Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells

Article abstract of DOI:10.1021/acschembio.9b00401

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, howev

Full text of DOI:10.1021/acschembio.9b00401

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Article
A fully Synthetic Heparan Sulfate-Based Neural Tissue Construct
that Maintains the Undifferentiated State of Neural Stem Cells
Pradeep Chopra, Meghan T Logun, Evan M. White, Weigang
Lu, Jason Locklin, Lohitash Karumbaiah, and Geert-Jan Boons
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00401 • Publication Date (Web): 07 Aug 2019
Downloaded from pubs.acs.org on August 8, 2019
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A fully Synthetic Heparan Sulfate-Based Neural Tissue Construct that Maintains the
Undifferentiated State of Neural Stem Cells
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Pradeep Chopra,1 Meghan T. Logun,2 Evan M. White,3 Weigang Lu,1,4 Jason Locklin,3,4
Lohitash Karumbaiah,2,* and Geert-Jan Boons1,4,5,
*
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1Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road,
Athens, GA 30602, USA
2Regenerative Bioscience Center, ADS Complex, University of Georgia, 422 River Road,
Athens, GA 30602, USA
3New Material Institute, University of Georgia, 220 Riverbend Road, Athens, GA 30602, USA
4Department of Chemistry, University of Georgia, 140 Cedar Street, Athens, GA 30602, USA
5Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, and Bijvoet Center for Biomolecular Research, Utrecht University, Universiteitsweg
99, 3584 CG Utrecht, The Netherlands.
*E-mail: gjboons@ccrc.uga.edu, g.j.p.h.boons@uu.nl, lohitash@uga.edu
Keywords: heparan sulfate, hydrogels, synthesis, neural stem cells
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ABSTRACT
Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due
to their ability to recruit and regulate the activity of growth factors. The structural and functional
heterogeneity of naturally derived heparin and HS is, however, an impediment for the
preparation of biomaterials for regenerative medicine. To address this problem, we have
prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human
induced pluripotent cell-derived neural stem cells (HIP™-NSCs) encapsulated in a
polyethylene glycol-based hydrogel modified by a pendent HS-disaccharide that is a known
ligand for fibroblast growth factor-2 (FGF-2), exhibited significant increase in proliferation and
self-renewal. This observation is important because evidence is emerging that undifferentiated
stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms.
Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological
half-live, from degradation. It is anticipated that by careful selection of a synthetic HS-
oligosaccharide, it will be possible to control retention and release of specific growth factor,
which in turn will provide control over cell fate.
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INTRODUCTION
Hydrogels, which are formed by chemical or physical crosslinking of water-soluble polymers,
provide attractive scaffolds for tissue regrowth and regeneration.1-3 The bulk properties of
hydrogels can influence stem cell fate and in particular mechanical properties and chemical
entities such as cell adhesion peptides have been explored to influence proliferation and
differentiation.4 Glycosaminoglycans, such as heparin and heparan sulfate (HS), are also
attracting considerable interest for the development of hydrogels for regenerative medicine.5
HS are highly sulfated linear polysaccharides that are ubiquitously expressed within the stem
cell microenvironment or niche where they interact with numerous signalling proteins, growth
factors, and extracellular matrix components.6, 7 The biosynthesis of HS occurs in the Golgi by
initially forming a linear polymer composed of alternating D-glucuronic acid (GlcA) and N-
acetyl-D-glucosamine (GlcNAc) moieties, which are modified by a series of enzymatic
transformations involving N-deacetylation/N-sulfation, epimerization of GlcA to L-iduronic acid
(IdoA), and sulfation of the C-2 hydroxyl of IdoA and the C-6 hydroxyl of glucosamine. These
transformations proceed only partially resulting in as many as twenty different disaccharide
moieties that can be arranged in different ways creating specific HS-epitopes.8
Evidence is emerging that cells can create unique HS-epitopes by regulating the expression
of isoforms of HS biosynthetic enzymes.9, 10 The so-called “HS sulfate code hypothesis” is
based on the notion that such epitopes can recruit or retain specific HS-binding proteins.
Indeed, the structure of HS changes during stem cell differentiation,11 which in turn is important
for the stage dependent recruitment of morphogens such the fibroblast growth factors (FGFs),
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bone morphogenetic protein (BMP) and wingless-type MMTV integration site family proteins
(Wnts).12
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Polymeric matrixes modified by heparin or heparan sulfate offer interesting vehicles for the
controlled delivery of growth factors.13 For example, the use of collagen modified by heparin
in combination with recombinant FGF-2 and VEGF, provided sustained release, induced
vascularization and reduced hypoxic conditions.14 In other studies, it has been found that
functionalization of hyaluronic acid based hydrogels with heparin diminished burst release of
BMP-2 and prolonged osteogenic activity.15 Despite these promising results, many challenges
remain for the use of heparin or HS for biomaterial construction. In particular, the structural
and functional complexity of these biopolymers is impedement because it can cause adverse
effects due to binding of many different proteins.16 In an interesting approach, this problem
was addressed by using partial desulfated heparin, which could modify growth factor
presentation and influenced cell fate in a PEG-based hydrogel.17 In another study, affinity
purification was employed to obtain an HS fraction that bound potently to BMP-2, and greatly
enhances BMP-2-induced bone formation in vitro and in vivo and was much more efficacious
than mucosal HS or heparin.18 These approaches are promising but still offer limited
capabilities to control the binding properties of HS.
Recent progress in the chemical19 and chemoenzymatic20 synthesis of HS oligosaccharides
is making it possible to probe ligand requirements of HS binding proteins. These studies are
revealing that relatively small structures, such as di- and tetra-saccharides, can bind with high
affinity and selectivity with HS-binding proteins.21-23 Furthermore, the attachment of such
compounds to polymeric backbones can provide neoglycopolymers that recapitulate
properties of HS.24-27 Therefore, we were compelled to investigate whether small synthetic HS-
oligosaccharides can be employed for hydrogel formation to influence cell fate.
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In a proof-of-principle study, we have synthesized a HS disaccharide that is a known ligand of
FGF-228 and used it to fabricate a neural tissue mimic that can encapsulate, recruit and
maintain the undifferentiated state of neural stem cells (NSCs). These findings are important
because NSCs have tremendous therapeutic potential to treat neurological deficits that
manifest after injury or disease of the central nervous system (CNS). Although the focus on
promoting the neuronal differentiation of NSCs to facilitate functional repair of neurological
deficits has its merits, emerging evidence suggests that NSCs in their undifferentiated state
promote more favorable functional outcomes via their paracrine signaling mechanisms rather
than through differentiation and cell replacement per se.29 It is therefore plausible that
strategies to promote “bystander” signaling by undifferentiated NSCs could carry significant
therapeutic benefit.
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RESULTS AND DISCUSSION
Chemical synthesis. Conventional approaches to incorporate heparin into biomaterial
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scaffolds rely on modification of the saccharide backbone with a reactive functional group.30,
31
Such an approach is unsuitable for presenting small synthetic oligosaccharides because
vital functional groups would be destroyed. Therefore, we developed a so-called pendant-
approach in which synthetic HS oligosaccharides (e.g. 10 and 11, Scheme 1) are modified
with an artificial anomeric aminopentyl linker, which can be reacted with an NHS activated
triazide PEG linker such as 12 to yield HS-oligosaccharides that possess three azide-moieties
(e.g. 13 and 14). It was anticipated that these azide-modified compound could be cross-linked
with a 4-armed PEG derivative modified with dibenzylcyclooctynol (17, DIBO) to yield stable
hydrogels.32 This approach would appropriately place the HS-disaccharides in the hydrogel
lattice to facilitate functional interactions with HS-binding proteins. For the purpose of this
study, compound 14 was selected as a high affinity ligand of FGF-228 whereas compound 13,
which is devoid of sulfate groups, was used as a negative control. We anticipated that a
hydrogel modified with a FGF-2 binding HS-oligosaccharide will retain and stabilize this growth
factor, which in turn would promote NSCs self-renewal and maintenance.
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Functionalized HS-disaccharides 13 and 14 were assembled from glycosyl acceptor 1 and
idosyl donor 2, which were coupled using NIS/TMSOTf as the promoter system to give
disacharide 3 in good yield as only the a-anomer (Scheme 1A).33 The benzylidene acetal of
3 was removed by a mixture of trifluoroacetic acid, dichloromethane and water to give a diol,
which was subjected to TEMPO/BAIB to selective oxidize the primary alcohol to a carboxylic
acid which was esterified by treatment with diazomethane to yield methyl ester 4. The C-4’
hydroxyl of 4 was protected as Fmoc by treatment with FmocCl in pyridine in the presence of
a catalytic amount of DMAP to give disaccharide 5. Next, the levulinoyl (Lev) esters of 5 were
removed by treatment with hydrazine acetate and the resulting hydroxyls were sulfated using
sulfur trioxide/pyridine complex in DMF, followed by Fmoc removal to provide 7. The methyl
ester of 7 was saponified with H2O2 and LiOH in THF. The azide of the resulting compounds
was reduced under Staudinger conditions using PMe3 and the resulting amine was N-sulfated
employing sulfur trioxide/pyridine complex in MeOH in the presence of Et3N and NaOH to
provide 9.
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OLev
O
OLev
O
A
OLev
O
OBn
O
SEt
O
O
(ii)
(i)
BnO
BnO
HO
BnO
OBn
O
Bn
OBn
Bn
MeO
N3
N3
O
Bn
O(CH2)5NCbz
O
O(CH2)5NCbz
N3
O
OLev
O
O(CH2)5NCbz
O
1
2
3
4 R = H
RO
Ph
O
OLev
OLev
(iii)
5 R= Fmoc
Ph
8
9
OR1
O
OR1
O
OR1
O
O
O
O
BnO
HO
OBn
O
BnO
Bn
MeO
OH
+Na-O
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OBn
+Na-O
H
N3
Bn
(v)
(iv)
(vi)
NH
O(CH2)5NR3
NH
R2
R2
O(CH2)5NCbz
O
O
O(CH2)5NCbz
O
O
O
HO
OR1
HO
OR1
HO
OR1
O
10 R1 = H; R2 = Ac; R3 = H
11 R1 = R2 = SO3Na; R3 = H
R4
8 R1 = H; R2 = Ac
9 R1 = R2 = SO3Na
6 R1 = H
N
O
O
7 R1 = SO3Na
R4
(vii)
O
13 R1 = H; R2 = Ac; R3 = C(O)Ph(R4)3
14 R1 = R2 = SO3Na; R3 = C(O)Ph(R4)3
R4
12
R4 = (OCH2CH2)3N3
B
NH2
n
R
n
O
O
O
(viii)
H2N
R
O
O
O
O
O
NH2
N
H
n
O
O
n
n
n
O
O
O
n
n
16
17
15
NH2
R
R
NO2
Scheme 1. Chemical synthesis of HS-hydrogels precursors. (A) unsulfated HS-triazide (13)
and sulfated HS-triazide (14); (B) DIBO-4-arm PEG polymer (17). Reagents and conditions: (i)
NIS, TMSOTf, DCM, –20 °C, 0.5 h; (ii) a) DCM/TFA/H2O, 0 °C to rt, 1 h; b) TEMPO, BAIB,
DCM/H2O, 1 h; c) CH2N2, THF, 0.5 h; (iii) FmocCl, pyridine, DMAP (cat.), 2 h; (iv) a) hydrazine
acetate, EtOH/toluene (2/1, v/v), 2 h; b) SO3.pyridine complex, DMF, 8 h; c) Et3N/MeOH (1/1, v/v),
0.5 h; for 6, only step a on 4; for 7, step a, b and c on 5; (v) a) 1.0 M aq. LiOH, aq. H2O2 (50%), 8
h then pH~14 with 4.0 M NaOH, 16 h; b) 1.0 M PMe3 in THF, NaOH, 1 h; c) Ac2O, NEt3, MeOH;
d) SO3.pyridine complex, MeOH, Et3N, 0.1 M NaOH, 16 h; for 8, step a, b and c on 6; for 9, step
t
a, b and d on 7; (vi) a) Pd/C, H2 (atm.), BuOH/H2O (1/1, v/v), 2 h; b) Pd(OH)2/C, H2 (atm.),
tBuOH/H2O (1/1, v/v), 16 h, 10 from 8 and 11 from 9; (vii) DIPEA, DMSO, 0 °C to rt, 16h, 13 from
10 and 14 from 11; (viii) Et3N, DCM, 0 °C to 16h.
In parallel, disaccharide 4 was subjected to a similar sequence of reactions without O-sulfation
and N-acetylation instead of N-sulfation to provide 8. Finally, two step hydrogenation of 8 and
9 over Pd/C in a mixture of tBuOH/H2O (1/1) removed the protecting group of linker, followed
by hydrogenation over Pd(OH)2/C removed the benzyl ethers and provide, after purification
by size exclusion chromatography over a P-2 column, the disaccharides 10 and 11,
respectively. Treatment of the latter compounds with the NHS activated triazide PEG linker 12
(Supplementary Scheme 1)34 resulted in the formation of the desired derivatives 13 and 14,
respectively. The DIBO-functionalized 4-armed PEG 17 was synthesized (Scheme 1B) by
reaction of p-nitrophenyl carbonate of DIBO (16) with commercially available PEG tetra-amine
(15, 10 kDa).
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Preparation and characterization of HS-containing hydrogels. Stoichiometric quantities of
DIBO-functionalized 4-armed PEG 17 and triazide functionalized sulfated-HS disaccharide 14
(1:1 molar ratio of azide and DIBO) were mixed resulting in the formation of stable hydrogels
within 20 min. The absence of the N3 stretch vibration at 2095 cm-1 in the IR spectra of the
lyophilized sulfated-HS hydrogels indicated that the cycloaddition had proceeded to
completion (Figure 1A). By varying the concentration of the components, the storage modulus
of the sulfated-HS hydrogels could be tuned to match the material properties of the
physiological host tissue (Figure 1B). The gels displayed minimal strain-hardening at high
frequency (100 rad s-1) and high strain (5%). Sulfated-HS hydrogels with 3 wt.% of compound
17 had a storage modulus (0.5 – 1 kPa) appropriate for NSCs cultures, and was employed for
further studies.
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Figure 1. Physio-mechanical characterisation of HS-hydrogels (A) An overlay of FTIR spectra
of sulfated-HS triazide (14) and lyophilized sulfated-HS hydrogel showing consumption of azide
(2095 cm-1); (B) Storage moduli of 2 wt.%, 3 wt.%, 4 wt.% and 5 wt.% sulfated-HS hydrogels over
a frequency range of 0.1 to 100 rad s-1 at a constant strain of 5% at 25 °C; (C) Scanning electron
micrograph (SEM) of lyophilized 3 wt.% sulfated-HS hydrogel at magnification of 500x (inset
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represents magnified image of dashed box), pore size analysis of SEM images displays an
average pore size of 20.16 µm2; (D) Cumulative % release of FGF-2 encapsulated in sulfated-HS
and unsulfated-HS hydrogels determined by ELISAs.
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Swelling studies of this hydrogel showed an acceptable swelling ratio of 2.2 over a period of
10 days (Supplementary Figure 1). The microarchitecture observed using SEM and pore size
analysis of the SEM images revealed an average pore size of 20.16 μm2 indicating that the
material is sufficiently porous to facilitate the migration and proliferation of encapsulated NSCs
(Figure 1C).
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FGF-2 is a potent mitogen for neural progenitor cells, however, it is highly unstable and its
o
concentration rapid declines when incubated at 37 C probably due to thermal denaturation
proteolytic digestion, and cellular uptake. It has been shown that heparin can prevent
degradation and its presence leads to faster cell growth.35, 36 To evaluate FGF-2 stabilization
and retention capacity of the hydrogels, we quantified the cumulative % release of FGF-2 of
hydrogels modified by the sulfated (14) and control oligosaccharide (13) over a period of 72
h. For this experiment, human FGF-2, HS-triazide (13 or 14) and DIBO-4-arm PEG polymer
(17, 3 wt.% final concentration, 1:1 molar ratio of azide group and DIBO group) were mixed to
form a hydrogel, which was overlaid with PBS. At regular time intervals, the PBS was replaced
and the concentration of FGF-2 determined by enzyme-linked immunosorbent assay (ELISA,
Figure 1D). Total amount of FGF-2 released from the sulfated HS hydrogels was higher than
unsulfated HS hydrogel. The enhanced cumulative release of FGF-2 detected in the sulfated-
HS construct is likely due to significantly enhanced protection and lower heat induced
degradation of FGF-2 activity in sulfated-HS matrices when compared to unsulfated-HS
matrices.37-40
NSCs cell fate in HS-containing hydrogel. We performed a hydrogel contact viability assay
in order to evaluate the effects of HS-hydrogels on NSC viability. For these assays, NSCs
were cultured for 2 h on poly-D-lysine coated surface. Subsequently, HS-hydrogel
components reconstituted in neurobasal media was overlaid on top of NSC monolayer and
allowed to crosslink. After formation of stable hydrogels, hydrogels were overlaid with
expansion media and incubated for 24 h before staining with calcein AM and hoechst stain.
NSCs viability was assessed by comparing live cells (green fluorescence) to live or dead cells
(DAPI, blue fluorescence), and to bright field images of the NSCs. We observed that the
hydrogels formed from 14 and 17 did not induce cytotoxicity, and that the majority of the
encapsulated NSCs were alive 24 h post encapsulation (94%) (Figure 2A-E).
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Figure 2. Cytocompatibility and proliferation of NSCs encapsulated in HS-hydrogels. NSCs
were stained for nuclei (Hoechst) and live cell (Calcein AM) after culturing for 24 h. Bright field
image and an overlay of these images is also presented. Scale – 50 μm. (B) Total isolated DNA
from NSCs grown on Geltrex® coated culture plate (T0-day0 and T7-day7) and from NSCs
encapsulated in the sulfated (14)- and unsulfated (13, control)-HS hydrogel. Statistical differences
observed between groups are represented by **** indicating P < 0.001 and ** indicating P < 0.01.
In order to investigate the influence of HS-oligosaccharides on proliferation, NSCs were
encapsulated in the hydrogels modified by the sulfated (14) and control (13) oligosaccharide.
Unlike the contact viability assay, where NSCs were cultured in a monolayer, NSCs in these
assays were encapsulated by the hydrogel matrix after resuspending the NSC pellet with the
HS-hydrogel mix. This results in NSCs being distributed throughout the volume of the
hydrogel. Hydrogel encapsulated NSCs were cultured in FGF-2 containing media (conditioned
media) for 24 h, after which they were cultured in media without FGF-2 (unconditioned media)
for an additional 6 days. Next, the rate of proliferation was determined by quantifying total
DNA. Both sulfated and unsulfated-HS hydrogel encapsulated NSCs yielded significantly
greater quantities of total DNA, indicating higher proliferation when compared to NSCs
cultured under standard 2D culture conditions. Amongst the hydrogel encapsulated NSCs, those
cultured in sulfated HS matrices demonstrated significantly greater proliferation when compared
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to unsulfated HS controls (Figure 2F), as inferred from the total DNA recovered. Our results also
demonstrate that FGF-2 deprived NSCs grown under standard 2D culture conditions began
differentiating into neural lineage cells within 48h post-seeding (Supplementary Figure 3) when
compared to hydrogel encapsulated NSC, which suggests that hydrogels can efficiently engage
endogenously released growth factors and promote NSCs proliferation. Although the
cytocompatible crosslinking chemistry and optimum mechanical properties of our synthetic
heparin constructs ensure cell viability (Figure 2E), the absence of cell-adhesion peptides in
the 3D matrix results in the NSCs exhibiting a rounded morphology41, which is typical of the
proliferative state of hydrogel encapsulated NSCs.
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(A)
(D)
(H)
(B)
(E)
(I)
(C)
(G)
DAPI
B-III
O-4
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(F)
(J)
GFAP
DAPI
B-III
Sox-1
Merged
(N)
(J)
DAPI
O-4
(H)
(K)
(L)
(M)
Merged
Merged
GFAP
DAPI
Sox-1
(O)
80
****
**
****
ns
60
40
20
0
-III
O-4
GFAP
Sox-1
Unsulfated-HS
Sulfated-HS
Figure 3. Immunocytochemistry results of NSCs encapsulated in HS-hydrogels. (A−G) A
representative image (Z-stack) of NSCs encapsulated in a 3 wt.% unsulfated-HS hydrogel
conditioned for 24 h with FGF-2 and cultured for 6 days without FGF-2; Group-1 unsulfated-HS
hydrogels were stained for nuclei, neuronal, oligodendrocyte, and astrocyte markers, represented
by DAPI (A), β−III tubulin (B), O-4 (C), and GFAP (D), respectively. An overlay of these images (A-
D) is also presented (G); Group-2 unsulfated-HS hydrogels were stained for nuclei and stem cell
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marker, represented by DAPI (E) and Sox-1 (F), respectively. An overlay of these images (E-F) is
presented in Supplementary Figure 4; (H−N) NSCs encapsulated in a 3 wt.% sulfated-HS hydrogel
conditioned for 24 h with FGF-2 and cultured for 6 days without additional FGF-2. Group-1 sulfated-
HS hydrogels were stained for nuclei, neuronal, oligodendrocyte, and astrocyte markers and are
represented by DAPI (H), β−III tubulin (I), O-4 (J), and GFAP (K), respectively. An overlay of these
images (H-K) is also presented (N); Group-2 sulfated-HS hydrogels were stained for nuclei and
stem cell marker, represented by DAPI (L) and Sox-1 (M), respectively. An overlay of these images
(L-M) is presented in Supplementary Figure 4; Scale – 20 μm. (O) Quantification of markers β-III
tubulin, O-4, GFAP and Sox-1 expression in NSCs encapsulated in unsulfated-HS and sulfated-
HS hydrogel. Statistical differences observed between groups are represented by ns indicating-
not significant, **** indicating P < 0.001, ** indicating P < 0.01.
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Next, we investigated whether the sulfate moieties of the HS-disaccharide influences cell
differentiation. For this purpose, relative expression of relevant phenotypic markers of stem
cell differentiation was performed by immunocytochemical staining using appropriate
antibodies. These studies demonstrated that sulfated-HS oligosaccharide 14 , which can
stabilize FGF-2, greatly impacted cell differentiation (Figure 3). Thus, NSCs cultured in this
hydrogel displayed significantly higher expression of the self-renewal biomarker Sox-1
(P<0.001) and the astrocytic marker GFAP (P<0.001). Furthermore, we detected a
significantly (P=0.02) lower expression of the oligodendrocyte marker O-4. Although no
significant difference in neuronal differentiation as determined by β-III tubulin was observed,
the higher expression of the Sox-1 is indicative of NSCs that are maintaining their
undifferentiated state. In addition, we believe higher GFAP expression is indicative of glial
lineage NSCs that can differentiate into neurons and glial cells.42 Furthermore, the lower
expression of the oligodendrocyte marker O-4 indicates NSCs self-renewal and maintenance.
The maintenance of self-renewal and proliferative capacity of NSCs is likely due to recruitment
and/or stabilization of FGF-2 in the hydrogel containing a HS-ligand for this growth factor.
CONCLUSIONS
An important goal in regenerative medicine is to create three dimentional matrices that
resemble properties of the extracellular matrix to guide cell fate.43 Integrins are
transmembrane receptors that facilitate adhesion of extracellular matrix components such as
laminin, collagen and fibronectin. Peptides derived from these extracellular matrix proteins,
such as RGDS, IKVAV and, YIGSR have found wide use in the construction of functional
materials for cell adhesion and their presence can also influence cell fate.44, 45 HS is also an
important component of the extracellular matrix that is involved in growth factor retention and
signaling, and efforts have been made to incorporate heparin into biomaterials.5, 16, 30, 31 The
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pleiotropic nature and binding promiscuity of heparin can, however, result in undesired effects.
For example, growth differentiation factor 5 (GDF5) which is involved in cartilage/skeletal
formation and homeostasis, is inhibited by heparin.46 In addition, heparin can cause
hemorrhagic side effects and induce thrombocytopenia. It is derived from animal products
exhibiting batch to batch differences and has been contaminated with other sulfated
molecules.47, 48 To address these liabilities of natural derived heparin, we demonstate here
that incorporation of a small and well-defined synthetic HS-oligosaccharide into a hydrogel
can influence cell fate. In particular, the use of a tailor made saccharide ligand for FGF-2
greatly increased self-renewal and proliferative capacity of NSCs.
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Chemical synthesis makes it now possible to identify HS-oligosaccharides that interact with
specific HS-binding proteins. Furthermore, a synthetic approach allows the incorporation of
an artifical linker to facilitate controlled attachment of cross linking functionalities for hydrogel
formation. Here, we modifed an HS oligosaccharide with a linker having three azido moieties
and a stable hydrogel could be formed by addition of a PEG-derivative modified by four DIBO
moieties. We developed DIBO for strain promoted azide-alkyne cycloaddition (SPAAC),49 and
this reagent reacts spontaneously with azides in the absence of a catalyst, and thus is
attractive for biomedical application. It was found that the presence of a sulfated
oligosaccharide did not impede hydrogel formation, and by tuning the concentration of the
reaction components, a hydrogel could be formed that has storage modulus resembling that
of neuronal tissue. It is likely that the placement of a HS-disaccharide in a pendant fashion
within hydrogel scaffolds creates a multivalent display which augment binding avidity for
growth factors owing to the cluster effect,50 and contributes to thermal stability and protects
40
against proteolytic cleavage.39,
It is anticipated that by careful selection of a HS-
oligosaccharide, it will be possible to control retention and release of specific growth factor
which in turn will give greater control over cell fate.
In this study, a HS disaccharide was employed that can bind FGF2 but not induce cell signaling
through the formation of a ternary complex with FGFR1.28 Future studies will focus on the
incorporation of an HS oligosaccharide that can induce the formation of such a ternary
complex and possible cellular responses will be investigated. Furthermore, PEG was selected
as the polymeric material because it is widely used in tissue engineering and approved by
FDA for human use. The synthetic approach gives, however, limited control over HS-
oligosaccharide spacing and valency. Future studies will focus on the use of alternative
polymeric backbones, such as dextran or hyaluronic acid, that make it possible to attach HS-
oligosaccharides at different densities. Such an approach can be combined with the use of
other cell-responsive and cell-instructive components such as cell adhesion peptides and
protease–cleavable linkers that can promote degradation of a hydrogel.
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Traumatic Brain Injuries and stroke result in significant brain tissue loss and atrophy, which
leads to lifelong disabilities and cost-of-care burden. There is a dire need for therapies for
these conditions in order to prevent rampant brain tissue atrophy and promote functional
repair. Our future studies will focus on the design of tailored synthetically derived
glycomaterials that can better regulate cellular repair cascades in order to facilitate functional
recovery following severe brain trauma.
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MATERIALS AND METHODS
Chemical synthesis. Preparation of HS-triazide, general procedure for amide coupling
(Scheme 1): To the stirring solution of amino compound (10 or 11) in DMSO (0.5 mL for 0.05
º
mmol) and DIPEA (4 equiv.) at 0 C was added compound 12 (2 equiv., in 0.5 mL DMSO).
The progress of reaction was monitored by TLC (EtOAc/pyridine/H2O/AcOH 8/5/3/1, v/v/v/v or
EtOAc/MeOH/H2O 7/2/1, v/v/v). After stirring for 16 h, the reaction mixture was diluted with
water (1.0 mL) and lyophilized. Solid residue obtained was purified by reverse phase
chromatography (RP-18 silica gel column, H2O/MeOH, 9/1→1/1, v/v). The appropriate
fractions were concentrated in vacuo, and the residue was passed through a column Dowex®
50 x 8 Na+ resin (0.6 x 5 cm) using MeOH/H2O, 9/1 as eluent to afford desired product (13,
0.038 g, 67.9% and 14, 0.026 g, 71.8%).
DIBO-functionalized 4-armed PEG polymer (17): To the stirring mixture of 4-arm PEG-amine
polymer (15, MW 10K, 0.30 g, 0.03 mmol, CreativePEGworks) in anhyd. CH2Cl2 (3.0 mL) at
0 °C was added p-nitrophenyl carbonate of DIBO (16, 0.11 g, 0.3 mmol) and Et3N (0.15 mL).
The resulting reaction mixture was left stirring in melting ice bath for 16 h. After completion of
reaction, confirmed by TLC (Rf : 0.65, EtOAc/IPA/H2O/NH4OH 3/3/2/1, v/v/v/v) reaction
mixture was concentrated under vacuum to give a yellow oil which was further purified over
LH-20 column (CH2Cl2/CH3OH, 1/1, v/v) to give the desired compound 17 (0.27 g, 83.4%) as
a white solid. The detailed synthetic procedures and spectral data of intermediates
compounds are provided in Supporting Information.
Preparation of HS-hydrogels. Stochiometric equivalent of DIBO-4-arm PEG polymer (150
µL, 10 wt% in PBS, 17) and unsulfated or sulfated HS-triazide (150 µL, 13 or 14 in PBS, 1:1
molar ratio of azide group and DIBO group), were mixed by vortexing in a glass vial and left
º
cross-linking for 20 min at 37 C. The formation of stable hydrogels was confirmed by vial-
tilting method (Supplementary Figure 1).
Fourier transform infrared (FTIR) spectroscopy. To determine degree of azide functional
group consumption FTIR measurements of lyophilized sulfated-HS hydrogel (3 wt.%, final
concentration of DIBO-4-arm PEG polymer, 17) samples were taken with a Nicolet model
6700 with a grazing angle attenuated total reflectance (GATR) accessory at 64 scans with a
4 cm−1 resolution (Figure 1A).
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Rheological evaluation of HS-hydrogels. The pendant approach circumvents interference
of HS disaccharide in strain promoted cycloaddition between triazide and DIBO polymer.
Although, the strategic placement of HS has little influence on rheological properties, we
nevertheless conducted mechanical characterization on hydrogels that were functionalized
using sulfated HS-triazide. Hydrogels (300 µL each of 2 wt.%, 3 wt.%, 4 wt.%, and 5 wt.% final
concentration of DIBO-4-arm PEG polymer, 17) were prepared by mixing equal volume of 17
(150 µL) and sulfated HS-triazide (14, 150 µL, 1:1 molar ratio of azide group and DIBO group)
in a PDMS mold (16 mm diameter and 3 mm thickness), swelled in PBS for 24 h at 37 ºC. The
hydrogels were characterized by isothermal frequency sweep experiments in triplicate from
0.1–100 rad·s-1 and 5% strain using a parallel plate rheometer (Anton Paar) (Figure 1B).
Swelling study of HS-hydrogels. The swelling ratio of HS-hydrogels was measured over a
period of 10 days. Sulfated-HS hydrogels (3 wt.%, final concentration of DIBO-functionalized
4-armed PEG polymer, 17, 100 µL discs, in triplicate) were prepared on the surface of a 14
mm2 coverslip affixed glass bottom petri dishes (In Vitro scientific). Initial weight (Wto) of each
hydrogel was obtained by subtracting the weight of glass bottom Petri dishes before and after
casting the hydrogels. Each hydrogel disc was subsequently overlaid with 100 µL of PBS and
placed in a tissue culture incubator set at 37 ºC, 5% CO2, and 95% humidity. Swollen hydrogels
were weighed (Wt) immediately after the removal of excess media every 24 h for 7 days. The
hydrogels were overlaid with 100 µL of PBS post-weighing. The swelling ratios were calculated
by Wt/ Wto and plotted (Supplementary Figure 2).
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Scanning Electron Microscopy (SEM). The microarchitecture of HS-hydrogels was
observed using a Zeiss 1450EP scanning electron microscope (Carl Zeiss). Sulfated-HS
hydrogels (3 wt.%, final concentration of DIBO-functionalized 4-armed PEG polymer, 17) were
prepared in a polydimethysiloxane (PDMS) mold (16 mm diameter and 3 mm thickness),
swelled in PBS for 24 h at 37 ºC, flash frozen in liquid nitrogen, then lyophilized for 48 h. The
lyophilized gels were mounted on 10 mm SEM stubs and sputter coated (Structure Probe Inc.)
with gold for 60 seconds before being imaged under an accelerated voltage of 20 kV. Images
were acquired at 1000x and 1500x magnifications to observe the porosity and structure of
hydrogels. For pore size analysis 1000x magnification images were processed using ImageJ
software (NIH) (Figure 1C).
FGF-2 retention in HS-hydrogels. HS-hydrogels were crosslinked with human FGF-2 and
its cumulative % release profile was established using ELISA. DIBO-functionalized 4-armed
PEG polymer (25 µL of 6 wt.%, 17) was dispensed into each well of a 96-well plate (in
triplicate) followed by the addition of either unsulfated-HS triazide (13, 25 µL) or sulfated-HS
triazide (14, 25 µL) (1:1 molar ratio of azide group and DIBO group) containing FGF-2 (10 ng,
R&D Systems, Inc.), mixed by pipetting, left cross-linking at 37 ºC for 30 min. Upon hydrogel
formation, the 50 µL of hydrogel was overlaid with 50 µL of PBS, incubated at 37 ºC, 5% CO2,
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and 95% humidity. At 6h, 12h, 24h, 36h, 48h and 72h, the overlaid PBS was collected and
stored at –80 ºC, and replaced with fresh 50 µL of PBS. Due to non-degradable nature of HS-
PEG hydrogels, the hydrogel bound FGF-2 cannot be retrieved. As a result, the extent of FGF-
2 retention was determined as the difference between initial FGF-2 loaded and cumulative
FGF-2 released. The FGF-2 released by unsulfated-HS and sulfated-HS hydrogels was
determined by human basic FGF Quantikine ELISA kit (R&D Systems, Inc.) according to
manufacturer’s instructions, and the cumulative % recovery of FGF-2 plotted (Figure 1D).
NSC expansion. Human induced pluripotent neural stem cells (HIP™ NSCs, MTI-
GlobalStem) were expanded on a Geltrex™ coated plate in expansion media consisting of
Neurobasal® medium (Life Technologies), B-27® supplement (Life Technologies), MEM non-
essential amino acids (Life Technologies), GlutaMAX™ supplement (Life Technologies), and
basic fibroblast growth factor (20 ng mL–1, R&D Systems, Inc.), incubated at 37 ºC, 5% CO2,
and 95% humidity. Cells were fed with expansion media every other day unless passaged or
extracted for use in assays.
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Viability assessment of hydrogel overlaid NSCs. Coverslip affixed glass bottom Petri
dishes (In Vitro scientific) were coated with poly-D-lysine hydrobromide (100 µg mL–1, Sigma-
Aldrich) overnight at 37 ºC, washed with PBS before platting 20 x 103 HIP™ NSCs. After 1 h,
expansion media was aspirated and sulfated-HS hydrogels (3 wt.%, final concentration of
DIBO-4-arm PEG polymer, 17, 100 µL discs) were prepared on top of adhered NSC’s.
Alternatively, NSC’s were encapsulated in HS-hydrogels by mixing 25 µL of 6 wt.% DIBO-
functionalized-4-armed PEG polymer (17, containing 20 x 103 NSCs) and sulfated-HS triazide
(14, 25 µL) (1:1 molar ratio of azide group and DIBO group), left cross-linking at 37 ºC for 20
min. The NSCs encapsulated hydrogels obtained were overlaid with expansion media and
incubated at 37 ºC, 5% CO2, and 95% humidity for 24 h before being stained with Calcein AM
(Thermo Fisher), and Hoechst stain (Thermo Fisher) according to manufacturer’s instructions.
Live cells (green fluorescence) were compared to live or dead cells (blue fluorescence), and
to bright field images of the cells. Images were analysed using a Leica DM IRB series
microscope (Leica Microsystems, Inc.). Cell viability was assessed using co-localization of
Calcein green fluorescence to Hoechst blue fluorescence in 40X images for at least four
images per hydrogel sample, using cell colocalization tools associated with Volocity software
(PerkinElmer) (Figure 2A-E).
Total DNA isolation from hydrogel encapsulated NSCs. In order to evaluate the
proliferation capacity of hydrogels and effect of sulfation on proliferation, total DNA isolated
from NSCs grown on Geltrex™ coated culture plate was compared with those grown in
unsulfated-HS and, sulfated-HS hydrogels. 25 µL of 6 wt.% DIBO-functionalized-4-armed
PEG polymer (17, with 20 x 103 NSCs) was mixed either with unsulfated-HS triazide (13, 25
µL) or sulfated-HS triazide (14, 25 µL) (1:1 molar ratio of azide group and DIBO group), mixed
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by pipetting, left cross-linking at 37 C for 30 min. In addition, a culture plate was Geltrex™
coated and seeded with NSCs (20 x 103). Culture plates and plates containing HS-hydrogels
were overlaid with 2.0 mL of expansion media, incubated at 37 ºC, 5% CO2, and 95% humidity.
After 24 h, expansion media was replaced with basal media (expansion media without FGF-
2) and NSCs incubation was continued in FGF-2 deprived condition. After 7 days, total amount
of DNA present in NSCs grown on Geltrex™ coated culture plates (at time zero, T0 and time
day 7, T7), unsulfated-HS and, sulfated-HS hydrogels was determined by ZymocleanTM gel
DNA recovery kit (Zymo research corp.) according to manufacturer’s instructions, and the total
DNA isolated was plotted (Figure 2F).
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Immunocytochemistry. NSCs encapsulated in unsulfated-HS (two groups, in triplicate) and
sulfated-HS hydrogels (two groups, in triplicate) were fixed with 4% paraformaldehyde
containing 0.4 M sucrose in PBS. After 20 min, the hydrogels were rinsed thrice with PBS and
permeabilized in blocking buffer (PBS containing 4% goat serum, 0.5% Triton-X100) for 1 h.
Subsequently, the group-1 hydrogels (both unsulfated- and sulfated-HS hydrogels) were
incubated at 4 ºC with blocking buffer containing primary antibodies (at dilutions of 1:500)
against b-III tubulin (Millipore), a neuronal cytoskeletal marker; O-4 (R&D Systems, Inc.), an
oligodendrocyte marker; glial fibrillary acidic protein (GFAP, R&D Systems, Inc.), which marks
astrocytes, and the group-2 hydrogels (both unsulfated- and sulfated-HS hydrogels) were
incubated with a neural stem cell marker; Sox-1 (R&D Systems, Inc.). After 16 h, the hydrogels
were rinsed thrice with PBS, and incubated with blocking buffer again for 1 h followed by the
addition of appropriate AlexaFluor 488 (Life Technologies), AlexaFluor 555 (Life
Technologies), and Cy5 (Life Technologies) labelled secondary antibodies and incubation for
1 h. After rinsing hydrogels thrice with PBS, a nuclear stain (NucBlue, Life Technologies, CA)
was added to the hydrogels, incubated for 5 min, and rinsed thrice with PBS. The hydrogels
were subsequently mounted with fluoromount-G (SouthernBiotech) and cover-slipped next
day. NSCs were imaged using a Leica DM IRB series microscope (Leica Microsystems, Inc.).
Representative high-magnification images (15 in total, from three replicates) of unsulfated-HS
and sulfated-HS hydrogels were analysed using cell population tools in Volocity software
(PerkinElmer) (Figure 3). The Manders overlap coefficient was used to measure the degree
of colocalization between fluorescent images, as described previously.51 Each separate signal
intensity was quantified against DAPI signal to provide percentages of cells positive for
markers found in the FITC, TRITC, or Cy5 channels.
Statistical analysis. All statistical analysis were performed using one-way analysis of
variance (ANOVA) for significance with appropriate post-hoc tests using GraphPad PrismÒ or
SigmaPlot software. Direct mean comparisons were evaluated using a Student’s t-test. All
studies were performed in triplicate at the minimum. Statistical differences observed between
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groups are represented by ns indicating- not significant, **** indicating P < 0.001 and ** indicating
P < 0.01.
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Supporting information
9
The Supporting Information is available free of charge on the ACS Publications website at
DOI: 10.1021/acschembio.xxxxxxx.
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Full experimental details, characterization and spectral data of the compounds, hydrogel
swelling data, 2D culturing of NSCs, and immunocytochemical results for Sox-1.
ACKNOWLEDGEMENTS
This research was supported by the National Institute of General Medicine (NIGMS; Research
Resource for Integrated Glycotechnology P41GM103390 to G.-J.B.) and the National Institute
of Neurological Disorders and Stroke (NINDS; R01NS099596 to LK) of the National Institutes
of Health (NIH).
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