The glycans of stem cells

Abstract

Glycans cover all cellular surfaces and, not surprisingly, are involved in many facets of stem cell biology and technology. For instance, coaxing stem cells to either proliferate or differentiate into the specific cell types needed for transplantation requires intricate glycan-dependent modulation of signalling molecules such as FGF-2, Wnt, and Notch. Moreover, owing to their prominent cell-surface localization and lineage-specific signatures, glycan epitopes such as the stage-specific embryonic antigens (Lewis X/SSEA-1, SSEA3-4) and tumor-rejection antigens (TRA1-60, 1-81) are ideally suited for identifying and isolating specific cell types from heterogeneous populations. Finally, the non-human sialic acid Neu5Gc has been detected on the surface of human embryonic stem cells because of metabolic incorporation from animal products used for their culture. Transplantation of Neu5Gc-contaminated cells poses immunological risks due to the presence, in humans, of circulating antibodies recognizing this glycan epitope.

Figure 1. Hypothetical paradigm highlighting the use of glycans in stem cell preparation for therapeutic transplantation

FGF-2 driven proliferation of isolated neural stem cells is critically dependant on heparan sulfate proteoglycans (HSPG). Enrichment of this heterogeneous population can then be achieved by selecting Lewis X+ cells through flow activated cell sorting (FACS). Differentiation involves modulation of signaling pathways such as Notch, Wnt and FGF-2, which are all regulated by various glycans. After induction of neuronal differentiation, another FACS-based purification step could select only cells expressing PSA-NCAM, a known marker of the neuronal lineage which is also involved in neurogenesis. Finally, the stem cell preparation could theoretically be injected locally in conjunction with various glycosidases (e.g. hyaluronidase), helping clear the way for proper integration into the matrix.

Figure 2. Schematic representation of some glycans markers used for the identification and purification of stem cells

Monosaccharides components of various glycans epitopes are presented in the boxed legend. Several pertinent glycan epitopes used for the identification and purification of stem cells are shown. They are TRA 1–60, NG2, 473HD, SSEA-3 and SSEA-4, Lewis X, PSA-NCAM, CD34, GalNAc. Whenever possible the exact glycan structure is highlighted by a blue box.

Figure 3. Schematic representation of mechanisms proposed for Neu5Gc contamination of human embryonic stem cells

Neu5Gc-containing glycoproteins and glycolipids/GPIs from culture medium and mouse embryonic fibroblasts become expressed on the surface of human embryonic stem cells. The first proposed mechanism involves direct incorporation of glycolipids/GPIs into the membrane. The second, more complex mechanism, involves macropinocytosis of Neu5Gc-glycoconjugates. These contaminants are internalized to the lysosome where sialidases release free Neu5Gc and this is delivered to the cytoplasm through a lysosomal sialic acid transporter. Free Neu5Gc is then activated to CMP-Neu5Gc in the nucleus and sent back to the cytoplasm. Another sialic acid transporter then delivers CMP-Neu5Gc into the trans-Golgi where sialyltransferases (which do not discriminate much between CMP-Neu5Ac and CMP-Neu5Gc) add Neu5Gc from the latter to newly synthesized stem cell glycoconjugates.