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1. (WO2019045649) METHODS FOR ENRICHING MESENCHYMAL STEM CELLS
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METHODS FOR ENRICHING MESENCHYMAL STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application No. 10201707052R, filed 29 August 2017, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to method of identifying and enriching non-senescent mesenchymal stem cells.

BACKGROUND OF THE INVENTION

[0003] Mesenchymal stem cells (MSCs) can differentiate into a range of cell types. Many clinical trials are under way to evaluate their potential to treat ailments such as myocardial infarction, paralysis and graft-versus-host disease. However, current modes of cell delivery are inefficient, often resulting in low cell retention. This requires the delivery of large numbers (on the order of 109) of cells, which in turn necessitates a high degree of in vitro expansion of donor cells. During this expansion process, some cells will lose their ability to proliferate by entering a state called senescence. Although there are numerous markers and staining methods for detecting when a cell is in a proliferative state, such as detecting proliferating cell nuclear antigens, Ki-67, cyclin D and BrdU staining, the existing methods require access to the cell's organelles or require damaging the cell's DNA, and are thus incompatible with the bioprocessing of living cells. Accordingly, there is a need to provide a live cell-compatible method for identifying and enriching senescent cells from non-senescent cells.

SUMMARY

[0004] In one aspect, the present invention refers to a method of enriching non-senescent mesenchymal stem cell (MSCs), the method comprising contacting a sample comprising MSCs that have been subjected to an in vitro expansion process for MSCs, or a sample comprising MSCs that has not been subjected to an in vitro expansion process for MSCs, or clinical sample comprising MSCs, with a probe that binds to a glycan expressed on the cell surface of MSCs to allow binding of the probe to the glycan; separating non-senescent MSCs bound to the probe from senescent MSCs; removing probe from separated non-senescent MSCs; and collecting non-senescent MSCs; wherein separation of senescent MSCs from non-senescent MSC is based on a difference in expression of glycan at the cell surface of MSCs, wherein non-senescent MSCs express more of the glycan than senescent MSCs.

[0005] In another aspect, the present invention refers to a method for performing quality control for non-senescent mesenchymal stem cell (MSCs), the method comprising contacting a sample comprising MSCs with a probe that binds to a glycan expressed on the cell surface of MSCs, determining the amount

of non-senescent MSCs present in the sample; determining the amount of senescent MSCs in the same sample; wherein the determination of senescent MSCs from non-senescent MSC is based on a difference in expression of glycan at the cell surface of MSCs, wherein non-senescent MSCs express more of the glycan than senescent MSCs; wherein a high amount of non-senescent cells present in the sample is indicative of a sample of acceptable quality; wherein a low amount of non-senescent cells present in the sample is indicative of a sample of inferior quality.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The invention will be better understood with reference to the detailed description when considered in conjunction with the non -limiting examples and the accompanying drawings, in which:

[0007] Fig. 1 shows a workflow of mesenchymal stem cell lysate screening on lectin arrays and the results hereof. (A) Mesenchymal stem cells are lysed in buffer containing Triton X-100. The resulting glycoproteins are then biotinylated using NHS-PEG4-biotin (Thermo Fisher Scientific) in 50: 1 molar excess and cleaned up on Zeba spin desalt columns. The resulting biotinylated glycoproteins are then hybridized with Glycotechnica lectin arrays using their recommended protocol (with modifications to the method based on the assumption that the average size of glycoproteins present in the cell lysate is 30 kDa for the calculation of the molar ratio) and visualized using Streptavidin-Cy3. (B) is an image showing the results of LecChip experiment. Titration of biotinylated protein concentration was performed, showing that 5 μg/μl is the appropriate concentration for the experiment. This is with agreement with the standard protocol. 500 μg/μl and 50 μg/μl concentration were saturated. There was a high amount of variability between two conditions (Proliferative & senescent). In general, every lectin showed a binding difference, therefore thresholding was required.

[0008] Fig. 2 shows different lectin array hits. Binding signal of proliferating and senescent Mesenchymal stem cell lysates on lectin arrays were in general different for each lectin, thus making thresholding necessary to extract useful signal. 6 lectins showed at least 30% difference in binding signal between the two conditions at p < 0.05. GSL II binds to agalactosyl glycans; GSL-I A4 to a-GalNAc; LCA to core fucose; LTL to Lewis X and H-type 2; PNA to T-antigen; and PSA to core fucose. Of these hits, only PNA was successfully validated using live cell immunostaining. (A) shows a column graphs depicting the results of the referenced assay when analysed with the criteria of a 20% difference cut-off at p value< 0.05 (Student T-Test), showing that there were a large number of results to validate. (B) shows a column graphs depicting the results of the referenced assay when analysed with the criteria of a 30% difference threshold at p value< 0.05 (Student T-Test), showing a reduced number of results. Taken together, these results indicate that senescent cells may have more core fucose than proliferating cells.

[0009] Fig. 3 shows the results of the staining of live mesenchymal stem cells with peanut agglutinin (PNA). In the absence of free lactose, which acts as a competitive competitor of PNA binding, robust PNA staining (light grey) counterstained by DAPI nuclear staining (dark grey) was observed in proliferating mesenchymal stem cell cultures, but not in senescent mesenchymal stem cells. Brightfield images confirmed that senescent Mesenchymal stem cell cultures expressed the established intracellular senescence- associated marker β-galactosidase (darker regions), whereas proliferating cells were free of staining. In the presence of free lactose, PNA staining was largely abolished, confirming that the observed PNA binding was sugar-specific. Images were taken at 20x magnification and the cells had been fixed using formalin. Senescence associated β-galactosidase staining was performed overnight and same field images were generated the next day. Of the hits identified from the lectin experiment, the results indicated that PNA could be validated using live-cells immunostaining. To show if any of the observed PNA binding was non-specific, 100 mM of lactose was used as a competitive inhibitor of PNA's specific binding. 100 mM lactose abolished all observable binding, indicating that the binding was sugar-specific.

[0010] Fig. 4 shows a schematic of the N and O-glycomics workflow. MSC cells are lysed in buffer containing CHAPS detergent under reducing conditions and digested into glycopeptides using trypsin. N-glycans are then specifically liberated using PNGase-F, and separated from the remaining O-glycopeptides using C-18 solid phase extraction. O-glycopeptides are then liberated as reduced alditols through beta elimination using published procedures. N-glycans are tagged with 2-AB fluorescent dye prior to analysis on the Waters UNIFI Xevo system for superior quantification and increased ionization efficiency. O-glycan alditols are permethylated to increase separability and ionization efficiency prior to analysis on an Agilent 6550 iFunnel UPLC-qToF. N-glycan signals were quantified by integrating the area under the fluorescence peak associated with each glycan. N-glycans were identified based on normalized retention time matching against GlycoBase as well as observed MSI signal generated from each peak. O-glycan alditol signals were isolated by molecular feature extraction, and quantified by total ion count.

[0011] Fig. 5 shows data of the N-glycan analysis results. Panel A shows representative fluorescence traces for 2-AB labelled N-glycans isolated from proliferating and senescent cells. The major glycan species are shown. In total, approximately 70 structures were identified. No significant difference in any glycan structure or class of glycan structures (Panel B) was observed between proliferating and senescent cells.

[0012] Fig. 6 shows data of the O-glycan analysis results. Representative extracted ion chromatograms of permethylated O-glycan alditols are shown for both proliferating and senescent cells. Peaks that were putatively identified as T-antigen (15.9 minutes) and sialyl-T-antigen (20.3 minutes) are marked with arrows, and were found to be expressed at significantly different levels in the two experimental conditions. Subsequently, experiments were conducted using O-glycan standards to verify the identities of these peaks. Note that the HexNAc peak at 13.12 minutes was not included in the analysis since the analytical technique is not capable of distinguishing GlcNAc from GalNAc, and the sample preparation method used is not optimized to retain monosaccharides, thus leading to potentially large variations in monosaccharide signal.

[0013] Fig. 7 shows the MS-MS fragmentation of various standards, which have been named accordingly. (A) shows the MS-MS fragmentation of Gal-β 1,3 -GlcNAc standard. This permethylated

glycan alditol standard exhibited a retention time of 15.2 minutes, and showed a very small (non-observable) dehydrated GlcNAc fragment at 230.14, and a significant hydrated GlcNAc fragment at 276.18. The relatively low intensity of 230.14 relative to 276.18 is known to be characteristic of a β1-3 linkage. (B) shows the MS-MS fragmentation of Gal- i,4-GlcNAc standard. This permethylated glycan alditol standard exhibited a retention time of 15.5 minutes, and showed a dehydrated GlcNAc fragment at 230.14 of similar intensity as a hydrated GlcNAc fragment at 276.18. The comparable intensity of 230.14 relative to 276.18 is known to be characteristic of a βΐ -4 linkage. (C) shows the MS-MS fragmentation of Gal- i,3-GalNAc standard (T-antigen). This permethylated glycan alditol standard exhibited a retention time of 15.9 minutes, which matches the retention time of the putative T-antigen peak in the MSC samples. Furthermore, it showed a very small (non-observable) dehydrated GlcNAc fragment at 230.14, and a significant hydrated GlcNAc fragment at 276.18. The relatively low intensity of 230.14 relative to 276.18 is known to be characteristic of a β 1 -3 linkage. (D) shows the MS-MS fragmentation of Neu5Ac-a2,3-Gal- i,3-GalNAc standard from Fetuin O-glycan library (sialyl-T-antigen). This permethylated glycan alditol standard exhibited a retention time of 20.3 minutes, which matches the retention time of the putative sialyl-T-antigen peak in the MSC samples. Sialic acid fragments showing progressive loss of methanol were observed. (E) shows the MS -MS fragmentation of Hex-HexNAc peak eluting at 15.5 minutes in a mesenchymal stem cell sample. This minor Hex -HexNAc peak exhibited a retention time that matched that of the Gal- i,4-GlcNac standard. Furthermore, it showed dehydrated HexNAc fragment at 230.14 of comparable intensity to the hydrated HexNAc fragment at 276.18, which is characteristic of a β 1-4 linkage. Therefore this peak is identified as Gal- i,4-GlcNac. (F) shows the MS-MS fragmentation of Hex-HexNAc peak eluting at 15.9 minutes in a mesenchymal stem cell sample. This major Hex-HexNAc peak exhibited a retention time that matched that of the Gal- i,3-GalNac standard (T-antigen). Furthermore, it showed dehydrated HexNAc fragment at 230.14 of much lower intensity compared to the hydrated HexNAc fragment at 276.18, which is characteristic of a β1-3 linkage. Therefore this peak is identified as Gal- i,3-GalNac (T- antigen). (G) shows the MS-MS fragmentation of Neu5Ac-Hex-HexNAc peak eluting at 20.3 minutes in a mesenchymal stem cell sample. This peak exhibited a retention time that matched that of the Neu5Ac-a2,3-Gal- i,3-GalNac standard (sialyl-T-antigen). Furthermore, Neu5Ac fragments showing progressive loss of methanol at 376.20 and 344.17 were observed, confirming that this peak is sialyl-T-antigen.

[0014] Fig. 8 shows the result of the quantitation of O-glycan analysis. The column graph depicts the volume percentage of a particular glycan in comparison between proliferative and senescent cells, according to the neutral mass of the glycan. There was a statistically significant (p < 0.05) 10% decrease in T-antigen in senescent cells relative to proliferating cells (p < 0.05, one-tailed Student's t-test). There was also a statistically significant (p < 0.025) 10% increase in sialyl-T-antigen in senescent cells relative to proliferating cells. Statistical tests were performed using one -tailed Student's T-test (p<0.05, one-tailed Student's t-test; n=7).

[0015] Fig. 9 shows data generated using qPCR quantitation on genes encoding for key glycosyltransferases in the synthesis of T and sialyl-T-antigens. ST3Gal-I and ST3Gal-IV, which are glycosyltransferases involved in extending T-antigen with sialic acid, were expressed at biologically similar levels in proliferating and senescent cells (2 -fold difference is the minimum required for biological significance). With regard to the cut-off chosen for significant difference, a two-fold qPCR result was consistent with the idea that the T-antigen is down-regulated in senescent cells, resulting in a higher proportion of sialyl-T-antigen. In contrast, ClGalT-1 expression (which is responsible for making the Core I structure) was 4.1 -fold higher in proliferating relative to senescent cells. These gene expression results are consistent with the gly comics results, which indicate CIGalTl to be down-regulated in senescent cells: a lower ClGalT-1 expression level would decrease the percentage of T- antigen in the overall glycan pool in senescent cells. Since ST3Gal-l and ST3Gal-IV expression levels are unchanged, a higher proportion of T-antigen glycans will be extended with sialic acid in senescent cells.

[0016] Fig. 10 shows images of Western Blot analyses performed on mesenchymal stem cell lysates using peanut agglutinin (PNA) as a probe. Western blot staining that peanut agglutinin binds to discrete bands, indicating that there are a limited number of glycoproteins expressing T-antigen at sufficient density for peanut agglutinin binding. Bands at 15 kDa, 50 kDa, 100 kDa and 120 kDa were bound more intensely in proliferating cell lysate as compared to senescent cell lysate, indicating differential staining.

[0017] Fig. 11 shows the results of mesenchymal stem cell N-Glycomics and 0-Glycomics analysis. The column graph depicts the percentage of N-glycans in proliferating and senescent cells. No differences in N-glycans were observed when classified according to type of N-Glycans or when using hierarchal clustering.

[0018] Fig. 12 shows the results of the 0-Gly comics analysis. Mesenchymal stem cell (MSC) O-glycomics was performed on Agilent C18-qToF. (A) shows the extracted O-glycans on chromatograph and the structures annotated are putative based on what is known of O-glycans structures. These are considered to be compositions until verification using glycans standard. T-antigen is marked with an arrow (15.9 min), and the sialyl T-antigen also with an arrow (20.3 min). These structures are related structures. (B) shows column graphs depicting the quantification of O-glycans. It is further shown that the T-antigen can be bound by peanut agglutinin (PNA), and that the T-antigen and the sialyl T-antigen are statistically different between proliferative and senescent cells. In other words, (B) shows the results of quantification of O-glycan alditol relative volume percentages (N = 7). * indicates p < 0.05, 2-tailed Student's T-test assuming unequal variances. A legend of the glycan structural symbols according to the Oxford nomenclature can be found in Fig. 13.

[0019] Fig. 13 shows a schematic of the glycan structural symbols (according to the Oxford nomenclature) and the lines used to denote a- and β-linkages.

[0020] Fig. 14 shows the results of the analysis of fetal bone marrow -derived mesenchymal stem cells. (A) shows cells that were cultured to senescence by repeated 1 :30 passaging on tissue culture plastic. Passaged MSCs exhibited a linear increase in cumulative population doublings and a constant

doubling time over time in culture until P21. Here, cells showed a sudden increase in doubling time from 20+/X to 250+/-X hours, indicating that a large proportion had entered a senescent state. (B) shows the beta-galactosidase staining and (C) shows quantitative nuclear DNA staining, both of which confirm that the cells had entered a senescent state. Quantitative nuclear DNA staining data showed that there were very few P21 cells in the proliferative S-phase, and most cells were arrested in the G0/G1 or G2 phase. (D) shows cells cultured under differentiation conditions, depicting that the cells showed impaired differentiation towards the bone, adipocyte and chondrocyte lineages compared to proliferating mesenchymal stem cells.

[0021] Fig. 15 shows representative fluorescence chromatogram profiles of 2AB -labelled N-glycans separated by HILIC-UPLC. Typical of whole cell lysates, the profiles of both proliferating and senescent cells are dominated by high mannose glycans.

[0022] Fig. 16 shows a Western blot image of Collagen type I expression is reduced in senescent hMSCs. Western blot was carried out on non-reduced proliferating (P8) and senescent (P25) human mesenchymal stem cell (hMSC) whole cell lysates using anti -collagen type I antibody as a probe.

[0023] Fig. 17 shows the results of a Lectin array screening and live cell immunostaining, showing that reduced T-antigen expression is present in senescent mesenchymal stem cells (MSCs). (A) shows column graphs depicting the relative fluorescence in whole-cell lysates from either proliferating or senescent mesenchymal stem cells. These whole cell lysates were biotinylated, allowed to hybridize with Glycotechnica lectin arrays, and then visualized with streptavidin-Cy3. The displayed results are the subset of lectin probes that showed > 30% difference between proliferating and senescent groups at p < 0.05. Of the six "hits", only peanut agglutinin (PNA, dotted box) could be validated using live cell immunostaining. (B) shows micrograph images of 4x magnification image of cells 120 hours after seeding, showing structures in proliferating cell cultures stained by PNA-488 (top panel). Senescent mesenchymal stem cell monolayers showed no staining. (C, i - iii) Proliferating MSC monolayers stained with PNA-488 at 30 minutes, 6 hours and 120 hours after trypsinization and seeding onto tissue culture plastic. It is noted that increasing amounts of PNA staining could be seen with time (C, top panels i to iii). Images were taken at lOx magnification, and mean fluorescence intensity from three independent experiments is quantified in panel (C, iv). Abbreviations - GSLII: Griffona Simplicifolia Lectin II. GSL I-A4: Griffona Simplicifolia Lectin I. LCA: Lens Culinaris Agglutinin. LTL: Lotus Tetragonolobus Lectin. PNA: Peanut agglutinin. PSA: Pisum Sativum Agglutinin.

[0024] Fig. 18 shows information regarding the expression of glycosyltransferase genes ("glycogenes"). (A) shows a schematic showing the glycosidic linkages formed by the glycosyltransferases involved in T- and sT-antigen synthesis. (B) shows a column graph depicting the fold-change in glycogene expression in proliferating relative to senescent mesenchymal stem cells. Dotted line: threshold of biological significance (2-fold). * indicates p < 0.05, 2-tailed Student's T-test assuming unequal variances.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0025] Mesenchymal stem cells (MSCs) are heterogeneous populations of cells that are capable of both self-renewal and differentiation into a multitude of lineages, including but not limited to, osteoblasts, adipocytes and chondrocytes. Mesenchymal stem cells are considered to be vital for tissue homeostasis, haematopoiesis and immunomodulation, and can be isolated from various sources including bone marrow aspirate, adipose tissue and peripheral blood based on, for example, the positive expression of CD73, CD90 and CD105; and/or, for example, the negative expression of CD14, CD19, CD34, CD45, and HLA class II; and other criteria, for example, adherence to tissue culture plastic. Mesenchymal stem cells are therefore highly attractive for cell therapies, and there have been many clinical trials in recent years exploring their use particularly in the treatment of heart disease, graft versus host disease, diabetes and peripheral arterial disease. Although some clinical trials have found benefits to mesenchymal stem cell treatments, the evidence is by no means unequivocal and there are doubts regarding the efficacy of mesenchymal stem cell injections. There exists a wide variability in quality and reproducibility of mesenchymal stem cells, which is understood to be a contributing factor to the irreproducibility of clinical outcomes.

[0026] To increase the quality and safety of mesenchymal stem cell based cell therapies, there is increasing demand for reproducible, rigorous bioprocessing methods for scalable in vitro expansion and critical quality attribute characterization. Cell therapies often require very large numbers of cells - for example, a phase II clinical trial evaluating the efficacy of mesenchymal stem cells in graft-versus-host-disease infused approximately lxlO8 cells per patient. In another example, a clinical trial evaluating mesenchymal stem cells in myocardial infarction infused more than lxlO8 cells per patient. In vitro culture and expansion are therefore unavoidable parts of the mesenchymal stem cell supply chain, and there are perennial concerns that these methods risk the introduction of defects into the cellular product such as, but not limited to, chromosomal abnormalities, xenogenic material, allergenic substances or microbial contaminants.

[0027] It has been shown that senescent mesenchymal stem cells can be an unintentional by-product included with injected cells. As cells age and divide, they acquire senescence-associated phenotypes characterized by, for example, cell cycle arrest, chromatin reorganization, altered gene expression and the secretion of pro-inflammatory and matrix-degrading molecules. Naturally-occurring senescent cells are associated with age-related organ degradation and failure, thus their inclusion with cell therapies is thought to contribute to poor efficacy. Depleting senescent cells using cell sorting is one possible way to overcome this problem, however there is currently no reliable cell surface marker of proliferation or senescence. The most commonly-used senescence marker is Senescence -Associated β-Galactosidase, which can be visualized by the chromogenic substrate X-gal. However, the disadvantage of this approach is that X-gal stains accumulate in intracellular lysosomes, which requires fixing of the cells prior to staining. Consequently, while this X-gal method can be used for quality control, it cannot be used to deplete senescent cells in living samples.

[0028] Cell surface glycans are highly complex carbohydrate molecules whose structures are exquisitely sensitive to changes in the spatial and temporal expression of protein -modifying enzymes, such as but not limited to, glycosyltransf erases. Therefore, cell surface glycans can be used as a source of aging and disease-related biomarkers. Given that senescence is known to strongly influence cellular metabolism and gene expression, the inventors have shown that there would be glycan changes associated with senescence. As shown herein, the glycomes of proliferating and senescent fetal bone marrow-derived mesenchymal stem cells have been studied using a variety of orthogonal approaches, including lectin microarrays and high-resolution LC-MS to determine the existence of glycan biomarkers associated with senescence, and the use of the same in conjunction with glycan-specific lectins for the depletion of senescent cells.

[0029] Thus, mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine, as they can differentiate into a range of cell types including, but not limited to bone, cartilage and fat. However, in order to be effective in therapy, cells used in such therapies need to be provided in a high number. Due to the small size of heterologous and autologous samples, expansion of cells in vitro and prior to use in therapy is required. There are, however, cellular mechanisms which limit the number of times a (for example, human or mammalian) cell will divide before entering a state called senescence. The number of times a cell will divide before arresting is commonly described as the Hayflick limit.

[0030] Senescence is also referred to as cellular or replicative senescence, and describes a state in which cells lose their proliferative capabilities and cease to divide. This state is characterised by, for example, hypertrophic cell morphology, low cell mobility, and the secretion of inflammatory paracrine factors that produce or accentuate some of the effects commonly observed in, for example (whole organism) aging. This can lead to symptoms of aging, such as, but not limited to, the impairment of organ function or outright organ failure. Having said that, it is not to say that senescent cells are devoid of life. Terms such as "dormant" or "arrested" can also be used to describe cells which are in a senescent state. The presence of senescent cells has been shown to affect, for example, tumour suppression, wound healing and potentially embryonic/placental development, and, without being bound by theory, is thought to play a pathological role in age-related or geriatric diseases

[0031] It has been shown that the elimination of senescent cells can mitigate the effects of, for example, aging. Thus, eliminating senescent mesenchymal stem cells from a donor cell population prior to cell delivery is understood to result in superior therapeutic efficacy of said donor cell population.

[0032] Disclosed herein is a method for identifying non-senescent mesenchymal stem cells. Also disclosed herein are methods for separating and/or enriching non-senescent mesenchymal stem cells. These methods, as disclosed herein, are based on the presence of one or more cell surface markers, in other words, cell surface marker expression. In one example, the cell surface marker is a glycan. As used herein, the term "glycan" refers to compounds consisting of a large number of monosaccharides linked glycosidically. That is to say that the monosaccharides are linked between the hemiacetal or the hemiketal group of one saccharide and the hydroxyl group of another compound.

[0033] As used herein, the term "glycan" is used synonymously with the term polysaccharide. Glycans can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched. In general, glycans are found on the exterior surface of cells, whereby O- and N-linked glycans are very common in eukaryotes. For example, glycans can comprise solely of O-glycosidic linkages of monosaccharides. As another example, cellulose is considered to be a glucan (a specific form of a glycan), composed of P-l,4-linked D-glucose. As another example, chitin is defined to be a glycan composed of β- 1,4-linked N-acetyl-D-glucosamine. Thus, in one example, the glycan disclosed herein is an N-linked or an O-linked glycan. In another example, the glycan is an O-linked glycan. In yet another example, the glycan is linked via an a-glycosidic bond or a β-glycosidic bond. In a further example, the glycan is linked via a β-glycosidic bond. In yet another example, the glycan is an O-linked glycan linked via a β-glycosidic bond. In another example, the glycan is a 1,3-linked glycan or a 1,4-linked glycan. In yet another example, the glycan is a 1,4-linked glycan. In a further example, the glycan is a β 1,3 -linked glycan or a β 1,4-linked glycan. In one example, the glycan is a β 1,3 -linked glycan. Glycans as disclosed herein can be, but are not limited to, Galβl-4GalNAcβl-R (N-(2,4-dihydroxy-6(hydroxymethyl)-5-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3-yl)acetamide), Gal- pi,3-GalNAc (N-(2,5-dihydroxy-6-(hydroxymethyl)-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3-yl)acetamide; also known as T-antigen), Ga^l-3GalNAcal-Ser/Thr (0-(3-acetamido-5-hydroxy-6-(hydroxymethyl)-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)serine/threonine), (Sia)Ga^l-3GalNAcal-Ser (5-acetanndo-2-((2-((3-acetamido-2-(2-amino-2-carboxyethoxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)-4-hydroxy-6-(l,2,2-trihydroxyethyl)tetrahydro-2H-pyran-2-carboxylic acid), (Sia)Ga^l-3GalNAcal-Thr, GalNAca-Ser/Thr (0-(3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)serine/threonine), or combinations thereof. In one example, the glycan is Gal^l,3-GalNAc (N-(2,5-dihydroxy-6-(hydroxymethyl)-4-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetr ahydro-2H-pyr an-3 -yl) acetamide) .

[0034] The present application shows that, for example, the glycan T-antigen (also known as Gal-βl,3-GalNAc), which is ubiquitously expressed on mesenchymal stem cell surfaces, is down -regulated during senescence.

[0035] Although there are numerous markers and staining methods for cell proliferation, such as proliferating cell nuclear antigen, Ki-67, cyclin D and BrdU staining, all the existing methods require access to the cell's organelles or require damaging the cell's DNA, and are thus incompatible with the bioprocessing of living cells. In contrast, T-antigen (Gal^l,3-GalNAc) is present on the cell surface and is thus readily bound by extracellular probes such as, but not limited to lectins. Lectin binding to the glycan as disclosed herein is simply reversed by washing with free lactose. This simplicity also makes the method disclosed herein compatible with cGMP cell manufacturing processes.

[0036] As used herein, the term "lectin" refers to carbohydrate -binding proteins, in other words macromolecules, which are highly specific for sugar moieties of other molecules. Lectins serve many different biological functions in animals, but in light of the present disclosure, they bind soluble extracellular and intercellular glycoproteins, for example, a soluble carbohydrate or to a carbohydrate moiety that is a part of a glycoprotein or glycolipid. Most lectins do not possess any enzymatic activity. They typically agglutinate certain animal cells and/or precipitate glycoconjugates.

[0037] Thus, in one example, the probe used for detecting the glycan as disclosed herein is lectin. In another example, the lectin is an agglutinin. This means that binding of the lectin causes the bound proteins or glycans to clump or coagulate together. Non-limiting examples of the lectins, as disclosed herein, are, but are not limited to, peanut agglutinin (PNA), jacalin (AIL), ricinus communis agglutinin (RCA; Ricin), hairy vetch lectin (VVL), agaricus bisporus lectin, abrus precatorius agglutinin, agropyrum repens lectin, amaranthin, amaranthus leucocarpus lectin, frutalin (derived from the same plant as jacalin), bauhinia purpurea lectin, codium fragile agglutinin, gleheda, lactarius deliciosus lectin, lactarius deterrimus lectin, laelia autumnalis lectin, maclura pomifera agglutinin, bitter gourd seed lectin, morniga-G, sophora japonica lectin, vateirea macrocarpa lectin, vicia graminea lectin, and mistletoe lectin I, and combinations thereof. In one example, the lectin is peanut agglutinin (PNA).

[0038] By way of an example, the lectin peanut agglutinin has been shown to specifically and reversibly bind to the glycan T-antigen (Gal- i,3-GalNAc). It has been shown that peanut agglutinin (PNA) stains proliferating cells more strongly than senescent cells. Thus, without being bound by theory, it is thought that the use of peanut agglutinin as a counter-stain that can be used in conjunction with, for example, senescence-specific fluorescent small molecules. For example, peanut agglutinin can be competitively removed by including, for example, free lactose in the culture medium.

[0039] The above offers a method of defining proliferating and senescent cell populations in donor mesenchymal stem cell populations, based on which such donor cell samples could be further purified, giving significant benefits to mesenchymal stem cell-based regenerative cell therapies.

[0040] There are methods known in the art, for example mass spectrometry, which can be used to identify the presence of cell-surface bound glycans. However, using such a method in routine quality control screening of mesenchymal stem cells can be a costly and time inefficient method. Thus, the methods disclosed herein detect changes in mesenchymal stem cell surface glycans by using lectin arrays. Also disclosed herein are methods using lectin probes (for example, western blots of whole cell lysates) to detect distinctive changes in the "glycan signature" on the surface of mesenchymal stem cells, as these cells progress towards senescence.

[0041] In another example, the method disclosed herein is used to distinguish proliferating and senescent mesenchymal stem cells based on the differential expression of the O-linked glycan T-antigen (Gal- i,3-GalNAc), a marker present on the cell surface. This method improves cell therapies in two ways: firstly, as a routine quality control screening methodology, secondly, by allowing senescent cells to be separated out prior to treating subjects in need thereof.

[0042] Also disclosed herein is a method for distinguishing proliferating and senescent mesenchymal stem cells based on the differential expression of the O-linked glycan T-antigen (Gal- i,3-GalNAc), a marker present on the cell surface, predominantly on proliferating cells. The Gal- i,3-GalNAc glycan has been found to be down-regulated in mesenchymal stem cells during senescence.

[0043] The methods disclosed herein are based on, for example, the capability of lectin to bind to a specific glycan found on the surface of proliferating cells. For example, when enriching samples to contain substantially only non-senescent cells, the binding of lectin to the glycan is sufficient for detecting the presence of non-senescent cells. In other words, if no lectin-glycan binding can be detected in the sample, then the sample is considered to contain no proliferating cells, or, if at all, a negligible amount of proliferating cells.

[0044] The presently described methods are performed on intact, viable cells. The methods disclosed herein do not result in the destruction of cells, meaning that once isolated and/or enriched, these cells can be further expanded (that is allowed to grow in culture in order to increase cell numbers).

[0045] Thus, in one example, the method as disclosed herein refers to a method of enriching non-senescent mesenchymal stem cells (MSCs), the method comprising contacting a sample comprising mesenchymal stem cells that have been subjected to an in vitro expansion process for mesenchymal stem cells, or a sample comprising mesenchymal stem cells that have not been subjected to an in vitro expansion process for mesenchymal stem cells, or clinical sample comprising mesenchymal stem cells, with a probe that binds to a glycan expressed on the cell surface of mesenchymal stem cells to allow binding of the probe to the glycan; separating non-senescent mesenchymal stem cells bound to the probe from senescent mesenchymal stem cells; removing probe from separated non-senescent mesenchymal stem cells; and collecting non-senescent mesenchymal stem cells; wherein separation of senescent mesenchymal stem cells from non-senescent mesenchymal stem cells is based on a difference in expression of glycan at the cell surface of mesenchymal stem cells, wherein non-senescent mesenchymal stem cells express more of the glycan than senescent mesenchymal stem cells.

[0046] In one example, the method as disclosed herein is used to perform quality control for non-senescent mesenchymal stem cells in a sample. As previously mentioned, peanut agglutinin stains proliferating cells more strongly than senescent cells, thereby indicating that a higher concentration of lectins can be found on the cell surface of proliferating cells. Accordingly, it is understood that a low concentration of lectins indicates senescent cells. Thus, based on this understanding, in the methods disclosed herein, a difference in expression or presence of the glycan on the cell surface of the mesenchymal stem cells can refer to an increase or decrease of the concentration of glycans on the cell surface. This increase or decrease is a relative measure determined in comparison to cells of a known passage number. Thus, in one example, non-senescent mesenchymal stem cells express at least 10%, at least 25%, at least 50%, at least 100%, at least 200% or at least 300% more or more of the glycan than senescent mesenchymal stem cells. In another example, non-senescent mesenchymal stem cells express between 10% to 50%, between 20% to 60%, between 30% to 70%, between 40% to 80%, between 50%

to 100%, between 90% to 150%, or between 100% to 200% more of the glycan than senescent mesenchymal stem cells.

[0047] Thus, in another example, a method for performing quality control for non-senescent mesenchymal stem cells is disclosed. Such a method comprises the steps of contacting a sample comprising non-senescent mesenchymal stem cells with a probe that binds to a glycan expressed on the cell surface of non-senescent mesenchymal stem cells, determining the amount of non-senescent mesenchymal stem cells present in the sample; determining the amount of senescent non-senescent mesenchymal stem cells in the same sample.

[0048] The determination of senescent non-senescent mesenchymal stem cells from non-senescent mesenchymal stem cells is based on a difference in expression of glycan at the cell surface of non-senescent mesenchymal stem cells, whereby non-senescent mesenchymal stem cells express more of the glycan than senescent non-senescent mesenchymal stem cells. In addition, a high amount of non-senescent cells present in the sample is indicative of a sample of acceptable quality. Conversely, a low amount of non-senescent cells present in the sample is indicative of a sample of inferior quality.

[0049] Thus, in on example, there is disclosed a method for performing quality control for non-senescent mesenchymal stem cells comprising the steps of contacting a sample comprising non-senescent mesenchymal stem cells with a probe that binds to a glycan expressed on the cell surface of non-senescent mesenchymal stem cells, determining the amount of non-senescent mesenchymal stem cells present in the sample; determining the amount of senescent non-senescent mesenchymal stem cells in the same sample, wherein the determination of senescent non-senescent mesenchymal stem cells from non-senescent mesenchymal stem cells is based on a difference in expression of glycan at the cell surface of non-senescent mesenchymal stem cells, wherein non-senescent mesenchymal stem cells express more of the glycan than senescent non-senescent mesenchymal stem cells: wherein a high amount of non-senescent cells present in the sample is indicative of a sample of acceptable quality; wherein a low amount of non-senescent cells present in the sample is indicative of a sample of inferior quality.

[0050] In one example, the sample quality is determined by quantifying the percentage of non-senescent mesenchymal stem cells in the sample. This percentage, by way of an example, is based on determining the total number of cells in a sample, along with the number of cells to which the lectin as disclosed herein has bound to the glycan as disclosed herein. Based on this information, a person skilled in the art would be able to determine the percentage of both the non-senescent mesenchymal stem cells, as well as the percentage of senescent mesenchymal stem cells in the sample. Thus, a sample of acceptable quality is considered to have a high percentage of non-senescent mesenchymal stem cells.

[0051] In terms of quality control, the percentage of non-senescent mesenchymal stem cells in a sample can be, but is not limited to, about 75% to about 100%, from about 89% to about 100%, from about 96% to about 100%, from about 75% to 95%, from about 80% to 90%, from about 89% to 94% from about 86% to 99%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least about 99.9% of non- senescent mesenchymal stem cells to senescent mesenchymal stem cells. In another example, the percentage is at least 90% non-senescent mesenchymal stem cells in a sample.

[0052] That is to say, in one example, that a sample is considered to be of high quality when the amount of non-senescent mesenchymal stem cells exceeds the amount of senescent mesenchymal stem cells that may be or are present in the sample. In another example, a sample is considered to be of high quality when the amount of non-senescent mesenchymal stem cells is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the total number of cells present in the sample.

[0053] As used herein, the term sample refers to liquid or solid samples thought or suspected to contain the targeted cells. In one example, the sample can be a sample known to contain mesenchymal stem cells. In another example, the sample comprises mesenchymal stem cells, whereby the sample has been subjected to an in vitro expansion process for mesenchymal stem cells. In another example, the sample comprises mesenchymal stem cells, whereby the sample has not been subjected to an in vitro expansion process for mesenchymal stem cells. Such samples which have not undergone any in vitro expansion process can also be referred to as natural samples. As used herein, the term "clinical sample" refers to biopsy samples, samples retained after surgery, donor tissue, including but not limited to autologous and heterologous donor tissue, and the like.

[0054] In one example, the cells isolated using the methods disclosed herein are to be used as therapy.

That is to say that the cells are to be used in treatment. Such treatments include, but are not limited to, cell transplantation; autologous stem-cell transplantation; Graft-versus-host-disease (GVHD); multiple sclerosis; cardiovascular disease (for example, myocardial infarction); rheumatoid arthritis; acute respiratory distress syndrome (ARDS); diabetes; chronic wounds, including chronic wounds associated with diabetes; and neurodegenerative diseases, such as, but not limited to, stroke and Parkinson's disease.

The symptoms that are to be treated using the cells disclosed herein can be, but are not limited to, symptoms associated with aging; chronic wounds, including chronic wounds associated with diabetes; and cardiovascular disease.

[0055] Although mesenchymal stem cells (MSCs) hold great promise for regenerative medicine, clinical trials have shown variable results from MSC-based cell therapies in a variety of disease settings. Recent work suggests that senescence, acquired by mesenchymal stem cells because of repeated cell division during ex vivo expansion, adversely affects mesenchymal stem cell potency and thus represents a significant confounding factor in many cell therapies. Notably, current methods for qualifying potent, proliferative mesenchymal stem cells rely on negative staining for intracellular β-galactosidase, which requires fixed samples and are thus incompatible with living cells. Cell-surface biomarkers of senescence would be highly attractive because of their potential compatibility with affinity reagents such as lectins, thus enabling the enrichment of potent, living mesenchymal stem cells. Here, it is shown that expression of the O-linked glycan Gal- i,3-GalNAc (T-antigen) was significantly reduced in senescent MSCs, whereas the expression of its sialylated derivative, Neu5Ac-a2,3-Gal- i,3-GalNAc, was significantly

increased. This finding was corroborated using a variety of different methods, including lectin arrays, live cell immunostaining, and LC-MS2. It was further shown that the decrease in T-antigen was due to reduced expression of cell surface Collagen I, and that T-antigen was present at several novel sites on the collagen al chain. The results disclosed herein are consistent with previous findings showing that collagen production is reduced or impaired in senescent fibroblasts, and show that T-antigen is a useful quality attribute for monitoring mesenchymal stem cells proliferation and potency in culture.

[0056] In one example, fetal bone marrow-derived mesenchymal stem cells were cultured to senescence by repeated 1 :30 passaging on tissue culture plastic (Fig. 14A). Passaged mesenchymal stem cells exhibited a linear increase in cumulative population doublings and a constant doubling time over time in culture until P21. At this point, cells showed a sudden increase in doubling time from 20+/X to 250+/-X hours, indicating that a large proportion had entered a senescent state. This is supported by beta-galactosidase staining (Fig. 14B), gross morphological inspection and quantitative nuclear DNA staining (Fig. 14C). The quantitative nuclear DNA staining data showed that there were very few P21 cells in the proliferative S-phase, and most cells were arrested in the G0/G1 or G2 phase. When cultured under the appropriate differentiation conditions (Fig. 14D), P21 cells showed impaired differentiation towards the bone, adipocyte and chondrocyte lineages as compared to proliferating mesenchymal stem cells, indicating that they would be of limited use for regenerative medicine applications. P21 cells were therefore considered as senescent in subsequent experiments.

[0057] Glycan arrays have been shown to be a highly effective means of probing for gross changes in the cellular glycome. Accordingly, the cell lysates of proliferating and senescent mesenchymal stem cells were screened using lectin microarrays to determine if any class of glycan showed significant differences in expression. It was first determined that 5 μg/ml of biotinylated cellular protein was optimal for these assays (Fig. IB). Since many lectins showed at least some difference in binding signal between the two test groups, thresholding was then applied so that only lectins that showed > 30% difference in average binding intensity at a significance of p < 0.05 (N =3) were considered as potential "hits". Of the six identified hits (Fig. 14A), only Peanut Agglutinin (PNA) could subsequently be verified as a genuine "hit" using live cell immunostaining (Fig. 14B) as cultures of proliferating mesenchymal stem cells showed intense PNA staining, but senescent mesenchymal stem cells showed none. It was observed that trypsinized proliferating cells were negative for PNA staining (Fig. 14D(i)), and that the PNA binding signal increased over time in culture. At 6 hours post-seeding (Fig. 14C(ii)), only isolated clusters of proliferating mesenchymal stem cells stained positive for PNA, whereas at 120 hours post-seeding (Fig. 14C(iii)),proliferating mesenchymal stem cells were stained robustly with PNA. In addition, PNA-stained structures tended to traverse cell boundaries perpendicular to the local direction of cell alignment, indicating that PNA stained an extracellular matrix glycoprotein.

[0058] Since PNA binds to T-antigen, an O-linked disaccharide, an LC-MS analysis of chemically-released O-glycans was performed to confirm the presented observations. O-glycan alditols were released by alkaline β-elimination, permethylated using established procedures and subjected to C18-UPLC-MS.

Mass spectrometry signals were tentatively annotated using Glycobase (Campbell, M. P., Royle, L., Radcliffe, C. M, Dwek, R. A. & Rudd, P. M. GlycoBase and autoGU: tools for HPLC -based glycan analysis. Bioinformatics 24, 1214-1216 (2008)), and a subset of annotations deemed to be of statistical significance were then subsequently verified using the retention times and MS2 fragmentation spectra of chemically-defined glycan standards (Figs. 11, 12 and 15). Extracted ion chromatograms (Fig. 12A) showed clear separation of each molecular feature, most notably enabling baseline separation of even isomeric structures such as Gal- i,4-GlcNAc (peak at around 15.5 minutes) and Gal- i,3-GalNAc (peak at about 16 minutes). Relative quantification of ion counts (Fig. 12B, N = 7) clearly showed that senescent cells expressed 12.9% lower Gal- i,3-GalNAc (T-antigen) than proliferating cells, strongly corroborating the lectin array and peanut agglutinin live cell immunostaining results. In addition, senescent cells expressed 11.3% higher Neu5Ac-a2,3-Gal- i,3-GalNAc (sT-antigen) than proliferating cells.

[0059] Since mass spectrometry only provided relative quantification, it was not immediately clear whether the relative changes in T- and sT-antigen expression were primarily due to: (1) a decrease in T-antigen expression, or (2) an increase in sialylation of T-antigen structures. To this end, quantitative PCR was performed to examine changes in the expression levels of genes encoding the glycosyltransferases ClGalT-I, ST3Gal-I and ST3Gal-IV, which are collectively responsible for synthesizing the T- and sT-antigens43 (Fig. 12A). Expression differences greater than 2-fold were considered to be biologically significant. The results (Fig. 12B) indicate that ClGalT-I gene expression was significantly higher in proliferating cells (4.10+0.36 fold, N = 3), whereas ST3Gal-I and ST3Gal-IV gene expression did not exceed the required threshold for biological significance (1.98+0.09 fold and 1.09+0.10 fold, N = 3). Without being bound by theory, it is thought that this result is consistent with the theory that there is a decrease in T-antigen expression in senescent cells, whereas the rate of sialylation remains unchanged, thus resulting an overall decrease in the volume percentage of T-antigen and an increase in the volume percentage of sT-antigen.

[0060] High resolution LC-MS was performed on released N-glycans. PNGase-F released N-glycans were labelled with 2-aminobenzamide (2-AB) fluorescent dye and analysed using hydrophilic interaction chromatography coupled with high-performance liquid chromatography and mass spectrometry (HILIC-UPLC-MS). Fluorescent labelling has been shown to be a highly robust method for quantifying the relative abundance of glycan structures, since each glycan is labelled with only a single dye molecule. Glycan peak retention times were normalized against a dextran ladder (Fig. 15, top panel) and thus transformed into non-dimensional Glucose Unit (GU) values, which are relatively insensitive to variations in the instrumentation and methods used for analysis. Fluorescent peaks were assigned glycan identities based on two pieces of information: the Glucose Unit value, and mass spectrometric confirmation that the expected mass is observed in each peak. Glucose Unit value matching was performed against Glycobase.

[0061] Fluorescence chromatograms of 2-AB labelled N-glycans obtained from proliferating and senescent cells were generally similar to each other (Fig. 17A). It was possible to annotate between 58 to 70 structures per sample (see Fig. 13A for a full list of annotated N-glycan structures). As expected from whole -cell lysates, the most abundant N-glycans observed were of the high-mannose variety, which were likely released from intracellular compartments. There were no statistically significant changes in any observed N-glycan structure (N=4), nor were there statistically significant changes when N-glycans were categorized by their degree of sialylation, fucosylation, number of antennae and high mannose structures (Fig. 17B).

[0062] Mesenchymal stem cell-based cell therapies are often plagued by irreproducibility and a lack of proven efficacy, despite promising results in the treatment of graft-versus host disease, diabetes mellitus and peripheral arterial disease. There are many possible reasons for this, including a lack of standardization in cell isolation procedures, in vitro cultivation protocol, and cell delivery methods. Recently, one additional, often overlooked cause had been found, namely, the unintentional inclusion of senescent mesenchymal stem cells with injected cells. Cells have been shown to acquire senescence-associated phenotypes which promote deleterious inflammation, diminish organ functions and promote ageing-associated disorders, thus the presence of senescent cells may contribute to poor therapeutic efficacy. It has been shown herein that senescence may arise in mesenchymal stem cells of fetal origin within approximately 22 passages. Given that more than 10s cells may be required for a successful cell therapy, it is estimated that a fresh autologous cell explant containing 106 colony-forming mesenchymal stem cells would have to undergo at least 7 population doublings to achieve a therapeutically relevant quantity of cells. Thus, it is thought that a significant proportion of injected cells exhibit senescent characteristics at the time of injection, and it would therefore be desirable to be able to enrich for proliferating cells either during cultivation or prior to cell delivery into patients.

[0063] The approach disclosed herein used lectin microarrays to generate a broad overview of major glycomic changes between proliferating and senescent mesenchymal stem cell lysates. Although lectins are notorious for poor specificity, if properly validated, they are still highly useful indicators of glycomic changes and can be used as affinity reagents for the enrichment of cell populations. Since cell lysates generally bound with different intensities to each lectin on the array, thresholding (> 30% difference in binding intensity) was used to narrow the list of probable hits to six lectins. Of these, only one hit, peanut agglutinin, could be validated using live cell immunostaining. This is consistent with the information that lectin microarrays exhibit significant variations in binding signal intensity even under highly controlled conditions, underscoring the need for careful validation of lectin microarray results. Peanut agglutinin intensely stained confluent proliferating cell cultures in a pattern that ran perpendicular to the axis of local cell alignment (Fig. 14C, D). This staining pattern, and its susceptibility to enzymatic treatment, strongly indicated that peanut agglutinin stained an extracellular matrix protein. In contrast, senescent mesenchymal stem cells seeded at an equivalent cell density were not stained appreciably by peanut agglutinin.

[0064] In the performed glycomic analysis, two different sample preparation and detection methods were used for N-glycan and O-glycan analysis. N-glycan analysis was performed by hydrophilic

interaction chromatography coupled with high-performance liquid chromatography (HILIC-UPLC) separation of 2AB -labelled glycans followed by matching of normalized retention times against Glycobase and mass spectrometry for orthogonal confirmation, a highly quantitative method based on methods known in the art. On the other hand, it was chosen to chemically release O-glycans under reducing conditions using alkaline β-elimination, which has been shown to be a reliable and less hazardous method than hydrazinolysis. One drawback of this alkaline β-elimination method is that it reduces terminal saccharides to alditols, thus making it difficult to label the glycans with a fluorescent dye. Therefore, the released O-glycan alditols were permethylated to increase their ionization efficiency, stabilize labile sialic acids, and provide more structural information during MS-MS fragmentation.

[0065] High resolution analysis of released N-glycan structures revealed no significant changes between the N-glycomes of proliferating and senescent mesenchymal stem cells (Fig. 17). N-glycans have been shown to be sensitive indicators of stem cell differentiation and human aging. Firstly, whole cell lysates were analysed, thereby introducing quantities of high mannose glycans from intracellular compartments. High mannose glycans were the most abundant category of glycans in the samples tested. Secondly, quantification of 2AB -labelled N-glycans based on fluorescence intensity may be complicated by the co-elution of glycan structures, particularly in high complexity samples such as cell lysates. Other approaches, such as chromatographic separation on a Porous Graphitic Carbon (PGC) column followed by negative mode electrospray ionization mass spectrometry, are able to resolve the issue of co-elution by digitally extracting molecular features, however, at the cost of being less quantitative.

[0066] Analysis of chemically-released permethylated O-glycan alditols revealed significant differences in the expression of O-linked T-antigen (Gal- i,3-GalNAc) and sT-antigen (Neu5Ac-a2,3-Gal- i,3-GalNAc). When measured as a volume percentage of overall ion counts, senescent mesenchymal stem cells expressed 12.9% lower T-antigen and 11.3% higher sT-antigen as compared to proliferating mesenchymal stem cells. Significantly, T-antigen is a known ligand of peanut agglutinin, thus strongly corroborating the lectin microarray and live cell immunostaining results. Under the optimized chromatographic conditions as disclosed herein, it was found that it was able to effectively resolve compositionally identical neutral structures such as Gal -β 1,3 -GlcNAc, Gal-β 1,4 -GlcNAc and Gal- i,3-GalNAc, and compositionally identical sialylated structures such as Neu5Ac-a2,3-Gal- i,4-GlcNAc, Neu5Ac-a2,6-Gal- i,4-GlcNAc, and Neu5Ac-a2,3-Gal- i,3-GalNAc, into distinct peaks based on their retention times. It was also possible to distinguish β1,3 and β1,4 linkages based on diagnostic MS2 fragment ions, providing orthogonal confirmation of the results shown herein. It was found that structures containing Ηεχ-β1,4-ΗεχΝΑϋ fragmented to yield an observable HexNAc Y-ion with an additional loss of methanol (m z = 230.141), whereas this Y-ion was not observable with structures containing Hex-β 1,3 -HexNAc. It was therefore possible to annotate T- and sT-antigen structures with a high degree of certainty in the experimental samples.

[0067] An quantity (3.0 - 3.85%) of Gal^l,4-GlcNAc of uncertain origin in both proliferating and senescent mesenchymal stem cell (MSC) samples was found. One possibility is that these represent

"peeling" products of extended mucin type O-glycan structures, for example Gal- i,4-GlcNAc- i,6-(Gal- i,3-)GalNAc. An alternative possibility is that these represent extended O-GlcNAc structures. Although O-GlcNAc is commonly thought of as a non-elaborated monosaccharide that modulates intracellular signalling pathways, recent evidence suggests that O-GlcNAc can be extended with β-linked galactose on a least some glycopeptides, such as HLA class-I bound peptides. These saccharides have an entirely different biological function than T-antigen, and represent between 4% to 6% of the total Hex-HexNAc signal, thus the ability to distinguish these structures based on the method disclosed herein is potentially significant. It is noted that a common method of permethylated O-glycan analysis relies on Matrix-assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS), and would not have been able to distinguish these structures since they are isobaric. Furthermore, disaccharides are poorly retained on both hydrophilic interaction chromatography (HILIC) and porous graphitic carbon (PGC) columns, thus isomeric disaccharides are difficult to resolve using these column chemistries. The results show that C18-UPLC-MS is a successful strategy for high resolution profiling of small (di- and tri-saccharide) permethylated O-glycan alditols, particularly for recombinant glycoproteins produced in a Chinese hamster ovary (CHO) cell host, since Chinese hamster ovary cells are known not to produce significant quantities of extended core 1 and core 2 O-glycans.

[0068] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0069] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

[0070] As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/- 0.5% of the stated value.

[0071] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub -ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub -ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0072] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0073] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0074] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[0075] Unless otherwise stated, lx PBS refers to phosphate buffered solution, with a pH of 7.4, containing 1 mM CaCl2 and 1 mM MgCl2. The following chemically-defined glycan standards were purchased from Dextra Laboratories Ltd (Reading, UK): GN203 (Gai i-3GlcNAc), GN204 (Gai i-4GlcNAc), GN213 (Gai i-3GalNAc), SLN302 (Neu5Aca2-3Gai i-4GlcNAc) and SLN306 (Neu5Aca2-6Gai i-4GalNAc). A fetuin O-Glycan Library standard was purchased from Ludger Ltd (Oxfordshire, UK).

Cell Lysis and Protein Quantification

[0076] For N- and O-glycomics experiments, P7 and P26 MSC cell pellets containing 5xl06 to 15xl06 cells were re-suspended in 500 μΐ of 50 mM ammonium bicarbonate buffer, pH 8.5, containing 1% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate). For lectin microarray experiments, cell pellets containing lxlO6 cells were re-suspended in 500 μΐ of lx PBS, pH 7.4, containing 1% Triton -X.

[0077] To affect cell lysis, re-suspended cells in their respective buffers were placed in an ice bath and lysed using a Misonix 3000 ultrasonic cell disruptor (3 x 15 seconds, power setting 2). Insoluble debris was pelleted by centrifugation at 1400x g for 30 minutes at 4°C, following which the clarified supernatant was carefully transferred to a fresh tube. Where necessary, clarified cell lysate glycoprotein concentration was quantified using a bicinchonic acid assay (BCA assay, Bio-Rad).

Lectin Microarray

[0078] Clarified cell lysates were biotinylated using an EZ-Link NHS-PEG4-biotin kit (Thermo Fisher Scientific) at a 50: 1 molar excess, assuming the average molecular weight of glycoproteins in the lysate was 30 kDa. The biotinylated glycoproteins were buffer exchanged into lx PBS using Zeba Spin Desalting Columns (molecular weight cut-off (MWCO) of 7 kDa, Thermo Fisher Scientific), aliquoted, and frozen at -20°C for long-term storage.

[0079] Biotinylated glycoprotein aliquots were then diluted in LecChip Probing Solution (GlycoTechnica) at concentrations ranging from 5 μg/ml to 500 μg/ml. LecChips (GlycoTechnica) were defrosted and washed in probing solution according to the manufacturer's instructions. 100 μΐ of biotinylated glycoprotein solution was then applied to each LecChip well at 4°C overnight in a humidified chamber. Wells were washed three times in probing solution to remove unbound material, then incubated with Streptavidin-Cy5 (Biolegend) diluted 1 :200 in lx PBS for 45 minutes at room temperature in the dark. Wells were washed twice with probing solution and twice with lx PBS before glycoprotein attachment to the LecChips was visualized using a GenePix 4000B Microarray Scanner at an excitation wavelength of 635 nm. Fluorescence intensities from the resulting images were extracted using ScanArray Express software (Perkin Elmer).

N-Glycan Release

[0080] Clarified cell lysates in 50 mM ammonium bicarbonate + 1% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate) were digested with 20 μg of mass spectrometry-grade trypsin (Promega V5280) at 37°C overnight. Digested glycopeptides were then denatured by the addition of dithiothreitol to a final concentration of 5 mM, and residual tryptic activity was inactivated by three cycles of heating to 95°C for 5 minutes, followed by cooling to room temperature for 5 minutes. The glycopeptide mixture was supplemented with io* in volume of lOx G7 buffer (New England Biolabs), then 1500 U of PNGase-F (New England Biolabs) was added and N-glycan cleavage was allowed to proceed for 3 hours at 37°C.

[0081] To separate free N-glycans from the remaining peptides and O-glycopeptides, samples were passed through CI 8 Sep-Pak columns (Waters Corporation). Being hydrophilic, free N-glycans were not retained on the columns and were collected in the flow-through. Peptides and O-glycopeptides were eluted from Sep-Pak columns using 50% acetonitrile (CAN), dried in a centrifugal evaporator and used for the O-glycan release protocol (see below).

[0082] N-glycan labelling with 2AB (2-aminobenzamide) fluorescent dye was performed according to published methods. Briefly, labelling reagent consisting of 70% DMSO, 30% acetic acid, 1 M sodium cyanoborohydride and 0.3 M 2-aminobenzamide was freshly prepared. Purified N-glycans from each cell pellet were dissolved in 100 μΐ of labelling reagent and allowed to react overnight at 37°C. Excess dye and salts were removed by desalting on PD Minitrap G-10 size exclusion columns (molecular weight cut- off (MWCO) at 700 Da, GE Healthcare). Labelled N-glycans were collected in the void volume, then dried in a centrifugal evaporator and reconstituted in 50 μΐ of 70% CAN (acetonitrile) / 30% water for liquid chromatography coupled mass spectrometry (LC-MS) analysis.

O-Glycan Release and Permethylation

[0083] O-glycans were released from the dried O-glycopeptide fractions by adding 400 nL of a solution of 40 mg/ml sodium borohydride in 0.1 M sodium hydroxide and incubating at 45 °C for 16 hours. The reaction was terminated by adding glacial acetic acid drop-wise, followed by clean-up using Dowex 50W-X8(H) 50-100 mesh resin chromatography. O-glycans were eluted using 5 ml of 5% acetic acid and evaporated to dryness using a N2 sample concentrator. 500 nL of 10% acetic acid in methanol was then added and dried to remove borate (repeated five times). To ensure consistency, chemically-defined glycan standards (20 μg each) were prepared in an identical fashion.

[0084] To affect permethylation, sodium hydroxide dissolved in dimethyl sulfoxide and iodomethane was added to the dried O-glycan samples in glass tubes. The reaction was allowed to proceed under rotation at 30 rpm for about 3 hours. 1ml of deionised water was added drop-wise to quench the reaction. 2 ml of chloroform was then added and the mixture was mixed thoroughly. After allowing the mixture to separate into two layers, the upper aqueous layer was removed. Deionised water was added to the chloroform layer and this step of mixing and removal of aqueous layer was repeated several times until the chloroform layer was clear. The chloroform layer was then evaporated to dryness using a N2 sample concentrator.

Permethylated O-Glycan Analysis by Liquid chromatography-mass spectrometry (LC-MS)

[0085] The CI 8 Sep-Pak® cartridge from Water Corporation (Milford, MA) was primed sequentially with 5 ml methanol, 5 ml deionized water, 5 ml acetonitrile and 5 ml deionized water. The dried permethylated sample was re-dissolved in 200 μΐ of 50% methanol and loaded to the Sep-Pak® cartridge. Elution was done using 2 ml of 15%, 35%, 50% and 75% acetonitrile in water (v/v). Each elution fraction was collected and evaporated to dryness using a SpeedVac.

[0086] The permethylated O-glycans from the 35% and 50% fractions were combined and reconstituted in 50 μΐ of 80% methanol in water. Using an Agilent 6550 iFunnel QTOF MS (Santa Clara, CA) equipped with an Agilent 1290 infinity LC system, 2 μΐ of the reconstituted glycans were separated on an Agilent 50 x 2.1mm Zorbax Eclipse Plus CI 8 RRHD column with particle size of 1.8 micro. The elution gradient was delivered at 0.5 ml/min using solutions of (A) 0.1% formic acid (v/v) in water and (B) 0.1% formic acid (v/v) in acetonitrile at the following proportions and time points: 3-10% B, 0 to 10 minutes; 10-40% B, 10 to 25 minutes; 40-70% B, 25 to 30 minutes; 70-90% B, 30 to 38 minutes. The column was flushed with 90% B for 12 minutes before re-equilibrating with 3% B for 15 minutes.

[0087] Mass spectra were acquired in positive ion mode over a mass range of m z 100-2000 with an acquisition rate of 1 spectrum per second. The following parameters were used for the acquisition: drying gas temperature 150°C at 12 L/min, sheath gas temperature 300°C at 12 L/min, nebulizer pressure at

45 psi and capillary voltage at 2500 V. Mass correction was enabled using an infused calibrant solution with reference mass of m/z 121.0873 and 922.0098.

[0088] The LC-MS data was processed using Molecular Feature Extractor (MFE) algorithm of MassHunter Qualitative Analysis Software (version B.06.00 Build 6.0.633.10 SP1, Agilent Technologies). A permethylated mass list was generated based on the neutral masses of O-glycans found on the GlycoBase. This list with mass filter of 10 ppm was used to search the LC-MS data. Mass peaks were filtered with a peak height of at least 100 counts and resolved into individual ion species. Using a Glycans Isotopic distribution model, charge state of a maximum of 3 and retention time, all ion species with singly, doubly and triply protonated ions and their sodium adducted ions associated with a single compound were summed together. The neutral compound mass was then calculated and a list of all compound peaks in the samples and standards were generated with abundances depicted by chromatographic peak areas.

[0089] Targeted tandem MS was acquired in positive ion mode over a mass range of m/z 100-2000 with an acquisition time of 1.5 seconds per spectrum. A targeted mass list was generated based on the desired MFE compounds found on the samples for MS/MS analysis. The precursor masses of interest along with its charge state, retention time and peak width were indicated. The isolation width used was narrow (~1.3m/z) and the collision energy (CE) used for each precursor compound were automatically calculated by the acquisition software based on the following equation:

CE(eV) = 3(m/z ÷ 100) - 4.8

[0090] The targeted tandem MS data was processed using MFE algorithm with the same settings used for searching LC-MS data and the MS/MS spectrum were extracted from each of the targeted compounds.

Analysis of Extracellular Matrix Glycoproteins by LC-MS

[0091] For glycopeptide analysis, the lyophilized sample was reconstituted in 20 μL· of 0.1 % formic acid. 2 μΕ was then analysed by nanospray LC-MS/MS (liquid chromatography coupled mass spectrometry) on an Obitrap Fusion Tribrid (Thermo Scientific) with an Easy-Spray™ source coupled to a nano LC system (Dionex Ultimate 3000 RSLCnano, Thermo Scientific) with a CI 8 trap column (Acclaim PepMaplOO C18, 5 μπι pore size, 5 mm x 300 μπι, Thermo Scientific). Solvent A was 0.1% formic acid/99.9% water and Solvent B was 0.1% formic acid/99.9% acetonitrile. Glycopeptides were enriched on the trap column using 0.1% TFA/99.9% water at a flow rate of 15 μΕ per minute for 5 minutes before being resolved on a 50 μπι x 15 cm Easy-Spray™ CI 8, 2 μιη pore size nano column (Thermo Scientific) at a flow rate of 300 nL per minute using a gradient of 4% B for 5.5 minutes and then ramping 4% to 95% B in 29.5 minutes.

[0092] The mass spectrometry (MS) scan was acquired for the range of m/z 300-2000, source voltage set at 2000 V, ion transfer tube temperature at 300°C, resolution at 120,000, AGC target of 4 xl05 and maximum injection time of 50 ms. Tandem MS/MS was with high-energy collisional induced dissociation (HCD) with the following parameters: top speed 3 s, charge state 2-7, dynamic exclusion if precursor was detected once in 6 s and excluding for 15 s with mass tolerance of ±10 ppm, intensity greater than 5 x 104; isolation mode = quadrupole; isolation window = 1.6 m/z; collision energy = 30%; resolution = 30,000; AGC target = 1 x 105; maximum injection time = 100 ms. Data was acquired using Xcalibur (v3, ThermoScientific).

[0093] T antigen-containing glycopeptides were identified from the data using Byonic (v2.14, ProteinMetrics). The settings were: protein database was the total human proteome from Uniprot (at May 2017); allowing per peptide two O-glycans from the list: HexNAc(l), HexNAc(2), HexNAc(l)Hex(l), HexNAc(2)Hex(l), HexNAc(l)Hex(l)NeuAc(l) and HexNAc(l)Hex(l)NeuAc(2); cleavage sites C-terminal of R and K, fully specific Trypsin with three missed cleavages allowed; precursor and fragment mass tolerances of 25 ppm, lock mass of m/z 519.13882; allowing one rare modification of methionine oxidation; precursor isotope off by x set at 'too high (narrow)'; maximum precursor mass of 10,000 Da; precursor charge as originally assigned; smoothing width of m/z 0.01 ; and a 1% protein FDR tolerance. Glycopeptide spectrum matches were confirmed manually.

Western Blot

[0094] Cell lysate protein concentration was determined using BCA assay (Thermo Fisher Scientific 23225). 50 μg of cell lysate protein was denatured by adding lOx glycoprotein denature buffer and heating at 70°C for 15 minutes. Denatured cell lysate proteins were mixed with 4x LDS sample buffer (NuPAGE), then 30 μg of protein was loaded into each lane of Novex 4 - 12% Bis-Tris gels and run at 120 V for 2 hours at 4°C. Glycoproteins were transferred from their gels to polyvinylidene difluoride (PVDF) membranes using the iBlot dry blotting system (Thermo Fisher Scientific) following the manufacturer's recommended settings. Transferred blots were first washed in MilliQ water, then blocked for 1 hour with a 5% BSA solution in Tris-buffered saline + 0.1% Tween-20. Biotinylated Peanut Agglutinin lectin (Vector Labs) was applied at 0.5 μg/ml overnight at 4°C. Streptavidin-HRP polymer (Sigma Aldrich) was applied at a dilution of 1 :20,000 at room temperature for 1 hour. Blots were washed extensively in Tris-buffered saline + 0.1% Tween-20 between each of the preceding two steps to reduce background. Bands were visualized with SuperSignal West Pico Chemiluminescent substrate and imaged using an ImageQuant LAS 500 (GE Healthcare Life Sciences).

Quantitative Polymerase Chain Reaction

[0095] The following primers were ordered from IDT and diluted to 10 μΜ in 10 mM TE (Tris EDTA) buffer:

Gene Forward Primer SEQ ID Reverse Primer SEQ ID

NO: NO:

C1GALT1 TCCTCTGTGGATCAGCAA 1 TTAGGCTGGGTGTCAACC 2

TAGG TTT

ST3GAL1 GGAGGACGACACCTACC 3 CCACCGACCTCTTCTCCA 4

GAT G

ST3GAL4 CTTCCTGCGGCTTGAGGA 5 CTCACTCCCCTTGGTCCC 6

TTA ATA

Beta- CAAGATCAACCGGGAAA 7 TGGATGGCGACATACATG 8

Actin AGATGA GC

[0096] mRNA was extracted from cells using RNeasy mini kit (Qiagen), and reverse transcribed to cDNA using Superscript III First Strand Synthesis System (Thermo Fisher Scientific). cDNA was added to 2x Master Mix (Quantifast SYBR Green PCR Kit, Qiagen) in the following ratio: 900 μΐ master mix, 10 μΐ cDNA, 890 μΐ RNase-free water to form the reaction mix. 22.5 μΐ of reaction mix was dispensed into each well of Applied Biosystems Micro Amp Fast 96-well reaction plates together with 1.25 μΐ of the appropriate forward and reverse primers to make a total reaction volume of 25 μΐ per well. Each reaction condition was run in triplicate wells. After sealing with optically transparent film, the plates were centrifuged briefly. Quantitative PCR was run for 40 cycles on an Applied Biosystems 7500 Fast instrument. Signals were normalized against beta-actin for relative quantitation.

Peanut agglutinin (PNA) Staining

[0097] Cells were plated onto 6-well cell culture plate at density of 3000 cells/cm2 for proliferative cells and 6000 cells/cm2 for senescent cells. Three groups of cells were set up according to staining time (30 minutes, 6 hours or 120 hours after seeding). 20 mg/ml of peanut agglutinin (PNA) was added separately onto the cells at the respective staining time. This was followed by incubation at 37°C, 5% C02 for one hour. Media was then replaced by fresh media containing Hoechst stain for visualization of cell nuclei. The cells were incubated for 15 minutes at 37°C, 5% C02 for Hoechst staining. Cells were then visualized under Nikon Eclipse Ti-E inverted microscope (Nikon, Japan).

MSC lectin array protocol

[0098] Young and senescent MSC cells (Cell A and B respectively) have been provided at 2xl06 cells per pellet (4 pellets). The cells had been previously trypsinized.

Biotin conjugation of cell lysate

[0099] Cell pellets were lysed in 100 μΐ Triton-X buffer (0.1 M PBS (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) with 1.0% Triton X-100, and passed through a 0.22 μπι filter)) by passing cells up/down 30 x through the bore of a narrow needle. The lysed sample was centrifuged at 1400x g for 5 minutes to pellet debris and transfer the resulting supernatant to a fresh tube. The protein concentrations of Cell A & B lysates were measured using a BCA assay, whereby the standard bovine serum albumin (BSA) was dissolved in Triton-X buffer. The lysate was then reacted using the NHS-PEG4-biotin kit.