic ossification 16,17 . Therefore, understanding the bias in the bone remodelling balance is important to avoid the risk of these diseases. Osteoblasts are responsible for bone formation, and some protein markers, such as alkaline phosphatase (ALP), osteocalcin, and type I collagen, are thought to be useful as bone formation biomarkers 18 . However, using these markers has several practical problems: (1) ALP and type I collagen are not specific to bone; (2) Expression may change in response to environmental factors, including the time of day, season, food, diseases, and drugs 19,20 . To overcome these limitations, a new biomarker is needed for bone formation. As mentioned above, exosomal components can be various kinds of biomarkers. However, a limited number of studies have reported the role of exosomal glycans to monitor the cell state [21][22][23] .
Analysis of glycan patterns on EVs from various types of cells was reported by Batista et al. in 2011 24 . They showed that cell-specific and EV-enriched glycan patterns on EVs from T-cells, melanoma, colon cancer, and breast milk. Additionally, Liang et al. found that glycosylation is important for glycoprotein sorting into EVs 25 . Our previous study first showed that comprehensive glycan patterns on intact exosomes can be analysed using an evanescent field fluorescence-assisted (EFF) lectin array system 14 . The advantages of this method especially by using EFF lectin array system are as follows: (1) Glass slides spotted with dozens of lectins (an array with 45 lectins was used for this study) enable determination of glycan patterns simultaneously; (2) Washing steps to remove unbound samples are unnecessary because the area of the evanescent field is confined to the immediate vicinity of the glass (<150 nm); (3) Rapid and simple processing of a small amount of sample are superior to other processes such as those in mass spectrometry, high performance liquid chromatography, nuclear magnetic resonance, and capillary electrophoresis 26 .
Here, we hypothesized that specific glycan biomarkers for osteogenic differentiation of adipose-derived MSCs can be detected by profiling of exosome surface glycans by an EFF-lectin array system. Undifferentiated MSCand osteogenically differentiated MSC-derived exosomes were collected and their surface glycans were analysed using an EFF-lectin array method. We found that several lectins bound to exosomes from differentiated cells more strongly than to those from undifferentiated cells, indicating that monitoring exosomal glycans using an EFF-lectin array may discriminate not only cellular differentiation, but also reprogramming, pluripotency, and the cancer stage.

Results
Isolation and characterisation of MSC-and osteogenically differentiated MSC-derived exosomes. Differentiation from MSCs to osteoblasts was induced by culture in growth medium supplemented with dexamethasone, (+)-sodium L-ascorbate, and β-glycerophosphate disodium. ALP and Alizarin red staining after 21 days of osteogenic induction revealed that cells were successfully differentiated into mineralized osteoblasts ( Fig. 1).

Surface glycan patterns before and after osteogenic differentiation determined by an EFF-lectin array. Cy3 dye-labelled plasma membrane proteins from MSCs or osteogenically differentiated
MSCs and intact exosomes from both cell types were prepared to analyse glycan patterns using the EFF-lectin array. The samples were added to each well on a lectin array containing 45 lectins (Supplementary Table S1), and each fluorescence intensity was normalized to the average intensities of the 45 lectins. As we found in our previous study, exosomes were more strongly bound to α2-6 sialic-acid-recognizing lectins [Sambucus nigra (SNA), Sambucus sieboldiana (SSA), and Trichosanthes japonica (TJA-I)], which was common to both undifferentiated and differentiated MSCs ( Supplementary Figs S2 and 3). Next, we evaluated the difference between exosomes from MSCs and osteogenically differentiated MSCs. Four lectins (ECA (Galβ1-4GlcNAc), BPL (terminal β-GalNAc), WFA (terminal β-GalNAc), and SBA (terminal β-GalNAc)) had statistically higher binding affinities (>3-fold change, *P < 0.05 and **P < 0.01) to exosomes from osteogenically differentiated MSCs than to those from undifferentiated MSCs (Fig. 3). In cell membrane fractions, increasing BPL and WFA binding affinities were observed during osteogenic differentiation (>3-fold change, *P < 0.05 and **P < 0.01, Fig. 4).
To obtain further insights into the affinity for lectins, we investigated the binding curves for each of the 45 lectins with cells or exosomes as a function of the concentrations from undifferentiated and osteogenically differentiated MSCs ( Supplementary Figs S4 and 5). The resulting signals were normalized to the signal of one specific lectin, Lycipersicon esculentum (LEL), whose signal did not change during differentiation, as a reference 27 . www.nature.com/scientificreports www.nature.com/scientificreports/ Similarly to the results shown in Figs 3 and 4, four lectins (ECA, BPL, WFA, and SBA) strongly bound to osteogenically differentiated MSC-derived exosomes and cells with the increase in sample concentration (Fig. 5). Furthermore, other four lectins (PSA, AOL, GNA and HHL) showed higher binding to exosomes from osteogenically differentiated MSCs, and six lectins (AOL, MAL_I, PHA(L), TxLC_I, ABA, and MPA) showed higher binding to cells from osteogenically differentiated MSCs in accordance with the increase in sample concentration (Fig. 6).

Discussion
Because the characteristics of exosomes are known to reflect their cell of origin, they are attracting attention as biomarkers for diagnosis and treatment of diseases (especially cancer) and to determine the cell state. Most studies have concentrated on exosomal miRNAs or proteins, and there is little information about the role of exosomal glycans, mainly because glycans have much more complex structures than those of other biomolecules (e.g. DNAs, miRNAs, and proteins). Furthermore, specialized equipment is required, as well as complicated sample pretreatment, multiple samples, and long analysis time for their structure determination 28 . In our previous study, we found that an EFF-lectin array method is excellent for analysis of exosomal glycans in terms of both operability and sensitivity compared with typical approaches such as MS and HPLC. As another advantage, glycan-lectin interactions can be simply detected by adding intact fluorescence-modified exosomes to the array without any special processing.
Because exosomes, especially those derived from MSCs, are considered to be novel candidates for cell-free therapy 29 , we further showed that the surface glycans on exosomes play important roles in cellular uptake and distribution 14 . To discover additional functions of exosomal glycans, in this study, we examined differences in surface glycan patterns on exosomes before and after induced differentiation of MSCs. Some glycoproteins and glycolipids are known as stem cell markers 30 , and specific changes in surface glycans of stem cells have been reported. In mouse embryonic stem cells, the lectin binding profile is considered to be an ideal indicator of differentiation [31][32][33] . In MSCs, α2-6-sialylated N-glycans have been reported to be an index indicating whether MSCs have differentiation potentials 34 . Furthermore, it has been shown that glycan patterns of osteogenically and adipogenically differentiated MSCs are different from those of undifferentiated MSCs 35,36 . The number of reports on the roles of exosomes in bone remodeling have been increasing in recent years, which mainly focus on the differences in exosomal protein [37][38][39] and miRNA 40,41 profiles between pre-osteoblasts and mineralizing osteoblasts, or evaluate cellular interactions with them. Because growth factors released by osteoblasts promote the proliferation of prostate cancer cells 42 , Morhayim et al. found that osteoblast-derived exosomes are efficiently internalized into prostate cancer cells 38 , and Bilen et al. revealed involvement of cadherin-11 in cellular uptake of exosomes 39 . Furthermore, receptor activator of nuclear factor κB ligand (RANKL), which is expressed in osteoblasts, was identified in osteoblast-derived exosomes. RANKL-positive exosomes were shown to be taken up by osteoclasts, resulting in the induction of osteoclastic differentiation from monocytes 43,44 . These findings suggest that osteoblasts deliver their biological information through exosomes to target cells.
In the current study, we substituted exosomes for plasma membranes to identify novel biomarkers of osteogenic differentiation of MSCs. There were not many differences between exosomes before and after osteogenic differentiation of MSCs in terms of fundamental characteristics (size, morphology, and exosomal marker proteins), while specific lectins strongly bound to exosomes from differentiated cells. An EFF-lectin array method www.nature.com/scientificreports www.nature.com/scientificreports/ assists comprehensive analysis of multiple samples simultaneously and estimates the difference between them. We also evaluated the binding affinities of cell membrane fractions and exosomes using binding curves. This evaluation yielded the same results that both cell membrane fractions and exosomes from osteogenically differentiated MSCs displayed high affinity for four lectins (ECA, BPL, WFA, and SBA). Interestingly, higher affinities interactions were observed between some lectins and exosomes or cell membrane fractions, especially at a high concentration of samples, indicating that analysis of exosome surface glycans by the EFF-lectin array system may identify predictive indexes of osteogenic differentiation.
Few reports have focused on the change in the glycan pattern during biological events. Gerlach et al. examined glycan profiles of urinary extracellular vesicles (uEVs) by a lectin microarray and found that surface glycans on uEVs can be used as biomarkers of polycystic kidney disease 22 . Moyano et al. analysed glycolipids (sulfatides) in plasma exosomes from multiple sclerosis patients and found that C16:0 sulfatide levels were higher than in healthy samples 45    www.nature.com/scientificreports www.nature.com/scientificreports/ ALP and alizarin red S staining. To evaluate osteogenic differentiation, MSCs were seeded at a density of 5000 cells/cm 2 in a 24-well cell culture plate and cultured until 90% confluence. After 21 days of osteogenic induction, ALP and Alizarin Red S staining were performed using a TRACP & ALP double-stain Kit (TAKARA BIO Inc., Shiga, Japan) and Calcification Evaluation Set (Iwai Chemicals Co., Ltd., Tokyo, Japan), according to the manufacturers' instructions, respectively.

Isolation of MSC-and osteogenically differentiated MSC-derived exosomes.
For MSC-derived exosome isolation, subconfluent cells were cultured in fresh growth medium for 48 h before collecting the supernatant. For osteogenically differentiated MSC-derived exosomes, the medium was replaced with fresh medium at