Carbohydrate engineered cells for regenerative medicine

Abstract

Carbohydrates are integral components of the stem cell niche on several levels; proteoglycans are a major constituent of the extracellular matrix (ECM) surrounding a cell, glycosoaminoglycans (GAGs) help link cells to the ECM and the neighboring cells, and small but informationally-rich oligosaccharides provide a "sugar code" that identifies each cell and provides it with unique functions. This article samples roles that glycans play in development and then describes how metabolic glycoengineering - a technique where monosaccharide analogs are introduced into the metabolic pathways of a cell and are biosynthetically incorporated into the glycocalyx - is overcoming many of the long-standing barriers to manipulating carbohydrates in living cells and tissues and is becoming an intriguing new tool for tissue engineering and regenerative medicine.

Figure 1. Landscape of the cell surface

The cell surface is shown illustrating representative carbohydrates found in the ECM and attached to cell surface protein and lipids (note that the various components of the cell:cell and cell:matrix interface are not to scale).

Figure 2. Glycan-based stem cell markers

Examples of stem cell markers comprised of, or dominated by, carbohydrates include (A) the CD34 antigen, a highly glycosylated sialomucin, consisting of up to 80% carbohydrate by mass [32], (B) SSEA-1 consists of the (shaded) Lewis X (Le^X) trisaccharide, which can be found on CD34 as well as many other surface elements, that can be “masked” with sialic acid (to create the sLe^X tetrasaccharide) [45], and (C) SSEA-3 and SSEA-4 consist of similar glycolipid structures but with different antigenic saccharide structures [18].

Figure 3. Functional roles of carbohydrates

(A) Growth factors (e.g., FGF) are sequestered in the proteoglycan components of the ECM and also associate with GAGs attached to surface proteins to spatially organize receptors. (B) Notch signaling depends on O-fucosylated glycans to transmit signals from once cell type to another during development, thereby regulating the developmental fate of the target cell (additional details are provided elsewhere [138]).

Figure 4. Overview of metabolic glycoengineering

(A) Normally monosaccharides are obtained from the diet or salvage processes and metabolically assembled into the complex glycan structures that comprise the glycocalyx of mammalian cells. (B) Non-natural monosaccharides (e.g., a ManNAc analog, shown) used in metabolic glycoengineering intercept the glycosylation pathways of a cell and substitute for the natural metabolites, ultimately replacing the native surface sugars (e.g., the Neu5Ac form of sialic acid prevalent on human cells). Types of ManNAc used in metabolic engineering include analogs used to install (C) natural mammalian siaolosides Neu5Ac and the “non-human” Neu5Gc sialoside, (D) the first generation analogs developed by Reutter’s group, and (D) analogs with various chemical functional groups not naturally found in the glycocalyx.

Figure 5. Multiple, overlapping mechanisms monosaccharide analogs

Cellular responses emanating from metabolic glycoengineering can arise from one or more factors acting separately or in combination. These include (A) increased flux through glycosylation pathways that has been proposed to engage transcriptional mechanisms [96] (e.g., by activating transcription factors, TF); (B) changes to cell surface glycans such as increased N-glycan branching that facilitates formation of the galectin lattice [103]; (C) installation of abiotic sialic acid, fucose, or GalNAc residues into surface glycans that alter lectin binding (illustrated by the reduced binding of viruses to sialic acids with modified N-acetyl groups) [72], and (D) new chemical functionalities, such as thiolated sialic acids, that enable new modes of cell attachment to chemically compatible growth substrates, such as gold, that can trigger changes to cell fate, illustrated by neuronal differentiation of human embryonic cells [58].

Figure 6. The hexosamine as a template for drug discovery

(A) ManNAc (as well as GlcNAc and GalNAc, B) provides a versatile template for modulation of biological activity; by “mix and matching” groups at each of the R₁-R₅ positions (additional R₁ groups are shown in Figure 4 and provided in other publications [72, 76]), tens of thousands of compounds can be generated that have specific biological properties and pharmacologic characteristics [134].