Specific Glycosaminoglycans Modulate Neural Specification of Mouse Embryonic Stem Cells

Abstract

Mouse embryonic stem (mES) cells express a low sulfated form of heparan sulfate (HS). HS chains displayed by ES cells and their progeny become more complex and more sulfated during progression from pluripotency to neuroectodermal precursors. Sulfated epitopes are important for recognition and binding of a variety of ligands including members of the fibroblast growth factor (FGF) family. We demonstrated previously that mES cells lacking HS cannot undergo neural specification but this activity can be recovered by adding soluble heparin, a highly sulfated glycosaminoglycan (GAG). Therefore, we hypothesized that soluble GAGs might be used to support neural differentiation of HS competent cells and that the mechanisms underlying this activity might provide useful information about the signaling pathways critical for loss of pluripotency and early lineage commitment. In this study, we demonstrate that specific HS/heparin polysaccharides support formation of Sox1 1 neural progenitor cells from wild-type ES cells. This effect is dependent on sulfation pattern, concentration, and length of saccharide. Using a selective inhibitor of FGF signal transduction, we show that heparin modulates signaling events regulating exit from pluripotency and commitment to primitive ectoderm and subsequently neuroectoderm. Interestingly, we were also able to demonstrate that multiple receptor tyrosine kinases were influenced by HS in this system. This suggests roles for additional factors, possibly in cell proliferation or protection from apoptosis, during the process of neural specification. Therefore, we conclude that soluble GAGs or synthetic mimics could be considered as suitable low-cost factors for addition to ES cell differentiation regimes. STEM CELLS 2011;29:629-640 Disclosure of potential conflicts of interest is found at the end of this article.

6-O-Sulfated Chitosan Promoting the Neural Differentiation of Mouse Embryonic Stem Cells

“…It has been reported that the addition of NaClO 3 can specifically inhibit the bioactivity of ATP-sulfurylase, an important enzyme that affects the sulfation of glycosaminoglycan, and thus can prevent the sulfation of heparin. 26 Furthermore, desulfated heparin cannot induce differentiation of ESCs including neural differentiation, 11,12,27,28 which was confirmed by our results (see the Supporting Information, Figure S5). Therefore, in our subsequent experiments, 20 mM NaClO 3 was added to the differentiation medium containing heparin, 2S or 6S to eliminate the effect of autocrine heparin and HS on neural differentiation.…”

“…11 The K5 polysaccharide has a carbohydrate backbone similar to heparin and HS, but without sulfate groups, and could not promote the neural differentiation of ESCs. 12 This result indicated that the sulfate groups in sulfated glycosaminoglycans play a very important role in promoting the neural differentiation of ESCs. Furthermore, several common sulfated glycosaminoglycans (e.g., chondroitin sulfate and dermatan sulfate) have been shown to promote neural differentiation in vivo, albeit at a lower intensity than that of heparin and HS.…”

“…12 In addition to sulfation sites, the degree of sulfation (DS) of a sulfated polysaccharide also affects the neural differentiation of ESCs.…”

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

“…3A) into saccharide substituted peptide ions (Y-ions according to the nomenclature of Domon and Costello (40)) and poor peptide fragmentation. The presence of one sulfate group (SO 3 ) at the sub-terminal GalNAc residue was indicated by a weak but significant [⌬GlcAGalNAcϩHSO 3 ] ϩ oxonium ion at m/z 442.07.We have previously determined that this is a sulfate group (79.9568 amu) as opposed to phosphate (79.9663 amu) based on the Orbitrap mass measurement at 35,000 resolution (20). The identity and location of the other S/P substitution could not be unambiguously determined as it readily dissociated upon fragmentation.…”

“…In addition to their structural role as matrix components, eukaryote CSPGs are known to be involved in more specialized functions such as signal transduction, morphogenesis and regulation of stem cell behavior and differentiation (3)(4)(5)(6)(7)(8)(9). In many cases, their biological activity is mediated by selective binding of protein ligands to distinct glycan structural variants (10,11).…”