Comparative Assessment of the Effects of Gender-specific Heparan Sulfates on Mesenchymal Stem Cells

Abstract

We compare here the structural and functional properties of heparan sulfate (HS) chains from both male or female adult mouse liver through a combination of molecular sieving, enzymatic cleavage, and strong anion exchange-HPLC. The results demonstrated that male and female HS chains are significantly different by a number of parameters; size determination showed that HS chain lengths were ϳ100 and ϳ22 kDa, comprising 30 -40 and 6 -8 disaccharide repeats, respectively. Enzymatic depolymerization and disaccharide composition analyses also demonstrated significant differences in domain organization and fine structure. N-Unsubstituted glucosamine (⌬HexA-GlcNH 3 ؉ , ⌬HexA-GlcNH 3 ؉ (6S), ⌬HexA(2S)-GlcNH 3 ؉ , and N-acetylglucosamine (⌬HexA-GlcNAc) are the predominant disaccharides in male mouse liver HS. However, N-sulfated glucosamine (⌬HexA-GlcNSO 3 ) is the predominant disaccharide found in female liver. These structurally different male and female liver HS forms exert differential effects on human mesenchymal cell proliferation and subsequent osteogenic differentiation. The present study demonstrates the potential usefulness of gender-specific liver HS for the manipulation of human mesenchymal cell properties, including expansion, multipotentiality, and subsequent matrix mineralization. Our results suggest that HS chains show both tissue-and gender-specific differences in biochemical composition that directly reflect their biological activity.Heparan sulfate proteoglycan structure classically consists of a core protein to which one or more linear HS glycosaminoglycan (GAG) 2 chains are attached at specific serineglycine residues. Heparan sulfates have complex sulfated domain substructures that are initially synthesized as nonsulfated polysaccharides of D-glucuronic acid-N-acetyl-Dglucosamine (GlcA-GlcNAc) repeats (1-4). These linear, sulfated glycosaminoglycans have molecular masses that typically range from 10 to 100 kDa (5). Concurrent with the polymerization of an HS chain is a series of enzymatic modifications that generate the diverse sulfated domain clusters at intervals along the growing chain. The non-templatedriven diversity of HS structure is thought to be capable of giving rise to a wide range of biological functions through selective binding.Several studies have demonstrated that the binding of growth factors to HS that gives rise to mitogenic or adhesive activity occurs only when specific structural features are present within the HS chain (6). Such features include sulfation at specific positions within a given sequence of disaccharides; 6-O-sulfated N-sulfate glucosamine and 2-O-sulfated iduronic acid residues are thought to be particularly important, and minimum binding sequences are generally at least 5 or 6, and often 8 -14 disaccharides in length (7-9). The precise structures within HS sulfated domains that are involved in these interactions have remained elusive. Variations in composition and the organization of HS from different cells and tissues have confirmed the relationship between HS stru...

Affinity Selection of FGF2-Binding Heparan Sulfates for Ex Vivo Expansion of Human Mesenchymal Stem Cells

“…Having isolated the FGF2‐binding variant HS8, we next determined its disaccharide composition, which together with chain length and sulfation pattern, is characteristic of its ability to bind particular growth factors (Nurcombe et al, ; Murali et al, ; Murali et al, ; Wang et al, ). This is important because the disaccharide composition of HS chains governs their affinity for proteins (Lindahl et al, ; Maccarana et al, ; Pye et al, ).…”

“…A modified ELISA was used to determine the affinity between protein and HS, as described previously (Murali et al, ; Bramono et al, ). GAG‐binding plates (Iduron, Cheshire, UK) coated with heparin (Sigma–Aldrich) or HS (Celsus laboratories, Cincinnati, OH) were incubated with the indicated amounts of biotinylated peptides or growth factors (R&D Systems, Minneapolis, MN) in blocking buffer.…”

“…Human MSCs culture‐expanded in the presence of this embryonic HS variant show enhance bone forming ability when transplanted into a rat femoral defect (Helledie et al, ). Other HS variants extracted from mouse liver, a highly regenerative organ, can also increase hMSC expansion and osteogenic differentiation (Murali et al, ). Although these studies highlight the utility of using HS variants as culture supplements, such sources of raw material are not able to meet future demands for large‐scale production of MSCs destined for clinical use.…”

Advancing biomaterials of human origin for tissue engineering

“…Following demonstration of the necessity of HS for the growth factor-stimulated differentiation of osteoprogenitor cells, the significance of HS for endogenous FGF-2 signaling (as a coreceptor) was also confirmed, suggesting that purified GAGs maybe promising alternatives to certain growth factors for enhancing the ex vivo proliferation and differentiation of MSCs [417]. Moreover, it is suggested that HS chains exhibit both gender- and tissue-specific diversity in biochemical composition that straightforwardly reflect their biological activities, as demonstrated by the potential benefit of gender-specific liver HS in manipulating human MSC properties, including cell expansion, multipotentiality and subsequent matrix production [418]. …”

Syndecan Heparan Sulfate Proteoglycans: Regulation, Signaling and Impact on Tumor Biology

“…The average number of sulfates per 100 disaccharides in HS of these rat tissues also differed, with the highest-sulfation detected in liver, spleen, lung and heart tissues (32). Furthermore, HS chains from adult male and female livers differ in length, disaccharide composition and the ratio of N-sulfation to N-acetylation levels (33).…”