Photocontrolled Multidirectional Differentiation of Mesenchymal Stem Cells on an Upconversion Substrate

Article abstract of DOI:10.1002/anie.201803939

The effective guidance of mesenchymal stem cell (MSC) differentiation on a substrate by near-infrared (NIR) light is particularly attractive for tissue engineering and regenerative medicine. However, most of current substrates cannot control multidirectio

Full text of DOI:10.1002/anie.201803939

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Communications
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International Edition: DOI: 10.1002/anie.201803939
German Edition: DOI: 10.1002/ange.201803939
Tissue Engineering
Photocontrolled Multidirectional Differentiation of Mesenchymal
Stem Cells on an Upconversion Substrate
Zhengqing Yan, Hongshuang Qin, Jinsong Ren, and Xiaogang Qu*
Abstract: The effective guidance of mesenchymal stem cell
(MSC) differentiation on a substrate by near-infrared (NIR)
light is particularly attractive for tissue engineering and
regenerative medicine. However, most of current substrates
cannot control multidirectional differentiation of MSCs like
natural tissues. Herein, a photocontrolled upconversion-based
substrate was designed and constructed for guiding multidirec-
tional differentiation of MSCs. The substrate enables MSCs to
maintain their stem-cell characteristics due to the anti-adhesive
effect of 4-(hydroxymethyl)-3-nitrobenzoic acid modified
poly(ethylene glycol) (P1) attached on the upconversion
substrate. Upon NIR irradiation, the P1 is released from the
substrate by photocleavage. The detachment of P1 can change
cell–matrix interactions dynamically. Moreover, MSCs cul-
tured on the upconversion substrate can be specifically induced
to differentiate to adipocytes or osteoblasts by adjusting the
NIR laser. Our work provides a new way of using NIR-based
upconversion substrate to modulate the multidirectional differ-
entiation of MSCs.
compatible environment. However, the complex composition
of natural materials usually makes it difficult to elucidate
their corresponding effects on cell differentiation. In contrast,
smart artificial interface materials are usually easy to modify
with functional biomolecules, which can dynamically switch
stem cells from self-renewal to differentiation through
a simple chemical microenvironmental change.[3a] In partic-
ular, photo-switched substrates have attracted much attention
since they possess the advantages of high spatial and temporal
precision and are environmentally friendly.[5c,10] However,
most studies have focused on high-energy UV/Vis light, which
limits their further applications in vivo due to the harmful side
effects and poor tissue penetration of UV/Vis light. There-
fore, it is necessary to develop biocompatible substrates to
control cell differentiation dynamically.
Since lanthanide-doped upconversion nanoparticles
(UCNPs) possess the intrinsic properties of absorbing and
converting near-infrared (NIR) light into high-energy UV,
visible, or NIR irradiation, they have been widely applied in
biodetection,[11] nanomedicines,[12] and information secur-
ity.[13] In our previous studies, UCNPs have been used for
on-demand manipulation of cell behaviors, such as cell
capture and release[14] and neurite outgrowth of PC12
cells.[15] Bian et al. recently reported that UCNPs can be
used to trigger the release of small molecules (e.g., kartoge-
nin) and subsequently induce the chondrogenic differentia-
tion of MSCs.[16] Although promising, none of the reported
UCNPs-based materials can modulate the multidirectional
differentiation of mesenchymal stem cells like natural tissues.
Developing a UCNP-based platform that enables MSCs to be
differentiated into diverse cell types is still challenging.
In this work, we designed and constructed a UCNP-based
substrate by molecular engineering. The substrate properties
can be adjusted by NIR irradiation, which modulates multi-
directional differentiation of MSCs. As illustrated in
Scheme 1, the substrate (named UCNP/P1/P2-RGD) consists
of UCNPs, 4-(hydroxymethyl)-3-nitrobenzoic acid (ONA,
a commonly employed photo-cleavage molecule[17]) modified
poly(ethylene glycol) (PEG) (marked as P1) and arginine-
glycine-aspartic acid (RGD)-modified PEG (marked as P2).
The RGD moieties of P2 on UCNP/P1/P2-RGD can capture
MSCs, while the PEG moieties of P1 can block the interaction
between MSCs and substrate.[18] Under NIR laser irradiation,
the UCNPs can convert NIR light into UV irradiation. The
UV irradiation can control the detachment of P1 and
subsequently change cell–matrix interactions. Strong correla-
tions exist between the P1 detachment and the power density
of the NIR irradiation. Upon low-power NIR irradiation
(0.5 Wcmꢀ2), MSCs tend to differentiate into adipocytes due
to the fact that some P1 start to be released from the
A
s one of the widely studied pluripotent stem cells,
mesenchymal stem cells (MSCs) have been considered as
the most eligible cells for skeletal tissue engineering and
regenerative medicine.[1] Guiding the differentiation of stem
cells into specific cell-types on the matrix efficiently is crucial
for stem-cell-based regeneration. Traditionally, growth fac-
tors are often used to control stem-cell fate. However, this
approach is still facing disappointing clinical outcomes from
trials.[2] There is thus an urgent need to develop alternative
strategies to control stem-cell differentiation. Engineering
cell-culture matrices is a good way approaching this issue[3]
because the complex signaling pathways of stem cells can be
regulated by the physicochemical properties of the extracel-
lular matrix, such as surface composition,[1a,4] adhesive
ligands,[5] topography,[6] smoothness/roughness,[7] and flexibil-
ity/rigidity.[8] To date, some natural materials, such as alginate
and hyaluronic acid[9] and synthetic materials,[3b,d] have been
developed as matrices. Natural materials can provide a bio-
[*] Z. Yan, H. Qin, Prof. J. Ren, Prof. X. Qu
Laboratory of Chemical Biology and
State Key Laboratory of Rare Earth Resource Utilization
Changchun Institute of Applied Chemistry
Chinese Academy of Sciences, Changchun, Jilin 130022 (China)
E-mail: xqu@ciac.ac.cn
Z. Yan
University of Chinese Academy of Sciences
Beijing, 100039 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201803939.
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Figure 1. a) TEM and b) HRTEM images of NaYF4: TmYb. c) TEM
image of UCNP@SiO2. d) Dark-field TEM image of UCNP@SiO2 and
its corresponding TEM elemental mappings of Si-K, F-K, Y-L, Yb-L, and
Tm-L edge signals.
Scheme 1. Schematic illustration of modulating cell–matrix interactions
and fate commitment of mesenchymal stem cells (MSCs) by using
a UCNP-based substrate.
1
3
1
3
uted to I6! F4 (345 nm) and D2! H6 (368 nm) transitions
of Tm3+ ions,[20] which could activate photo-cleavage of P1
molecule. When light from an NIR laser traveled through the
UCNPs and UCNP@SiO2 colloidal solutions, purple-blue
photoluminescence was also observed (Figure S5, inset). To
obtain amino-functionalized nanohybrids (UCNP@SiO2-
NH2), the surface of UCNP@SiO2 was modified with (3-
aminopropyl)triethoxysilane. The corresponding modifica-
tion processes were evaluated by Fourier transform infrared
spectra and zeta potential (Figure S6). Next, the substrate
UCNP/P1/P2-RGD was prepared according to the proce-
dures shown in Scheme S2. The carboxyl-containing substrate
was first modified with UCNP@SiO2-NH2. Then P1 and P2
were introduced onto the surface together (UCNP/P1/P2)
through a carbodiimide-mediated wet-chemistry approach.
Then, in order to endow the smart substrate with the ability to
capture cells, an amino-group-containing RGD was cova-
lently conjugated on the carboxyl motif of P2. The substrate
UCNP/P1/P2-RGD was constructed. SEM images showed
the surface topography of the substrate (Figure S7a). The
absorption spectrum of the substrate in Figure S7b coincided
with the absorption spectrum of the photoactive molecule P1,
which confirmed that polymer P1 was attached onto surface
successfully. Moreover, both the photograph and UCL
emission spectroscopy of substrate UCNP/P1/P2-RGD were
obtained (Figure S7c). For demonstrating that RGD was
added onto the substrate, a cell-capture experiment was
carried out. As shown in Figure S7d,e, few cells were attached
on the unmodified surface. In contrast, more cells were
adherent on the modified substrate. Taken together, these
results suggest that UCNP/P1/P2-RGD was prepared suc-
cessfully.
substrate. Since more P1 are detached from the substrate
upon high-power NIR irradiation than that upon low-power
NIR irradiation, MSCs display high level of osteogentic
differentiation at 6 Wcmꢀ2 NIR irradiation. This work
provides evidence that an upconversion substrate can be
used to regulate multidirectional differentiation of MSCs by
adjusting NIR laser.
As a starting point for our work, P1 was synthesized
according to the procedures in Scheme S1. P1 exhibits an
obvious absorption band between 200 and 400 nm (Figure S1
in the Supporting Information), thus suggesting that ONA
was included in P1. UCNPs (NaYF4:TmYb) that can harvest
NIR were utilized as a UV source for photocleavage of P1.
The UCNPs were synthesized through thermal decomposi-
tion.[19] Transmission electron microscopy (TEM) images
demonstrated that the UCNPs possessed uniform hexagonal
shapes with an average diameter of 45 nm (Figure 1a and
Figure S2). The high-resolution TEM (HRTEM) images in
Figure 1b show that the typical d-spacing value was 0.52 nm,
thus suggesting a single hexagonal-phase crystal of the
UCNPs. To increase the water solubility and biocompatibility,
a silica shell was coated onto the UCNPs by using a micro-
emulsion method to give UCNP@SiO2. The overall size of
UCNP@SiO2 was approximately 75 nm and the thickness of
thin silica shells was 15 nm (Figure 1c and Figure S3). Mean-
while, TEM elemental mapping of Si, F, Y, Yb, and Tm
further confirmed the formation of UCNP@SiO2 nanoparti-
cles (Figure 1d). Power X-ray diffraction peaks of UCN-
P@SiO2 (Figure S4) were consistent with the standard card
No. 28–1192. The obvious UV emission in the upconversion
luminescence (UCL) emission spectrum (Figure S5) is attrib-
To explore whether an NIR laser could be used to
dynamically control P1 detachment from the upconversion
substrate, the model dye fluorescein isothiocyanate (FITC)
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was covalently modified onto the amino terminus of P1
Before studying the differentiation behavior of MSCs, we
moieties for visualization. As shown in Figure 2a, after
modification, the substrate emitted bright green fluorescence.
Then the substrate was immersed in water and treated with
low-power or high-power NIR laser. After the substrate was
treated with a low-power NIR laser, the fluorescence intensity
first explored the biocompatibility of UCNP/P1/P2-RGD
upon low-power or high-power NIR irradiation. MSCs were
cultured on UCNP/P1/P2-RGD and cell viability was assessed
with methyl thiazolyl tetrazolium (MTT) assay. As shown in
Figure S9, both UCNP/P1/P2-RGD + low power NIR laser
and the UCNP/P1/P2-RGD + high power NIR laser had little
effect on cell viability under our experimental conditions.
Next, in order to explore cell pluripotency after culturing on
the smart substrate, the expression of Sox2, Nanog, and Oct4,
important genes related to cell pluripotency (’stemmness“),
were measured by quantitative real-time PCR (qRT-PCR).[21]
The results in Figure 3a showed that the stemness markers
Figure 2. a) Schematic illustration and fluorescence images of sub-
strate (UCNP/P1-FITC/P2-RGD) exposed to no NIR, low-power NIR, or
high-power NIR irradiation. b) Fluorescent spectra of solutions of
UCNP/P1-FITC/P2-RGD upon exposure to no NIR, low-power NIR, or
high-power NIR irradiation. c) D PL intensity of solutions and the
percentage of P1 detachment. n=3.
Figure 3. MSCs were cultured on UCNP/P1/P2-RGD with exposure to
no NIR, low-power NIR, or high-power NIR irradiation in bipotential
differentiation media for 21 days. a) The expression of Sox2, Nanog,
and Oct4 (*p<0.05, n=3). b,c) Immunofluorescence imaging of
markers for osteogenic (RUNX2, green) and adipogenic (FABP4, red)
differentiation. Scale bar: 50 mm. d) Western blot analysis showing the
expression levels of osteogenic (RUNX2) and adipogenic (FABP4)
proteins in MSCs.
dropped slightly. The fluorescence of the substrate decreased
sharply upon exposure to high power NIR irradiation. Next,
to quantify the percentage of P1 detachment, the fluorescence
intensity changes of the surrounding solution upon different
power density and time duration were investigated. Fig-
ure S8a showed that the fluorescence intensity of the
solution, including UCNP/P1-FITC/P2-RGD increased
when the power density was increased from 0.5 Wcmꢀ2 to
6 Wcmꢀ2. Above 6 Wcmꢀ2, the increase was not significant.
So, in our following experiments, 6 Wcmꢀ2 and 0.5 Wcmꢀ2
were chosen as high and low power densities, respectively,
unless otherwise specified. Negligible changes in fluorescence
intensity were detected without NIR irradiation, which
confirms that almost no P1 was released from the substrate
(Figure S8b). Compared with the fluorescence intensity
changes in Figure S8b and S8c, the changes in Figure S8d
increased rapidly within 5 min, and then the fluorescence
intensity no longer changed after 5 min at 6 Wcmꢀ2 NIR
irradiation, which suggests that most of the P1 was released
within 5 min. Therefore, the irradiation time duration was set
as 5 min in the following experiments. As shown in Fig-
ure 2b,c, about 35% P1 was released from substrate upon
0.5 Wcmꢀ2 NIR irradiation for 5 min and 90% P1 released
upon 6 Wcmꢀ2 NIR irradiation for 5 min. The successful
detachment of P1 upon NIR irradiation provided a possibility
for the next step to control the differentiation behavior of
MSCs.
Sox2, Nanog and Oct4 were positive for MSCs cultured on the
smart substrate without NIR exposure. Upon both low-power
and high-power NIR irradiation, the expression of Sox2,
Nanog, and Oct4 was decreased significantly. This suggests
that the designed substrate maintained MSCs pluripotency in
the absence of NIR irradiation and the differentiation of
MSCs was triggered by P1 detachment in presence of NIR
irradiation.
To investigate the differentiation of MSCs cultured on
UCNP/P1/P2-RGD with different treatments, oil red O and
alizarin red S staining were carried out to measure neutral
lipids and calcium deposition respectively. Neutral lipids are
recognized as crucial indicators for mature adipocytes and
calcium deposition is an important marker for mature
osteoblasts. As shown in Figure S10, neither neutral lipids
(an adipogenesis marker) nor calcium deposition (an osteo-
genic marker) was found in MSCs on the substrate in the
absence of NIR exposure. With group of low-power NIR
irradiation, the MSCs exhibited primarily adipogenic differ-
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entiation (staining of neutral lipids) and a very low level of
osteogenic differentiation (staining of calcium deposition).
Whereas, for the high-power NIR group, the MSCs differ-
entiated into osteoblasts. Next, immunofluorescence and
western blot were carried out to further verify differentiation
of MSCs. The cells were stained with a cy5-conjugated
antibody against the adipogenic marker FABP4 (red fluores-
cence) and a FITC-conjugated antibody against the osteo-
genic marker RUNX2 (green fluorescence). A strong red
signal was found in cytoplasm and nucleus, thus indicating the
emergence of the FABP4 in the low-power NIR group
(Figure 3b). However, this adipogenic antigen was not over-
expressed when the substrate was treated with high-power
NIR irradiation. In addition, strong green fluorescence was
observed in the nucleus (Figure 3c), which suggested that
RUNX2 was upregulated in MSCs cultured on UCNP/P1/P2-
RGD upon high-power NIR irradiation. In contrast, expres-
sion of this osteogenic antigen expression was negligible in the
low-power NIR group. For the control group without any NIR
irradiation, neither red nor green fluorescence appeared.
Notably, lots of signals were observed in samples exposure to
no NIR or low NIR, which may attributed to the anti-
adhesion effects of P1 on UCNP/P1/P2-RGD. Once the
substrate was treated with the high-power laser, about 90% of
P1 (according to the data in Figure 2c) was detached from the
substrate, which could explain why few signals appeared on
the substrate. This shows that P1 played a vital role in the cell–
matrix interactions. Additionally, the expression of FABP4
and RUNX2 in MSCs were semi-quantified by using Image J
software (Figure S11). The mean fluorescence intensity of the
red signal (FABP4 expression) in the low-power NIR groups
was significantly higher than that in the groups with no NIR
irradiation or high-power NIR irradiation. The mean fluo-
rescence intensity of the green signal (RUNX2 expression) in
the high-power NIR group was significantly higher than that
in the no NIR or low-power NIR groups. Western blot
analysis of osteogenic (RUNX2) and adipogenic (FABP4)
protein expression in MSCs also showed a similar result
(Figure 3d). According to the results discussed above, we
concluded that NIR light can be used control cell differ-
entiation effectively. MSCs cultured on the substrate with
low-power NIR irradiation tended to differentiate into
adipogenic cells, while osteogenic differentiation was favored
when the substrate was treated with high-power NIR
irradiation.
Figure 4. Representative fluorescence microscopy images of cells cul-
tured on UCNP/P1/P2-RGD with exposure to no NIR (a), low-power
NIR (b) or, high-power NIR (c) irradiation. The cells were stained for F-
actin (green) to show the cell morphology. Scale bar: 50 mm.
whether cell differentiation was modulated by the departure
of polymer P1, the light-responsive polymer P1 was sub-
stituted with non-light-responsive PEG, marked as UCNP/
PEG/P2-RGD. Compared with MSCs on UCNPs/P1/P2-
RGD (Figure S12a), MSCs cultured on UCNPs/PEG/P2-
RGD (Figure S12b) exhibited low levels of cell differentia-
tion, no matter whether the substrate was treated with NIR
irradiation or not. In addition, to investigate the importance
of cell contractility to cell differentiation, the cell-traction
inhibitor 2,3-butanedione monoxide was used to treat MSCs
cultured on UCNPs/P1/P2-RGD and UCNPs/PEG/P2-RGD.
The MSCs on UCNPs/P1/P2-RGD were prone to differ-
entiate to adipogenic cells in the presence of 2,3-butanedione
monoxide, despite high-power NIR irradiation (Figure S12c).
As for MSCs on UCNPs/PEG/P2-RGD, they still displayed
low levels of cell differentiation in the face of 2,3-butanedione
monoxide, independent of NIR irradiation. (Figure S12d)
These results indicate that changes in cytoskeletal tension
play an important role in modulating cell differentiation,
which is consistent with previous reports.[5a,b,6a,c] That is to say,
when the polymer P1 are detached from the substrate, the
force between cell and substrate would change. Cells can
sense the force change[22] and transduce it into biochemical
signals such as b-catenin,[23] thereby leading to osteogenic and
adipogenic differentiation of MSCs.
In summary, we successfully prepared a new photocon-
trolled upconversion-based substrate. The substrate enables
MSCs to maintain their stem-cell properties due to the anti-
adhesive effect of P1 attached on the substrate. Upon NIR
irradiation, the P1 is released from the substrate by photo-
cleavage, with the level of NIR irradiation controlling the
percentage of P1 detachment and subsequently change in
cell–matrix interactions. Furthermore, western blot analysis,
immunofluorescence, RT-PCR, and oil red O/alizarin red S
staining were carried out. The results demonstrate that MSCs
tend to differentiate into adipogenic cells under low-power
NIR irradiation, whereas osteogenic differentiation is favored
under high-power NIR irradiation. Our work provides a new
way to modulate multidirectional differentiation of MSCs by
using a NIR-based upconversion substrate.
The relationship between cell morphology and fate were
next examined. The morphological changes on the upconver-
sion substrate were explored by phalloidin staining for
filamentous actin (F-actin) after different treatments.
Figure 4 showed that strong correlations were observed
between cell morphology and NIR irradiation. Figure 4a
showed that MSCs retained a round shape or spindle
fibroblast-liked shape without NIR exposure. Upon NIR
irradiation, the cells tended to spread on the substrate
(Figure 4b and Figure 4c). Moreover, a larger round adipo-
cyte-like shape was observed following low-power NIR
irradiation (Figure 4b), and the cells took up polygon shape
following high-power NIR irradiation (Figure 4c), which is
the typical morphology of osteoblasts. To further study
Acknowledgements
Financial support was provided by NSFC (21210002,
21431007, 21533008) and Key Program of Frontier of
Sciences, CAS QYZDJ-SSW-SLH052.
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The authors declare no conflict of interest.
Keywords: cell adhesion · NIR irradiation · photocleavage ·
tissue engineering · upconversion nanoparticles
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Manuscript received: April 2, 2018
Version of record online: && &&, &&&&
6
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These are not the final page numbers!

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Chemie
Communications
Tissue Engineering
Z. Yan, H. Qin, J. Ren,
X. Qu*
&&&& — &&&&
Photocontrolled Multidirectional
Differentiation of Mesenchymal Stem
Cells on an Upconversion Substrate
Leading light: An upconversion-based
substrate was designed and constructed
for photocontrolled multidirectional dif-
ferentiation of mesenchymal stem cells
(MSCs). The detachment of 4-(hydroxy-
methyl)-3-nitrobenzoic acid modified
poly(ethylene glycol) moieties on the
substrate can be adjusted by near-infra-
red (NIR) irradiation, which dynamically
changes cell–matrix interactions and
induces adipogenic or osteogenic differ-
entiation of the MSCs.
Angew. Chem. Int. Ed. 2018, 57, 1 – 7
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!