Angewandte
Chemie
DOI: 10.1002/anie.201409258
Cell-Surface Engineering
Hot Paper
Long-Lived Engineering of Glycans to Direct Stem Cell Fate**
Abigail Pulsipher, Matthew E. Griffin, Shannon E. Stone, and Linda C. Hsieh-Wilson*
Abstract: Glycans mediate many critical, long-term biological
processes, such as stem cell differentiation. However, few
methods are available for the sustained remodeling of cells with
specific glycan structures. A new strategy that enables the long-
lived presentation of defined glycosaminoglycans on cell
surfaces using HaloTag proteins (HTPs) as anchors is
reported. By controlling the sulfation patterns of heparan
sulfate (HS) on pluripotent embryonic stem cell (ESC)
membranes, it is demonstrated that specific glycans cause
ESCs to undergo accelerated exit from self-renewal and
differentiation into neuronal cell types. Thus, the stable display
of glycans on HTP scaffolds provides a powerful, versatile
means to direct key signaling events and biological outcomes
such as stem cell fate.
cells,[2d] whereas highly sulfated HS is associated with differ-
entiated cells and has been proposed to promote interactions
between soluble FGF and BMP factors and their receptors.[2c]
However, the precise sulfated epitopes and mechanisms
involved in the generation of specific cell lineages remain
unclear. We postulated that the presentation of particular HS
GAG structures on ESC surfaces might enable the selective
activation of signaling pathways and thereby induce desirable
cell fates. Such an approach would also provide novel insights
into the structure-function relationships of HS GAGs and
their roles in stem cell biology.
Elegant studies have recently shown that the short-term
display of synthetic HS glycopolymers can promote stem cell
specification to form intermediate neural rosettes.[4] How-
ever, directing the generation of fully differentiated, mature
cell types will likely require the development of new methods
to enable the long-term, stable presentation of defined HS
GAGs. Two powerful approaches for cell-surface glycan
engineering have recently been reported that employ lipid-
functionalized polysaccharides[5] and synthetic glycopoly-
mers.[6] Although both strategies can elicit short-term cellular
responses, the lipid tail anchor limits the membrane lifetime
of the exogenous glycans to several hours. Herein, we
developed a method to tailor cell surfaces with specific HS
derivatives using membrane-bound HaloTag proteins (HTPs)
as anchors (Figure 1A). Molecules covalently attached to
HTPs displayed prolonged cell-surface lifetimes of more than
one week, circumventing the temporal limitation of lipid
anchors. Moreover, mouse ESCs remodeled with heparin/
highly sulfated HS underwent accelerated exit from self-
renewal and commitment to a neural lineage through early
activation of extracellular signal-regulated kinase (ERK)/
mitogen-activated protein kinase (MAPK) signaling path-
ways. These results highlight the potential to elucidate the
functional roles of HS GAGs and direct cell differentiation by
remodeling the glycocalyx of stem cells.
HTP is a modified alkane dehalogenase that forms
a covalent adduct with chloroalkane substrates.[7] Strategies
based on HTP have been adapted for diverse applications,
ranging from cancer diagnostics to chemical proteomics.[8]
However, most reported applications have used HTP meth-
ods to append molecules that serve as detection or capture
agents. We chose to exploit the HTP platform to modulate
biological processes in living cells.
We first investigated the membrane lifetime of molecules
conjugated to HTPs. N-tert-Butyloxycarbonyl (Boc)-pro-
tected 1-(2-(2-amino-ethoxy)ethoxy)-6-chlorohexane[8c] was
deprotected with trifluoroacetic acid and reacted with N-
hydroxysuccinimidyl levulinate to obtain chloroalkane linker
(CL) 1 (Figure 1B; see also the Supporting Information,
Scheme S1). Condensation of 1 with a fluorescein-hydrazide
derivative gave CL-conjugated fluorescein (F-CL; Figure 1B
T
he ability to control embryonic stem cell (ESC) differ-
entiation holds great promise as a renewable source of
replacement cells and tissues to treat diseases, including heart
disease, diabetes, and neurodegenerative disorders.[1] How-
ever, realizing the full potential of stem cells will require new
strategies for directing differentiation, as well as a better
understanding of the molecular mechanisms that guide the
development of specific cell lineages and fates.
Heparan sulfate (HS) glycosaminoglycans (GAGs) have
recently been identified as important regulators of stem cell
differentiation.[2] HS GAGs are a ubiquitous class of extra-
cellular polysaccharides consisting of uronic acid and glucos-
amine disaccharide units. The sugar backbone is further
modified by various sulfotransferase enzymes, giving rise
potentially to hundreds of sulfation patterns. This rich
structural diversity enables HS GAGs to interact selectively
with proteins, including those involved in stem cell differ-
entiation, such as fibroblast growth factors (FGFs), bone
morphogenic proteins (BMPs), and wingless-type MMTV
integration site family members (Wnts).[2c,3] Notably, specific
sulfation patterns of HS have been implicated in the
progression of ESCs from self-renewal to a differentiated
state. For example, undersulfated HS is found on pluripotent
[*] A. Pulsipher,[+] M. E. Griffin,[+] S. E. Stone, Prof. L. C. Hsieh-Wilson
Division of Chemistry and Chemical Engineering
California Institute of Technology
1200 E. California Blvd., Pasadena, CA 91125 (USA)
E-mail: lhw@caltech.edu
[+] These authors contributed equally to this work.
[**] This research was supported by a National Institutes of Health grant
(R01-GM093627; L.H.W.) and a National Science Foundation
Graduate Research Fellowship (DGE-1144469; M.E.G.). We thank
Greg Miller for assistance with microarrays. We also thank Fred Tan,
Elizabeth Jensen, and the Dervan laboratory for assistance with qRT-
PCR and helpful discussions.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!