Metabolic glycoengineering in hmsc-tert as a model for skeletal precursors by using modified azide/alkyne monosaccharides

Article abstract of DOI:10.3390/ijms22062820

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesen-chymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4Man-NAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the gly-coengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with flu-orescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-de-pendent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transi-ent cell surface modifications, and thus present a useful model to address different scientific ques-tions regarding glycosylation processes in skeletal precursors.

Full text of DOI:10.3390/ijms22062820

International Journal of
Molecular Sciences
Article
Metabolic Glycoengineering in hMSC-TERT as a Model
for Skeletal Precursors by Using Modified
Azide/Alkyne Monosaccharides
Stephan Altmann 1, Jürgen Mut 2, Natalia Wolf 2, Jutta Meißner-Weigl 1, Maximilian Rudert 1, Franz Jakob 1,
Marcus Gutmann 3 , Tessa Lühmann 3, Jürgen Seibel 2 and Regina Ebert 1,
*
1
Bernhard-Heine-Center for Locomotion Research, University of Würzburg, Friedrich-Bergius-Ring 15,
97076 Würzburg, Germany; stephan.altmann@klh.de (S.A.);
j-meissner-weigl.klh@uni-wuerzburg.de (J.M.-W.); m-rudert.klh@uni-wuerzburg.de (M.R.);
f-jakob.klh@uni-wuerzburg.de (F.J.)
Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany;
juergen.mut@uni-wuerzburg.de (J.M.); natalia.wolf@uni-wuerzburg.de (N.W.);
juergen.seibel@uni-wuerzburg.de (J.S.)
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg,
Germany; Marcus.Gutmann@uni-wuerzburg.de (M.G.); tessa.luehmann@uni-wuerzburg.de (T.L.)
Correspondence: r-ebert.klh@uni-wuerzburg.de; Tel.: +49-931-8031597
2
3
*
Abstract: Metabolic glycoengineering enables a directed modification of cell surfaces by introducing
target molecules to surface proteins displaying new features. Biochemical pathways involving glycans
differ in dependence on the cell type; therefore, this technique should be tailored for the best results.
We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal
stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-
modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and
N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering
process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency
(osteogenic and adipogenic differentiation potential) was maintained under these adapted culture
conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using
Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior
incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments,
the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived
metabolites were absent after two days. Taken together, these findings demonstrate the successful
metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications,
and thus present a useful model to address different scientific questions regarding glycosylation
processes in skeletal precursors.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Altmann, S.; Mut, J.; Wolf,
N.; Meißner-Weigl, J.; Rudert, M.;
Jakob, F.; Gutmann, M.; Lühmann, T.;
Seibel, J.; Ebert, R. Metabolic
Glycoengineering in hMSC-TERT as
a Model for Skeletal Precursors by
Using Modified Azide/Alkyne
Monosaccharides. Int. J. Mol. Sci.
2021, 22, 2820. https://doi.org/
10.3390/ijms22062820
Academic Editor: Grzegorz Wegrzyn
Received: 15 February 2021
Accepted: 8 March 2021
Published: 10 March 2021
Keywords: hMSC-TERT; metabolic glycoengineering; glycocalyx; modified monosaccharides; click
chemistry
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Bone marrow-derived human mesenchymal stromal cells (hMSC) are a heterogeneous
population of adult precursor cells, which have self-renewal and multilineage differentia-
tion capacities. Minimal criteria to define hMSC in vitro comprise their plastic adherence,
the expression of specific mesenchymal markers and the absence of hematopoietic markers,
as well as their capacity to differentiate toward the osteogenic, adipogenic, and chondro-
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Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
genic lineage [
1,
2]. The cultivation duration of primary hMSC is limited, because cells
in culture undergo the process of replicative senescence [3]. This characteristic, as well as
their heterogeneity, makes it difficult to use them for the establishment of standardized
procedures. A possibility to overcome these problems is to use an appropriate cell line,
Int. J. Mol. Sci. 2021, 22, 2820. https://doi.org/10.3390/ijms22062820
https://www.mdpi.com/journal/ijms

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for example telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT).
The overexpression of telomerase overrides replicative senescence and the cells display
a high proliferation rate while maintaining their mesenchymal differentiation capacity [4].
Therefore, hMSC-TERT are a suitable in vitro model for primary hMSC and can be used to
establish standardized methods.
As the outermost layer of the cell, the glycocalyx is a highly versatile network consist-
ing mainly of proteoglycans [
exhibits different important functions, e.g., from being a physical barrier regulating en-
docytotic processes [ ], mediating immunogenicity [ 12], cell adhesion [13 15] and
signaling [16], to the transduction of shear stress toward the cytoskeleton [17 19]. The func-
tions are mainly related to interactions with the cell environment, which also defines its
characteristics such as molecular composition, organization [20 21], and thickness [22].
5] and glycoproteins covering the membrane. The glycocalyx
6
–
8
9–
–
–
,
Due to its biochemical importance and its ease of accessibility for experimental manip-
ulations, the cell glycocalyx offers great potential as a target for questions in basic and
biomedical research. The method of choice here is metabolic glycoengineering, represent-
ing an easy-to-use and versatile tool for the investigation or modification of glycosylated
macromolecules such as proteins or glycans [23]. For this purpose, cells are fed with
monosaccharides bearing unnatural functional groups such as azides or alkynes, resulting
in their incorporation into macromolecules as part of the glycosylation process [24]. While
some of them remain in the cytosol, others are transferred to the cell membrane and become
parts of the glycocalyx [25]. Here, these modified monosaccharides are accessible for click
chemistry, which allows the introduction of specific molecules dependent on the respective
scientific question. Although the glycosylation process cannot be controlled per se, param-
eters such as the incorporation efficiency or cell viability can be optimized by adjusting
the methodic conditions [26]. This is even more important when glycoengineered cells are
used for in vivo purposes, in which the quality of the glycoengineering affects subsequent
cell behavior in specific environments or situations.
In cell therapy-based research, glycoengineering is applied to temporarily enhance
specific cell features. The transient surface expression of an E-selectin ligand could increase
the osteotropism of systemically administered hMSC in mice [27], and the glycoengineering
of human adipose-derived stromal cells (hASC) enabled the monitoring and tracking of cell
fate in mice [28]. Conjugation of glycoengineered hMSC with paclitaxel-loaded nanoparti-
cles and its subsequent systemically administration in mice led to enhanced tumor homing
and its growth inhibition [26], thus proving a therapeutic approach. Additionally, topics
such as glycomics [29], molecular imaging, immunomodulation, or extracellular commu-
nication, e.g., by small molecules or antibody-based modulators of glycosyltransferases
and glycosidases [30
,31] can be addressed with this technique. Many of the cell functions
that are mediated by glycans and glycosylation states are involved in cell differentiation
and development, as well as in stem cells of various differentiation stages and origins [32
34]. It has been reported that glycosylation impacts osteogenic differentiation of skeletal
precursors; hence, metabolic glycoengineering of skeletal precursors may offer an excel-
lent possibility to further dissect the influence of glycosylation on bone formation and
–
regeneration and open up new therapeutic targets [35,36]. Once the altered cells reach
their destiny, it is desirable that they return to a native cell state omitting any artificial
modifications. On the contrary, a certain stability of these alterations is necessary to define
the time window for further processing of glycoengineered cells, which is of interest for
research in tissue regeneration and engineering. The attachment capability of glycoengi-
neered macrophages resulted in a better in situ cellularization of a tissue scaffold implanted
in mice [37]. Additionally, our research, embedded in the biofabrication field, aims to
improve the cell adherence after bioprinting and also the cell stability by modifying the cell
glycocalyx.
In this study, we investigated the incorporation of N-azidoacetylglucosamine (Ac4Glc-
NAz), N-azidoacetylgalactosamine (Ac4GalNAz), N-azidoacetylmannosamine (Ac4Man-
NAz), and N-alkyneacetylmannosamine (Ac4ManNAI) into the glycocalyx of hMSC-TERT

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and their functional impact on the cells as the first step toward the application in biofabrica-
tion processes. hMSC-TERT were used as a model for primary bone marrow-derived hMSC
to overcome their heterogeneity and donor variabilities. Besides the comparative analysis
of the incorporation efficiency, we looked for the long-term stability of the introduced
modified monosaccharides. The complementary assessment of cell viability, differentiation
potential, and gene expression shall provide insights into the question of how the cells
adapt to the presence of modified monosaccharides. Finally, the results will help to evalu-
ate if and how far metabolic glycoengineering can be applied to TERT-modified skeletal
precursors.
2. Results
2.1. Impact of Click Reagents on the Cell Viability
Prior to the characterization of metabolic glycoengineering, we tested the impact of
click reagents on the cell features to define the optimal experimental conditions. All four
investigated modified monosaccharides showed only slightly increased apoptosis rates,
except Ac4ManNAz, which was 3.6-fold higher than the control, and significantly (p = 0.03)
decreased viability rates (40 to 60%) at 50
the 20 M concentrations showed viability rates above 90% for Ac4GlcNAz and Ac4GalNAz
and 67% for both mannose sugars, while there was no remarkably increased apoptosis.
µM concentrations (Figure 1A). The results for
µ
Figure 1. Cell viability of telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) after treatment with
click reagents. ( ) Viability and apoptosis rates after 72 h incubation with different modified monosaccharides relative to
DMSO-treated control (dashed line). Datasets (median with individual values) were tested for significance using two-tailed
paired t-test (p < 0.05 depicted as *). ( ) Live/dead staining after 5 min incubation with Cu(I)-containing click reaction
A
B
mixture. Green: fluorescence signal of living cells, magenta: fluorescence signal of dead cells. Representative images of
three independent experiments are shown.
The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction is cat-
alyzed by Cu(I) ions, which are stabilized by the THPTA ligand. Although the five-fold
ligand amount should bind all free Cu(I) species, we analyzed whether potential resid-

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ual free cytotoxic Cu(I) species have an impact on cell viability [38]. Live/dead staining
showed mostly living cells directly after incubation with the click reaction mixture (con-
taining different concentrations of CuSO4, the respective five-fold concentration of THPTA,
and 2.5 mM sodium ascorbate), with almost no dead cells, for all chosen concentrations
(Figure 1B). Despite a potentially higher reaction efficiency, we used 50
experiments to keep Cu(I) ion-mediated cytotoxicity as low as possible.
µM for all further
2.2. Evaluation of Different Modified Monosaccharides Regarding Their Incorporation and
Retention in the Glycocalyx
To assess the metabolic glycoengineering of hMSC-TERT, we tested all four modified
monosaccharides in two commonly used concentrations (20 and 50 µM) for their potential
to incorporate into the glycocalyx by clicking a detectable fluorescent dye using the CuAAC
click reaction.
Although there was no detectable fluorescence upon incubation with Ac4GlcNAz or
Ac4GalNAz compared to the DMSO-treated control, the mannose sugars showed a 2.4-
to 3.2-fold (Ac4ManNAz) or a 1.9- to 2.4-fold (Ac4ManNAl) higher fluorescence intensity
(Figure 2). Therefore, we decided to use both mannose sugars for all further experiments
and to refrain from applying Ac4GlcNAz or Ac4GalNAz.
Figure 2. Incorporation of four different modified monosaccharides into the glycocalyx of hMSC-TERT after 72 h incubation.
Yellow: fluorescence signal of Cy 3/5 after copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction, cyan:
fluorescence signal of cell nuclei. Representative fluorescence images of three independent experiments are shown.
The diagram shows the quantitative analysis of fluorescence micrographs from three independent experiments. Cy 3/5
fluorescence signal as a mean grey value is depicted relative to the DMSO-treated control (dashed line). Datasets (median
with individual values) were tested using two-tailed paired t-test for significance (p < 0.05 depicted as *, p < 0.01 depicted
as **).
The cell glycocalyx is subject to permanent turnover and remodeling due to intrin-
sic biosynthetic processes and extrinsic influences such as shear forces [39]; therefore,
we wanted to know how stable the chemical modifications, that were introduced by click

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reactions, were over time. For that reason, we evaluated the glycocalyx state at several time
points up to 6 d.
Although both mannose sugars revealed an initially strong decrease in fluorescence
intensity after 2 d (48% for Ac4ManNAz and 93% for Ac4ManNAl), their subsequent tempo-
ral course was different (Figure 3). In contrast to the missing fluorescence of Ac4ManNAl,
the azido mannose still showed a fluorescence signal, which continuously and evenly
declined until day six (from 1.8-fold to 1.4-fold). Interestingly, cells incubated for 2 d or
longer revealed small focal aggregates within the cytoplasm (section of Ac4ManNAz 20
2 d in Figure 3).
µM
Figure 3. Temporal course of the decline of glycocalyx modification in hMSC-TERT after 72 h incubation with Ac4ManNAz
or Ac4ManNAl. Yellow: fluorescence signal of Cy 3/5 after CuAAC click reaction, cyan: fluorescence signal of cell nuclei.
Representative fluorescence images of three independent experiments are shown. The diagram shows the quantitative
analysis of fluorescence micrographs from three independent experiments. Cy 3/5 fluorescence signal as a mean grey value
is depicted relative to the DMSO-treated control (dashed line). Datasets (median with individual values) were tested for
significance using two-tailed paired t-test (p < 0.05 depicted as *, p < 0.01 depicted as **).
2.3. Impact of Modified Monosaccharides on Mesenchymal Differentiation and Gene Regulation
Sugars are involved in many metabolic pathways; therefore, we analyzed the influence
of Ac4ManNAz or Ac4ManNAl on the mesenchymal differentiation potential of hMSC-
TERT as well as their impact on gene expression. Alizarin Red staining revealed the for-
mation of calcium phosphate aggregates for both modified monosaccharides (Figure 4A
left) after two weeks of osteogenic differentiation (it was less pronounced for Ac4ManNAz
50 µM). Oil Red staining resulted in evenly distributed lipid droplet formations in all
samples (Figure 4A right) under adipogenic conditions. Gene expression analyses of differ-
entiation marker genes (alkaline phosphatase (ALPL) and bone gamma carboxyglutamate
protein (BGLAP) (osteogenic markers), as well as fatty acid binding protein (FABP) and
peroxisome proliferator activated receptor gamma (PPAR
γ) (adipogenic markers)) showed
fold changes in the range from 0.4 to 1.3 after 72 h incubation with modified monosaccha-

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rides and subsequent differentiation (Figure 4B). In detail, stimulation with Ac4ManNAz
led to an increase of up to 1.3-fold for BGLAP (20 µM), ALPL, and both adipogenic marker
genes (50 µM), as well as for ALPL after incubation with 20 µM Ac4ManNAl. On the con-
trary, stimulation with Ac4ManNAl resulted in decreased gene expression after osteogenic
differentiation (0.6- to 0.8-fold for ALPL and BGLAP at 50
adipogenic differentiation (0.4- to 0.6-fold for FABP and PPAR
Additionally, stimulation with Ac4ManNAz (20 M) revealed a decrease of 0.6- to 0.7-fold
M) or BGLAP at 50 M. Overall, the fold changes
µM, or the latter at 20
µM) or
γ
at both concentrations).
µ
for both adipogenic marker genes (20
were scattered in all but two cases.
µ
µ
Figure 4. Differentiation capacity and gene expression of hMSC-TERT after 72 h incubation with Ac4ManNAz or
Ac4ManNAl. ( ) Alizarin Red and Oil Red staining after two weeks of osteogenic or adipogenic differentiation. Representa-
tive images of three independent experiments are shown. ( ) Gene expression analysis of alkaline phosphatase (ALPL) and
bone gamma carboxyglutamate protein (BGLAP) (osteogenic markers) as well as fatty acid binding protein (FABP) and
peroxisome proliferator activated receptor gamma (PPAR ) (adipogenic markers) relative to DMSO-treated control (dashed
line). Datasets (median with individual values) were tested for significance using two-tailed paired t-test (p < 0.05 depicted
as *). ( ) Gene expression analysis of fibronectin (FN1), laminin subunit gamma 2 (LAMC2), heparan sulfate proteoglycan
A
B
γ
C
(HSPG2), integrin subunit alpha V (ITGAV), thrombospondin 1 (THBS1), signal transducer and activator of transcription 1
(STAT1), and epidermal growth factor receptor (EGFR) relative to DMSO-treated control (dashed line). Extracellular matrix
(ECM). Datasets (median with individual values) were tested for significance using two-tailed paired t-test.
In addition, a set of seven genes responsible for different cell functions were analyzed
for fold changes after incubation with Ac4ManNAz or Ac4ManNAl. Functional areas were
represented by fibronectin (FN1), laminin subunit gamma 2 (LAMC2), and heparan sulfate
proteoglycan (HSPG2) linked to extracellular matrix components, integrin subunit alpha V
(ITGAV) and thrombospondin 1 (THBS1) linked to cell-cell interactions, as well as signal
transducer and activator of transcription 1 (STAT1) and epidermal growth factor receptor
(EGFR) linked to cell survival and signal transduction. Although most conditions did not

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result in significant fold changes compared to the DMSO-treated control, the expression of
three genes was slightly increased by using both modified monosaccharides (Figure 4C).
In detail, FN1 showed a slight increase of 1.3-fold (Ac4ManNAz 50
µ
M and Ac4ManNAl
M), HSPG2 an increase of 1.4- to 1.5-fold (Ac4ManNAz) or 2.6-fold for Ac4ManNAl
M, and STAT1 an increase of 1.3-fold (Ac4ManNAz). Additionally, ITGAV (1.2-fold
M.
Decreased gene expressions were seen for LAMC2 (0.9-fold for Ac4ManNAz and 0.6 to 0.8-
fold for Ac4ManNAl), ITGAV (0.6 to 0.7-fold at 20 M or 0.4-fold for Ac4ManNAl 50 M),
and THBS1 (0.6 to 0.8-fold for Ac4ManNAz). Furthermore, gene expression of STAT1
with 0.9-fold for Ac4ManNAl 50 M as well as EGFR (0.7- to 0.8-fold for Ac4ManNAz or
0.4-fold as a consequence of incubation with Ac4ManNAl used at 20 M) were diminished.
20
20
µ
µ
for Ac4ManNAz) and EGFR (1.4-fold for Ac4ManNAl) were slightly increased at 50
µ
µ
µ
µ
µ
Although the data showed high variances, it should be noted that there were often grouped
single values with one or two outliers representing natural biological variation.
3. Discussion
To set up the optimal conditions for metabolic glycoengineering prior to the click
experiments, we assessed the impact of modified monosaccharides on the phenotype of
hMSC-TERT. Regarding viability, sugar-treated cells revealed decreased viability rates
but no remarkably increased apoptosis rates, except partially for Ac4ManNAz at 50 µM
(Figure 1), which would indicate potential cytotoxicity. Modulation of both intrinsic and
extrinsic apoptosis pathways, such as via death receptors, cannot be excluded as a reason
for this phenomenon, although the influence of glycans and glycosylation patterns on cell
death induction is incompletely understood [40]. In 2004, Kim et al. showed a correlation
between diminished cell growth and enhanced sialic acid production after the treatment
of Jurkat cells with 500 µM Ac4ManNAc, while cell viability was similar to untreated
cells [41]. These findings are supported by our data from modified N-acetylmannosamine
(ManNAc) analogs using a different approach. Linked to the incorporation efficiencies,
the decreased viability rates for both mannose sugars would correlate with the respec-
tive sugar concentration, because a higher concentration results in a higher incorporation
efficiency (Figure 2). Despite no detection via click chemistry, we assume that the cells
rather use the azido variants of N-acetylglucosamine (GlcNAc) and N-acteylgalactosamine
(GalNAc) for O-linked GlcNAc processing [25]. This glycosylation type results in glycopro-
teins residing in the nucleus and the cytosol, which cannot be targeted by the alkyne-Cy3
dye during the click reaction, reflected by the absence of fluorescence signals (Figure 2).
Regarding the click reaction, cell viability was high even at Cu concentrations up to 100 µM
(Figure 1B), which can be explained by the short exposure time of 5 min with subsequent
washing steps removing residual Cu(I) ions and the stable complexing through the lig-
and [42]. This short-time resistance against Cu(I) ions has already been shown in viability
tests for NIH 3T3 fibroblasts and HEK293-F cells in 2016. In those experiments, the cells still
showed a survival rate of approximately 80% after 10 min incubation with up to 200
Cu (both), and even after 20 min for the HEK293-F cells [43].
µM
Additionally, we analyzed whether the stimulation of hMSC-TERT with modified
monosaccharides has an impact on their mesenchymal differentiation capacity. The assess-
ment of the osteogenic and adipogenic differentiation potential suggests no remarkable
impairment after incubation with Ac4ManNAz or Ac4ManNAl. The Oil Red staining
results revealed a highly regular lipid accumulation regardless of the test conditions
(Figure 4A, right), and the Alizarin Red staining also revealed positive results for all condi-
tions, confirming a successful mineralization as well (Figure 4A, left). The marker gene
expression data showed no significant inhibition or increased fold changes for adipogenic
and osteogenic marker genes after incubation with Ac4ManNAz at 20 µM, suggesting that
the differentiation process was not severely impaired (Figure 4B). To obtain more insight
into the molecular influence of both mannose sugars Ac4ManNAz and Ac4ManNAl, we an-
alyzed their impact on gene expression in hMSC-TERT. Genes were selected according to
a paper published by the group of Han et al. [44]. The authors showed that Ac4ManNAz,

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if applied in concentrations higher than 20
µM, led to a downregulation of genes related to
one of the following four subgroups: cell adhesion, cytokine-cytokine receptor interaction,
extracellular matrix (ECM)-receptor interaction, and cancer-related pathways. We selected
seven genes from these groups and analyzed their expression in hMSC-TERT after 72 h
incubation with Ac4ManNAz or Ac4ManNAl by qPCR. All examined gene expressions
were affected by high variances for all conditions, suggesting no relevant biological conclu-
sions for these datasets (Figure 4C). Considering that there were no obvious differences
(also not as trend findings) deriving from the functional click group or the concentration,
we assume that our data indicate no interference of Ac4ManNAz or Ac4ManNAl with
transcriptional processes during protein biosynthesis in hMSC-TERT as opposed to that
shown in endothelial progenitor cells.
Several modified monosaccharides exist and are used for metabolic glycoengineering;
therefore, we tested Ac4GlcNAz, Ac4GalNAz, Ac4ManNAz, and Ac4ManNAl as alkyne
analogs for their biochemical incorporation into the glycocalyx of hMSC-TERT. We could
detect only Ac4ManNAz and Ac4ManNAl in the glycocalyx as part of glycans (Figure 2),
however with a difference regarding their respective incorporation efficiency. Although
the indirectly derived fold changes for both mannose sugars in the range of 1.9 to 2.3 seem
to be small by numbers, we expect this level of incorporation efficiency to be sufficient for
our planned functional modifications in the biofabrication field, which has to be confirmed
by subsequent studies. Additionally, it is noteworthy that metabolic glycoengineering of
adherent cells might generally result in lower modification degrees because the apical side
of the cell layer is primarily exposed to the click reagents. We assume that the different
incorporation efficiencies do not result from processes prior to the glycosylation because
the uptake of the modified monosaccharides into the cell regulated by the diffusion rate
and their subsequent enzymatic deacetylation by unspecific esterases should work for
all four molecules. Therefore, we conclude similar intracellular sugar concentrations for
subsequent glycosylation pathways. Even though an enzyme exists for the direct conver-
sion of GlcNAc to ManNAc (N-Acylglucosamine 2-epimerase), the hMSC-TERT might use
Ac4GlcNAz to fill the UDP-GlcNAc pool for the cytosolic O-GlcNAc glycosylation instead.
GalNAc can be converted to UDP-GlcNAc via a three-step process; therefore, the similar
absent incorporation of Ac4GalNAz into the glycocalyx might be a consequence [25]. By far
the strongest fluorescence signals could be observed when using the mannose sugars.
In contrast to the other two isomers, these monosaccharides are direct precursors of sialic
acid, promoting its biosynthesis and related negative feedback loop [45,46]. Obviously,
the glycosylation process does not only depend on the type of sugar which is used, but also
on the cell type. In previous studies, we have shown that the glycoengineering of neu-
roblastoma (SK-N-MC) and osteosarcoma (U2OS) cell membranes resulted in the highest
density of labeled glycans for GalNAz glycoconjugates, followed by ManNAz-derived
glycoconjugates, while GlcNAz glycoconjugates showed the lowest density with a peculiar
dependence on the investigated cell type [47]. In further studies, treatment of HEK 293
cells with Ac4GlcNAz [43] or Ac4GalNAz [48] resulted in a proper incorporation as seen
in this study for the mannose sugars, while Ac4ManNAz showed different incorporation
efficiencies for pancreatic cell lines SW1990 and PANC-C1 as well as CHO and Jurkat
cells [49]. Moreover, quantification of whole cell sialic acid levels after incubation with
different modified monosaccharides by Dold et al. [48] revealed sugar analog-dependent
differences between HEK 293T and HeLa S3 cells, but also in the compartmentalization
of the respective sialic acids between cell membranes and cytosol. Interestingly, a study
from 2009 showed a higher conversion of Ac4ManNAl into the respective sialic acid than
Ac4ManNAz in six different cell lines [50], but without any investigation regarding their
compartmentalization. Taking into account the impact of the specific cell environment on
the glycocalyx development in terms of physical interactions (higher shear forces by fluid
flow as an example for endothelial cells [51]) and nutrition supply, these observations also
suggest specific enzyme expression patterns in the sialic acid biosynthesis pathway leading
to different metabolic fluxes.

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After detecting the successful incorporation of modified monosaccharides, we wanted
to know how long these introduced modifications persist in the glycocalyx, which will be
important for the applications of glycoengineered cells. The microscopic results revealed
an initial decrease in the fluorescence signal for both mannose sugars, meaning a loss of
the introduced glycocalyx modifications (Figure 3). While these alterations disappeared
after 2 d when using Ac4ManNAl, the fluorescence signal of the covalently bound alkyne-
Cy3 slowly decreased over time, but was still detectable after 6 d. In previous studies
with primary hMSC [26] or hASC [28], a constant decline of the fluorescence signal over
a period of 7 d or even 14 d in the case of hASC could be observed, with almost no signal
at the endpoint. With regard to our Ac4ManNAz data, it can be assumed that the modified
hMSC-TERT will behave similarly and return to their native glycocalyx state after a while.
The reason for the missing fluorescence signal after 2 d when using Ac4ManNAl is not
obvious because both functional groups (azide and alkyne) are biological inert and small
in terms of steric hindrance [52]. The only difference lies in their polarity; the azide exhibits
delocated partial charges, which could interfere at least with enzymatic cleavage. Finally,
the cells showed small focal signals (section of Ac4ManNAz 20 µM 2 d in Figure 3) after 2
d, which persisted until 6 d. These structures might be internalized glycoprotein fragments
as part of salvage processes in the light of glycocalyx turnover, which was also suggested
by Layek et al. [26].
Concerning the numerous application possibilities for metabolic glycoengineering due
to the feasibility of introducing any target molecule into the cell glycocalyx, it is important
to know which modified monosaccharides provide the best outcomes in terms of viability
and incorporation efficiency. In particular, if animals or patients are involved in the study,
the glycoengineering of cells needs to be optimized for the respective purposes. This study
further elucidates the surprisingly variable glycosylation of glycocalyx-located proteins
in dependence on the click group-bearing monosaccharide and the cell type. We could
demonstrate the successful incorporation of modified monosaccharides into the glycocalyx
of hMSC-TERT; therefore, further studies could focus on interesting applications for glyco-
engineered primary hMSC using hMSC-TERT as an easy-to-manage model before starting
time-consuming experiments with primary cells or even animals.
4. Materials and Methods
4.1. Cell Culture
hMSC-TERT were established from a 33-year-old male donor by the group of Kassem
(Odense, Denmark). These cells show high proliferation rates, while maintaining their
mesenchymal differentiation capacity in vitro and in vivo [53,54]. Cells were cultured
in Eagle’s Minimum Essential Medium (MEM) (Thermo Fisher Scientific, Waltham, MA,
USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (Bio & Sell, Ulm,
Germany) [55], 50 µ
g mL−1 gentamicin sulfate, and 100 nM sodium selenite (both Sigma-
Aldrich Chemie GmbH, Munich, Germany) [56] at 37 ◦C in a 95% humidified air and 5%
CO2 atmosphere.
4.1.1. Synthesis of 2-(N-4-Pentynoyl)-2-Deoxy-D-Mannopyranoside
The alkyne mannose was synthesized according to the literature [57], with slight mod-
ification. Briefly, 2-amino-2-deoxy-D-manno-pyranose hydrochloride (238 mg, 1.10 mmol,
1.00 eq.) was dissolved in dry methanol (12 mL). Na2CO3 (132 mg, 1.25 mmol, 1.13 eq.) and
O-succinimide-4-pentynoic acid were added to the solution and the mixture was stirred at
ambient temperature for 16 h. The solvent was evaporated and the crude product was pu-
rified by column chromatography (DCM:MeOH 9:1
Figure 5 is the structural formula of 2-(N-4-Pentynoyl)-2-Deoxy-D-Mannopyranoside.
→
8:2). Yield: 85% (243 mg, 937 µmol).

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Figure 5. Structural formula of 2-(N-4-Pentynoyl)-2-Deoxy-D-Mannopyranoside.
The 1H-NMR of a major anomer (400 MHz, MeOD) (Supplementary Figure S1): 5.00
(d, J = 1.6 Hz, 1H, H-1), 4.30 (dd, J = 4.7 Hz, J = 1.6 Hz, 1H, H-2), 4.00 (dd, J = 9.7
3
3
3
3
3
3
Hz, J = 4.9 Hz, 1H, H-3), 3.87–3.75 (m, 3H, H-5, H-6), 3.58 (dd, J = 9.6 Hz, 1H, H-4),
2.49–2.46 (m, 3H, H-20, H-30), 2.27–2.25 (m, 1H, H-50) ppm; 13C-NMR of a major anomer
(100 MHz, MeOD) (Supplementary Figure S2): 174.71 (C-10), 95.00 (C-1), 83.70 (C-40), 73.44
(C-5), 70.62 (C-3), 70.24 (C-50), 68.48 (C-4), 62.23 (C-6), 55.09 (C-2), 35.88 (C-20), 15.63 (C-30)
ppm; HRMS (ESI): m/z calculated for C11H17NO6 [M-Cl−] 294.0750, measured 294.0758,
(|∆m/z| = 2.8 ppm).
4.1.2. Synthesis of
2-(N-4-Pentynoyl)-2-Deoxy-(1,3,4,6)-Tetra-O-Acetyl-D-Mannopyranoside
Previous alkyne mannose (230 mg, 887
µ
mol, 1.00 eq.) was suspended in 3 mL
pyridine and cooled at 0 ◦C. Acetic anhydride (839
µL, 8.87 mmol, 10.0 eq.) was added
and the mixture was stirred at ambient temperature for 18 h. The mixture was diluted
with DCM (150 mL) and the organic layer was washed with saturated NaHCO3 (30 mL)
solution and brine (30 mL). The organic layer was dried over MgSO4 and the crude product
was purified by column chromatography (Cy:EE 1:1). Yield: 87% (330 mg, 772 µmol).
Figure 6 is the structural formula of 2-(N-4-Pentynoyl)-2-Deoxy-(1,3,4,6)-Tetra-O-Acetyl-D-
Mannopyranoside.
Figure 6.
Structural formula of 2-(N-4-Pentynoyl)-2-Deoxy-(1,3,4,6)-Tetra-O-Acetyl-D-
Mannopyranoside.
The 1H-NMR of a major anomer (400 MHz, CDCl3) (Supplementary Figure S3): 6.11
(d, 3J = 9.1 Hz, 1H, NH), 6.04 (d, 3J = 1.8 Hz, 1H, H-1), 5.32 (dd, 3J = 10.2 Hz, 3J = 4.4 Hz,
1H, H-3), 5.22 (dd, 3J = 10.0 Hz, 1H, H-4), 4.67 (ddd, 3J = 9.3 Hz, 3J = 4.4 Hz, 3J = 1.8 Hz,
1H, H-2), 4.29–4.24 (m, 1H, H-6), 4.11–4.01 (m, 1H, H-5, H-6), 2.56–2.45 (m, 1H, H-20, H-30),
2.17 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.00 (s, 3H, Ac) ppm; 13C-NMR of a major
anomer (100 MHz, CDCl3) (Supplementary Figure S4): 171.36 (C-10), 170.67 (CO(O)-6),
170.23 (CO(O)-3), 169.77 (CO(O)-4), 168.29 (CO(O)-1), 91.78 (C-1), 82.66 (C-40), 70.65 (C-50),
70.26 (C-5), 68.97 (C-3), 65.56 (C-4), 62.12 (C-6), 49.37 (C-2), 35.47 (C-20), 21.00-20.78 (4
CH3) 15.13 (C-30) ppm.
×
The 1H-NMR of a minor anomer: (400 MHz, CDCl3): 6.07 (d, 3J = 9.1 Hz, 1H, NH),
5.86 (d, 3J = 1.8 Hz, 1H, H-1), 5.16 (dd, 3J = 9.8 Hz, 1H, H-4), 5.04 (dd, 3J = 10.0 Hz, 3J =
4.0 Hz, 1H, H-3), 4.80 (ddd, 3J = 9.2 Hz, 3J = 4.0 Hz, 3J = 1.7 Hz, 1H, H-2), 4.29–4.24 (m,
1H, H-6), 4.11–4.01 (m, 1H, H-6), 4.80 (ddd, 3J = 9.7 Hz, 3J = 5.1 Hz, 3J = 2.5 Hz, 1H, H-5),
2.56–2.45 (m, 1H, H-20, H-30), 2.10 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.00 (s, 3H,
Ac) ppm; 13C-NMR of a minor anomer (100 MHz, CDCl3): 171.81 (C-10), 170.69 (CO(O)-6),

Int. J. Mol. Sci. 2021, 22, 2820
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170.30 (CO(O)-3), 169.77 (CO(O)-4), 168.50 (CO(O)-1), 90.69 (C-1), 82.73 (C-40), 73.58 (C-5),
71.46 (C-3), 70.65 (C-50), 65.38 (C-4), 62.00 (C-6), 49.60 (C-2), 35.64 (C-20), 21.00-20.78 (4
×
CH3), 15.21 (C-30) ppm. HRMS (ESI): m/z calculated for C19H25NO10 [M-Na+] 450.1371,
measured 450.1365, (|∆m/z| = 1.3 ppm).
4.2. Cell Viability and Apoptosis Assays
To examine cell viability and apoptosis, hMSC-TERT were seeded in a 96-well plate
◦
(1
×
103 cells per well) to adhere at 37 C and 5% CO2 overnight. Cells were treated
with peracetylated N-azidoacetylglucosamine (Ac4GlcNAz), N-azidoacetylgalactosamine
(Ac4GalNAz), N-azidoacetylmannosamine (Ac4ManNAz) (Jena Bioscience GmbH, Jena,
Germany), or N-alkyneacetylmannosamine (Ac4ManNAl) in 20 µM or 50 µM, or with
dimethyl sulfoxide (DMSO) (0.2% v/v) (AppliChem GmbH, Darmstadt, Germany) as
a negative control for 72 h. Viability and apoptosis rates were assessed using the CellTiter-
Glo Luminescent Cell Viability Assay and the Caspase-Glo 3/7 Assay, respectively (both
Promega GmbH, Mannheim, Germany), according to the manufacturer’s instructions.
Luminescence was measured with an Orion II Luminometer (Berthold Detection Sys-
tems, Pforzheim, Germany). Each reaction was performed in technical triplicates and
luminescence values were averaged.
4.3. Live/Dead Assay
To evaluate the ratio of viable to dead cells, hMSC-TERT were seeded in a 24-well plate
◦
(4
×
104 cells per well) to adhere at 37 C and 5% CO2 overnight. For preparation of the click
mixture, Dulbecco’s phosphate-buffered saline (DPBS) (AppliChem GmbH, Darmstadt,
Germany) was supplemented with 50 M, 75 M or 100 M CuSO4 (Merck KGaA, Darm-
stadt, Germany), 250 M, 375 M or 500 M of tris(3-hydroxypropyl-triazolylmethyl)amine
µ
µ
µ
µ
µ
µ
(THPTA) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), respectively, as well as 2.5 mM
sodium ascorbate (Sigma-Aldrich Chemie GmbH, Munich, Germany) and incubated at
37 ◦C for 10 min. Meanwhile, cells were washed with DPBS followed by a treatment of
one well with 70% (v/v) isopropanol (AppliChem GmbH, Darmstadt, Germany) for 5 min
as a dead control. Cells were incubated with the mixture at 37 ◦C and 5% CO2 for 5 min
and washed three times with DPBS. After staining by using the LIVE/DEAD Cell Imaging
Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA,
USA), cells were imaged with a LEICA fluorescence microscope (Leica Camera AG, Wetzlar,
Germany) using a 10× objective.
4.4. Adipogenic Differentiation
For adipogenic differentiation, hMSC-TERT were seeded at a density of 2 × 105 cells
per well in 6-well plates, cultivated until confluence and incubated in adipogenic differenti-
ation medium consisting of DMEM High Glucose, 10% FCS, 1 U mL−1 penicillin, 100
µg
mL−1 streptomycin, 1
g mL−1 insulin, and 100
µ
µ
M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX),
1
µ
M indomethacin. After two weeks, the cells were harvested
for RNA isolation and intracellular lipid droplet staining. Cells cultured in expansion
medium served as negative controls and the medium was changed twice per week.
4.5. Osteogenic Differentiation
hMSC-TERT were differentiated into the osteoblastic lineage by seeding 2
cells per well in 6-well plates, cultivated until confluence, and incubated in osteogenic
×
105
−1
differentiation medium consisting of DMEM High Glucose, 10% FCS, 1 U mL penicillin,
100
µ
g mL−1 streptomycin (all Life Technologies GmbH), 50
µ
g mL−1 L-ascorbic acid-2-
phosphate, 0.1
µM dexamethasone, and 10 mM β-glycerophosphate (all Sigma Aldrich
GmbH). After two weeks, the cells were harvested for RNA isolation and mineralized
matrix staining. Cells cultured in expansion medium served as negative controls and
the medium was changed twice per week.

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4.6. Histochemical Staining
For the detection of calcium hydrogen phosphate and hydroxylapatite in the extracel-
lular matrix, hMSC-TERT were fixed in methanol, stained with alkaline Alizarin Red S (1%
w/v) (Chroma-Schmidt GmbH, Stuttgart, Germany) for 2 min, and air dried. For the detec-
tion of intracellular lipid vesicles, adipogenic monolayer cultures were stained with Oil
Red O solution (Merck, Darmstadt, Germany). Images were taken with a digital camera
EX Z150 (Casio Computer Co, Tokyo, Japan).
4.7. RNA Isolation, Reverse Transcription and qPCR Analysis
For gene expression analyses, hMSC-TERT were seeded in 6-well plates (2
×
105
M of
cells per well) to adhere at 37 ◦C and 5% CO2 overnight and treated with 20 or 50
µ
Ac4ManNAz or Ac4ManNAl, or with DMSO (0.2% v/v) as negative control for 72 h. Total
RNA was isolated by using NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany)
according to the manufacturer’s instructions. For mRNA reverse transcription, 1
total RNA was used for first strand cDNA synthesis with MMLV reverse transcriptase
(Promega GmbH, Mannheim, Germany) in 25 L total volume. cDNA was diluted 1:10,
and 2 L were used for real-time qPCR with the GoTaq qPCR Master Mix (Promega GmbH,
Mannheim, Germany) in 20 L total volume. Primers were obtained from Eurofins MWG
µg of
µ
µ
µ
Operon (Ebersberg, Germany) or from Biomers GmbH (Ulm, Germany) (Supplementary
Table S1 for primer sequences and annealing temperatures). qPCR conditions were as
follows: 95 ◦C for 3 min; 40 cycles: 95 ◦C for 10 s; respective annealing temperature for
◦
10 s; 72 C for 10 s; followed by melting curve analysis for specificity of the qPCR products
by using the qPCR thermal cycler Professional Thermocycler Biometra (Analytik Jena
AG, Jena, Germany). Each reaction was performed in technical triplicates and Ct values
were averaged. Relative gene expression was calculated with the efficiency-corrected Ct
model [58] using RPLP0 as the reference gene.
4.8. Fluorescence Imaging after Click Reaction on the Cell Surface
For the evaluation of metabolic glycoengineering, hMSC-TERT were seeded in a 24-
well plate (1
afterwards in growth medium with Ac4GlcNAz, Ac4GalNAz, Ac4ManNAz, or Ac4ManNAl
at 20 M or 50 M, or with DMSO (0.2% v/v) as a negative control for 72 h. For the CuAAC
click reaction, DPBS was supplemented with 50 M CuSO4 (Merck KGaA, Darmstadt,
Germany) and 250 M THPTA (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), as
×
104 cells per well) to adhere at 37 ◦C and 5% CO2 overnight and incubated
µ
µ
µ
µ
well as 2.5 mM sodium ascorbate (Sigma-Aldrich Chemie GmbH, Munich, Germany), and
◦
incubated at 37 C for 10 min. Then, 20
µM alkyne-sulfo-Cy3 (Sigma-Aldrich Chemie
GmbH, Munich, Germany) or azido-sulfo-Cy5 [59] were added and cells were treated with
the mixture at 37 ◦C and 5% CO2 for 5 min after removal of the medium and one washing
step with DPBS. After the click reaction, cells were washed three times with DPBS and incu-
bated with growth medium for 30 min, 2 d, 4 d, or 6 d. For fluorescence imaging, cells were
washed once with DPBS, incubated with a mixture of 2% (v/v) formaldehyde (Merck KGaA,
Darmstadt, Germany) and 2.5% (v/v) glutaraldehyde (Sigma-Aldrich Chemie GmbH, Mu-
nich, Germany) at 4 ◦C for 10 min, washed three times with DPBS, and followed by an
incubation with 300 nM 40,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Chemie
GmbH, Munich, Germany) in DPBS at room temperature for 10 min for nuclei staining
and three washing steps with DPBS. Images were taken with a Zeiss Axio Observer 7 fluo-
rescence microscope (Carl Zeiss AG, Oberkochen, Germany) using a 10
×
objective, while
exposure time and gain for Cy3 or Cy5 fluorescence were kept constant. For qualitative
comparison, brightness and contrast of Cy3/5-related fluorescence signals were adjusted
according to the sample with the highest signal intensity. For indirect quantitation, grey
values of unaltered Cy3/5 related fluorescence signals from three different visual fields per
micrograph were measured using Fiji 1.53c (National Institutes of Health, Bethesda, MD,
USA) [60] and averaged.

Int. J. Mol. Sci. 2021, 22, 2820
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4.9. Data Processing
Numerical data were evaluated with Office Excel 2016 (Microsoft Corporation, Red-
mond, WA, USA) and graphs were created in Prism 7 (GraphPad Software, San Diego, CA,
USA), while image processing was performed with Fiji [60] and its plug-in ScientiFig [61].
For statistical analysis, datasets were compared to DMSO-treated controls for significance
in Office Excel 2016 using two-tailed paired t-tests, with which p-values less than 0.05 were
considered to be statistically significant.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006
7/22/6/2820/s1.
Author Contributions: Conceptualization, R.E. and J.S.; methodology, S.A., J.M.-W., J.M., N.W.,
M.G., and T.L.; formal analysis, S.A., J.M.-W., N.W. and J.M.; investigation, S.A., J.M.-W., J.M. and
N.W.; resources, R.E., J.S., F.J. and M.R.; data curation, S.A., J.M.-W. and J.M.; writing—original
draft preparation, S.A., R.E. and J.M.; writing—review and editing, all authors; visualization, S.A.;
supervision, R.E. and J.S.; project administration, R.E. and J.S.; funding acquisition, R.E. and J.S. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research
Foundation)—Project number 326998133–TRR 225 (subproject B05).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data presented in this study are available from the corresponding
author on reasonable request.
Acknowledgments: We thank Moustapha Kassem (Odense, Denmark) for providing hMSC-TERT.
Conflicts of Interest: The authors declare no conflict of interest and the funders had no role in the de-
sign of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,
or in the decision to publish the results.
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