Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs

This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac₅ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac₅ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac₅ManNTProp and Ac₅ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac₅ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac₅ManNTProp and Ac₅ManNTBut suppressed adipogenic differentiation, with Ac₅ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac₅ManNTProp and Ac₅ManNTBut) for manipulating human stem cells.