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This journal is ©The Royal Society of Chemistry 201 7 J. Mater. Chem. B, 2017, 5, 7927--7938 | 7927
Cite this: J. Mater. Chem. B, 2017,
5,7927
Heterogeneity of mesenchymal and pluripotent
stem cell populations grown on nanogrooves
and nanopillars†
Peng-Yuan Wang, *
ab
Sheryl Ding,
a
Huseyin Sumer,
a
Raymond Ching-Bong Wong
c
and Peter Kingshott
a
Surface nanotopographies are an important way of mimicking the stem cell niche on biomaterial
surfaces. Previous studies have focused on the differentiation of stem cells into a defined lineage using
nanotopographies, but they have rarely considered the homogeneity of cell populations produced.
We examined the impact of two types of substrates (i.e. nanogrooves and nanopillars made by soft
lithography) on the surface-induced differentiation of human amniotic membrane-derived mesenchymal
stem cells (hAM-MSCs) and mouse embryonic stem cells (mESCs) without the use of additional chemical
induction medium components. Cell morphology and proliferation were analysed at day 1 and day 3.
Gene expression was analysed at day 14 for hAM-MSCs and at day 7 for mESC-derived embryoid bodies
(mEBs) using quantitative real-time polymerase chain reaction (qPCR). The substrates with nanogrooves
had a noticeable effect on cell alignment in a depth dependent manner with both cell types showing
strong alignment along the deep grooves. On the other hand, the nanopillar substrates showed
inhibition of cell spreading for both cell types. The nanogrooves showed inhibition of hAM-MSC growth
but enhanced mEB proliferation, especially on the deeper grooves. The nanopillars did not significantly
affect hAM-MSC growth, but can modulate mEB growth depending on the pillar density, indicating that
mEBs are more sensitive to nanotopographies in terms of proliferation, while hAM-MSCs are only
sensitive to specific structures and sizes. Genes associated with bone, cartilage, and fat were
investigated for hAM-MSCs, whereas genes of the endoderm, mesoderm, ectoderm, and pluripotency
were investigated for mEBs. In general, gene expression for hAM-MSCs was not enhanced significantly
by the nanotopographies compared to the flat control. On the other hand, genes of bone, cartilage,
skeletal muscle, heart, and liver were up-regulated on both nanopillars and nanogrooves, especially
OP65 (ordered pillars with 65% density) and SG40 (shallow grooves with 40 nm depth) in a feature size
dependent manner. We found that a small portion of mEBs was composed of cardiac-like beating cells
(i.e. GFP-NKX2.5 positive) and a bone cell marker (i.e. OCN) indicating a heterogeneous cell population
being generated on those types of surfaces. This work highlights the importance of nanotopographies in
stem cell differentiation and how studying multiple properties of the substrate and cells is needed as we
strive to generate homogeneous and mature cell populations using biomaterials.
Introduction
Stem cell technology is an area of great excitement, with the
potential to revolutionise regenerative medicine and biomedical
research.
1
Advanced stem cell technologies have the potential
for future development of patient-specific cell therapies, tissue
regeneration, and/or more accurate research models. Stem cells
are characterised by their prolonged proliferation and ability
to differentiate into a range of cell lineages, depending on
the potency and source of the cells. Pluripotent stem cells, such
as embryonic stem cells (ESCs) derived from the blastocyst
inner cell mass, are able to differentiate into any cell type.
a
Department of Chemistry and Biotechnology, Faculty of Science, Engineering and
Technology, Swinburne University of Technology, Hawthorn, Victoria 3122,
Australia. E-mail: pengyuanwang@swin.edu.au; Tel: +61 3 9214 5939
b
Graduate Institute of Nanomedicine and Medical Engineering, College of
Biomedical Engineering, Taipei Medical University, Taipei 110, Taiwan
c
Centre for Eye Research Australia & Ophthalmology, Department of Surgery,
The University of Melbourne, Victoria 3004, Australia
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7tb01878a
Received 12th July 2017,
Accepted 22nd August 2017
DOI: 10.1039/c7tb01878a
rsc.li/materials-b
Journal of
Materials Chemistry B
PAPER
7928 |J. Mater. Chem. B, 2017, 5, 7927--7938 This journal is ©The Royal Society of Chemistry 201 7
Multipotent stem cells, such as amniotic membrane-derived
mesenchymal stem cells (AM-MSCs), are more limited in their
differentiation potential, but have their own advantages such as
safety.
2
Although stem cell technologies including in vitro
expansion have been investigated over the last decade, there
are still major barriers to be overcome such as the maintenance
of stemness during expansion and maturation of differentiated
stem cells before stem cells can become widely used in clinical
or commercial applications.
3
One such challenge is the ability
to modulate stem cell behaviour and differentiate stem cells
into a pure and mature cell population on a large scale with
high efficiency and cost-effectiveness.
Biomaterials modified with surface nanotopographies are
showing huge potential in directing stem cell behaviour and
developing better cell culture materials along with biomechanical
stimulation.
4
Surface nanotopographies can be well-defined for
stem cell manipulation compared to traditional biochemical
methods. This biophysical approach has some advantages such
as a longer lifetime, easier characterisation, higher stability,
and better reproducibility with little batch-to-batch variation
compared to biochemical approaches.
5
Nanotopographical
features can be used to mimic the in vivo stem cell niche present
on the extracellular matrix (ECM), which plays a role in the
coordination of cell morphology, self-renewal, migration, differ-
entiation, and many other functions.
1
Studies have shown that
surface nanotopographies can have striking influences on
almost all stem cell behaviour in vitro.
6–10
However, systematic
studies are still required, owing to the vast range of possible
topographical patterns being discovered, different stem cell
sources, and combinational stimulations using both biochemical
and biophysical cues, resulting in difficulties comparing results
between studies. Also, studies have often focused on one specific
cell type generated from one type of stem cell using one type of
surface structure, and often lacks information such as the homo-
geneity of surface-induced differentiated cell populations.
For example, Yim et al. used 350 nm-width PDMS nano-
grooves to study neurogenic differentiation of human mesenchymal
stem cells.
11
A positive outcome was found using 350 nm nano-
grooves compared to 10 mm-width microgrooves and flat controls
in neural differentiation. Another independent study showed that
microgrooves can improve cardiac differentiation from either
cardiac progenitors
12
or MSCs.
13
In addition, there was other
evidence showing that aligned MSCs expressed higher markers
of anisotropic tissue lineage such as tendon.
14
The exact effect of
nanogrooves on lineage commitment is unclear. Studies have
also used diverse nanotopographies, feature sizes, materials,
surface coatings, and cell sources resulting in difficulties in
elucidating the exact effects of the nanotopography.
In this study, we examined the effect and homogeneity of
surface-induced differentiation of stem cells, i.e. human amniotic
membrane-derived mesenchymal stem cells (hAM-MSCs) and
mouse embryonic stem cells (mESCs), on nanotopographies
consisting of two types of nanogrooves and four types of nano-
pillars. These were fabricated using a combination of colloidal
self-assembly, spin coating, reactive ion etching, electron beam
lithography, and soft lithography (Fig. 1). The morphology,
proliferation, and gene expression of the two stem cell lines
were analysed on the various surface topographies without
additional chemical or growth factor stimulation. Thus, the
role of surface-only induction of stem cell differentiation can be
ascertained and this systematic study explores this concept,
which is often overlooked in biomaterials and stem cell engi-
neering research.
Materials and methods
Materials
Polystyrene (PS) was purchased from Nihon Shiyaku Industries
(Tokyo, Japan). Polydimethylsiloxane (PDMS; Sylgard 184) was
received from Dow Corning (Midland, MI, USA). 40,6-Diamidino-
2-phenylindole dihydrochloride (DAPI) and phalloidin-tetra-
methylrhodamine B isothiocyanate (phalloidin-TRITC) were
purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Human mesenchymal stem cells were obtained from amniotic
membranes (hAM-MSCs) during delivery by caesarean section,
using a previously described protocol,
15
and were provided by
Prof. Richard Manasseh at Swinburne University of Technology.
hAM-MSC culture medium was composed of mesenchymal stem
cell growth medium (MSCGMt, Lonza, AU) supplemented with
MSCGMtSingleQuotst(Lonza). Murine embryonic stem cells
(cardiac-specific NKX2.5-eGFP transgenic mESCs) were generated
according to a previous study,
16
and were provided by Dr Huseyin
Sumer at Swinburne University of Technology. mESC cell culture
medium was composed of 90% (v/v) Dulbecco’s modified Eagle’s
medium (DMEM, 10569077, Thermo Fisher) supplemented with
10% ES screened fetal bovine serum (FBS; Thermo Fisher),
1mlmL
1
leukemia inhibitory factor (LIF; PMC9484, Sigma-
Aldrich, Castle Hill, NSW, AU), 1MEM NEAA (11140050,
Thermo Fisher), 1 mlmL
1
2-mercaptoethanol (21985023, Thermo
Fisher), and 1antibiotic antimycotic solution (A5955, Sigma-
Aldrich). 1st anti-vinculin (V19131), FITC-2nd-antibody (F2012),
and TRITC-2nd-antibody (T5393) were purchased from Sigma-
Aldrich. 1st anti-OCN (AB10911) was purchased from Merck, AU.
For the formation and culture of embryoid bodies (EBs) from
mESCs, the cell culture medium composition was identical except
that LIF was excluded.
Fabrication of surface nanotopographies
Nanogrooved substrates were fabricated using a combination
of electron beam lithography (EBL) and soft lithography as
described in a previous study.
17
In brief, grooved substrates
with 800 nm width/400 nm depth or 800 nm width/40 nm depth
were used and named ‘‘deep nanogrooves’’ (DG400) and ‘‘shallow
nanogrooves’’ (SG40), respectively. Nanopillar substrates
were fabricated using colloidal lithography (CL) and then soft
lithography. An ordered colloidal mask was fabricated using
self-assembly of 820 nm PS colloids into close packed mono-
layers on Si wafers according to a previous protocol.
18
Random
colloidal masks were produced by spin coating of different
concentrations of colloidal solutions on Si wafers (1 :4, 1 : 8 and
1 : 12 in water/ethanol) at 2500 rpm for 1 min. Reactive ion
Paper Journal of Materials Chemistry B
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etching (RIE; Oxford Instruments PLASMALAB100 ICP380,
MCN, Melbourne, Australia) was then applied on four different
colloidal masks with a gas flow of Cl
2
(50 sccm), SF
6
(3 sccm)
and O
2
(3 sccm) for 60 s, resulting in four nanopillar patterns:
ordered nanopillars (OP65), high density nanopillars (HP50),
medium density nanopillars (MP20), and low density nano-
pillars (LP10) with a 500 nm pillar height characterized using
contact model profilometry (Ambios XP 200, MCN, Melbourne,
Australia). Any residual PS colloids were removed from the
surfaces by using acetone and then ethanol under ultrasonic
agitation.
Soft lithography was then used to replicate the master patterns
according to a previous study.
19
In brief, master silicon patterns
were coated with octadecyltrichlorosilane (OTS)/toluene for
15 min, and then rinsed with toluene, dried in air, and rinsed
in 100% ethanol. The OTS-coated wafers were heated for
2 minutes at 120 1C. An approx. 5 mm layer of PDMS solution
(1-to-10: curing-to-base agent) was poured over the master
patterns, degassed in a vacuum desiccator for 1 h, and then
cured at 60 1C for 2 h. The PDMS stamps were removed from
the master patterns for making nanopatterns. Nanopatterns
were fabricated on either poly(ethylene teraphthalate) (PET) or
glass slides with a size of approx. 1.5 cm 1.5 cm. For glass
substrates, a thin layer of PS was first spin coated, and then
heated at 120 1C for 5 min. A drop of 5% PS/toluene was
imprinted by PDMS stamps on either PET or glass slides and
dried overnight in a hood. Surface nanotopographies were
examined using field emission scanning electron microscopy
(FE-SEM, ZEISS SUPRA 40 VP, Carl Zeiss, Germany) at 5 keV and
atomic force microscopy (Dimension Icon, Bruker, Germany) at
0.5 Hz (tapping model). The density and dimensions of nano-
pillars and nanogrooves were determined using SEM and AFM
images (n= 5).
Stem cell culture and analysis
Prior to cell culture, all substrates were treated with air
plasma (air flow rate of 2.0 10
2
mbar and 50 W power)
for 2 min, sterilised in 70% ethanol, and washed twice in
Fig. 1 (A) Fabrication of nanopillars and nanogrooves. Colloidal lithography and spin coating based approaches were used for nanopillars, while electron
beam lithography was used for nanogrooves. (B) Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of different
nanotopographies. AFM images were scanned over a 5 5mm area.
Journal of Materials Chemistry B Paper
7930 |J. Mater. Chem. B, 2017, 5, 7927--7938 This journal is ©The Royal Society of Chemistry 201 7
phosphate-buffered saline (PBS). All substrates were placed in
12-well plates for cell culture. Each surface was seeded with
hAM-MSCs at a density of 1 10
4
cells per cm
2
in 1 mL media.
Embryoid bodies of mESCs (mEBs) were formed by culturing
mESCsfor3daysinlow-attachment well plates (Corning, USA)
without LIF. Prior to cell culture, substrates were coated with
0.1% gelatin for 1 h at room temperature, and approx. eight
EBs were seeded on each surface.
Cell morphology was examined after 1 and 3 days using
fluorescence microscopy (Eclipse Ti-E microscope, Nikon, Japan),
confocal laser scanning microscopy (FV3000, FLUOVIEW,
Olympus, Japan) and FE-SEM. For fluorescence staining,
samples were fixed with 4% paraformaldehyde for 30 minutes,
permeabilised by 0.2% Triton X-100/PBS, and then washed with
PBS. After 24 h, focal adhesions of cells were stained using 1st
anti-vinculin (1 : 100 in PBS) and FITC-2nd antibody (1 : 200 in
PBS) for 1 h. After 1 week, immunostaining of osteocalcin
(OCN) of mEBs was done using 1st anti-OCN (1 : 100 in PBS)
and TRITC-2nd antibody (1 : 200 in PBS) for 1 h. Cell nuclei
and F-actin were stained using 3 mM DAPI and 0.165 mM
phalloidin–TRITC, respectively. Samples were imaged at excitation/
emission wavelengths of 346/442 nm for DAPI, 405/519 nm for
FITC, and 555/565 nm for TRITC.
For SEM imaging, samples were fixed with 4% paraform-
aldehyde and dehydrated by immersion in graded ethanol
at concentrations of 50%, 70%, 80%, 90%, 95%, and absolute
ethanol. Samples were dried and sputter coated with a 5 nm
gold layer for SEM imaging.
The alignment of cells was analysed using ImageJ according
to a previous study.
20
In brief, the cell or colony outline was
circled. The major and minor axes of cells or colonies were
determined automatically, so that an angle of major axis between
0 and 180 degrees was obtained. All angles were converted
between 0 and 45 degrees, where 0 degrees indicates prefect
alignment, while 45 degrees indicates a random distribution of
cells or colonies.
To determine proliferation, cell numbers or colony sizes were
determined after 1 and 3 days using fluorescence microscopy.
Growth rate is the number of cells at day 3 divided by that at day 1.
For hAM-MSCs, the cell number was counted using DAPI-stained
images. For mEBs, colony areas were measured using phalloidin-
TRITC-stained images. Five images were taken randomly from
each sample at a magnification of 10(n=5).
Quantitative real-time polymerase chain reaction (qPCR)
Gene expression of the different lineages was analysed at
specific time points. hAM-MSCs were analysed on various
nanotopographies in normal culture media after 14 days, while
mEBs were analysed on various surfaces in normal culture
media without LIF after 7 days. In normal culture medium,
surface-induced differentiation of MSCs was conducted for a
longer time (i.e. 2 weeks), while that of EBs was conducted for a
shorter time (i.e. 1 week) because spontaneous differentiation
of EBs was expected. At each time point, mRNA was extracted
using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany).
qPCR was conducted using an iTaqtUniversal One-Step
RT-qPCR kit (Bio-Rad Laboratories, California, USA) and a
CFX96 TouchtReal-Time PCR Detection System thermocycler
(Bio-Rad Laboratories, California, USA). The primers of selected
genes are listed in the ESI†(Table S1). Three lineage genes of
hAM-MSCs were analysed and the various genes selected
included aggrecan (ACAN), type II collagen (COL2) and SOX9
for chondrocytes, type I collagen (COL1), alkaline phosphatase
(ALP) and RUNX2 for osteoblasts; and PPARgfor adipocytes. Five
lineage genes of mEBs were analysed and the various genes
selected included COL1 and OCN for osteoblasts, COL2 for
chondrocytes, GATA6,GATA4,NKX2.5 and myosin heavy chain
(MHC) for cardiomyocytes, myogenin (MYOG) for skeletal
myoblasts, albumin (ALB) and alpha-fetoprotein (AFP) for
hepatocytes, NESTIN for neural cells, platelet-derived growth
factor receptor-A (PDGFR) for mesodermal tissues, and Kruppel-
like factor 4 (KLF4) and OCT4 for pluripotency. GAPDH was used
as the housekeeping gene. Gene expression on nanopatterns
was normalised to tissue culture polystyrene (TCPS; value = 1)
and compared to the flat control. Experiments were repeated
at least three times. Each sample was triplicated with three
technical repeats (n= 3). The DDCt method was used to
calculate and compare relative mRNA levels according to a
previous study.
21
Statistical analysis
All experiments were repeated at least three times. Data
are expressed as mean standard deviation (STDEV) unless
otherwise specified. Statistical analysis was performed for each
experimental group by one-way ANOVA and Student–Newman–
Keuls multiple comparison tests using GraphPad InStat V.3
(GraphPad Software, Inc.).
Results
Nanopatterns
The surface nanotopographies were fabricated using a combi-
nation of approaches and characterized using SEM and AFM
imaging (Fig. 1). The nanogrooves were designed to have a
width of 800 nm (equal groove-to-ridge ratio) and a depth of
either 40 nm or 400 nm. However, the obtained nanopattern
sizes were slightly different due to the fabrication process.
Nevertheless, the difference in groove depth was still clearly
visible. Two types of nanogrooves were created: shallow grooves
(SG40) and deep grooves (DG400).
Similarly, the size of nanopillars was slightly different after
RIE and soft lithography. Colloidal masks were made from
820 nm PS particles. After fabrication, the diameter of nano-
pillars was approx. 500 nm and the height of nanopillars was
approx. 450–750 nm depending on the particle density (Fig. 1
and Table 1). A higher particle density appears to protect the Si
substrate during etching resulting in a shorter pillar height.
The density of nanopillars decreased from 65% to 10% (Fig. 1
and Table 1). Thus, four types of nanopillars were created:
ordered nanopillars (OP65), high density nanopillars (HP50), medium
density nanopillars (MP20), and low density nanopillars (LP10).
Paper Journal of Materials Chemistry B
This journal is ©The Royal Society of Chemistry 201 7 J. Mater. Chem. B, 2017, 5, 7927--7938 | 7931
The surface roughness of OP65 was the lowest (approx. 73 nm in
Rq), while that of MP20 was the highest (approx. 273 nm in Rq;
Table 1).
Two stem cell types, hAM-MSCs and mEBs, were studied
on the nanopatterns. The cell morphology and cell–surface
interactions were analysed after 1 and 3 days (Fig. 2 and
Fig. S1, ESI†). Fluorescence staining showed that cell alignment
of hAM-MSCs was induced only by the deep nanogrooves
(i.e. DG400), and not others (Fig. 2A). The density of nanopillars
on the surfaces also had an effect on cell morphology, where
cell spreading was inhibited on lower density patterns, resulting
in more elongated cells (arrows in HP50 and LP10 in Fig. 2A).
Similarly, mEBs elongated and aligned with the deep nanogrooves
(DG400,Fig.2B),andthemEBshadmore3D-likemorphologies
on the nanopillars compared to the flat control (arrows in Fig. 2B).
That is, they extended from the tops of the pillars to the bare
substrate regions.
Morphological analysis of the two cell types showed that
the DG400 substrate caused alignment of hAM-MSCs (DG400:
angle B171, Fig. 2C) with a more elongated morphology
(DG400: elongation B5, Fig. 2D), which was significantly
different from the flat and other surfaces (po0.001 vs. flat,
Fig. 2C and D). mESC colonies showed cell alignment (DG400:
angle B191, Fig. 2D) and elongation (DG400: elongation
B2, Fig. 2D) along the DG400 substrate (Fig. 2D). With both
hAM-MSCs and mEBs, there was no significant difference in
cell alignment or elongation associated with the nanopillar
patterns (except for hAM-MSCs on LP10) compared to the flat
control.
The growth of hAM-MSCs, not mEBs, was inhibited on the
DG400 substrate compared to the flat control (Fig. 2E and
Fig. S3, ESI†). On nanopillars, hAM-MSC growth was slightly
affected by the nanotopographies without significance. The
mEB size was however inhibited by OP65 and LP10 nanopillars,
but increased on the HP50 nanopillars (Fig. 2E and Fig. S3, ESI†).
The details of cell–surface interactions were imaged on day 3
(Fig. 3). The filopodia or extensions of the hAM-MSCs and
mEBs ‘‘touched’’ the floor of the DG40 surfaces because the
height of the grooves was shallow. The majority of filopodia
and cell bodies of the two cell types attached to the top of ridges
of the DG400 surfaces due to the high aspect ratio and narrow
ridge spacing (Fig. 3A). This phenomenon resulted in the
alignment and elongation of the two cell types on the DG400,
not DG40, surfaces. Interestingly, hAM-MSCs and mEBs cultured
on the OP65 and HP50 surfaces attached only to the uppermost
surfaces of the nanopillars (solid arrows, Fig. 3B). On the HP
surfaces, the main body of the cells remained on the top of
nanopillars and the filopodia seemed to be guided by the pillar
arrangement (HP50, Fig. 3A and B). On the other hand, on the
MP20 and LP10 surfaces, both cell types interacted with both
the nanopillars and substrate floor because the pillar density
was too low to allow focal adhesions to form on the nanopillars
alone (empty arrows, Fig. 3A and B). Nanopillars can be seen
through the cells in the thinner parts of the cell body indicating
that nanopillars were accommodated by attached cells and
possibly semi-penetrated the cells.
Vinculin staining showed that focal adhesion was affected
by the underlying nanotopographies (Fig. 4). Both hAM-MSCs
and mESCs had clear aligned F-actin and vinculin distributions
on the DG400 surfaces. hAM-MSCs had a similar vinculin
distribution on OP65 and the flat control (Fig. 4A). mESCs
had a clear vinculin distribution at the periphery of cells, while
the distribution on the flat control was not obvious (Fig. 4B).
Gene expression of stem cells on nanotopographies
Nanotopography-induced differentiation was characterized
using qPCR. Various genes from different lineages were analysed.
Changes greater than 2-fold compared to the flat control were
considered significant and highlighted. For hAM-MSCs, ALP and
PPARgexpression was slightly enhanced on nanopatterns, while
other genes either had no change (i.e. COL1,RUNX2,andSOX9)or
decreased (i.e. ACAN) without significant changes compared to the
flat control (Fig. 5A). The COL2 gene was not expressed (i.e. most
of Ct values were 438; Fig. S2, ESI†).
For mEBs, the expression of OCN,MYOG and ALB increased
on the nanogrooves and the fold was dependent on the groove
depth (Fig. 5B). On nanopillars, the expression of COL1,OCN,
COL2,MYOG, and ALB increased and the fold was dependent
on the pillar density (Fig. 5B). Interestingly, the expression of
GATA4 decreased on DG400, HP50, and LP10 significantly
(Fig. 5B). Two pluripotent genes, i.e. OCT4 and KLF4, and
the hepatocyte marker AFP were downregulated on nanotopo-
graphies compared to TCPS, but no significant difference
occurred compared to the flat control. Two neural genes, i.e.
PDGFR and NESTIN, were upregulated on nanogrooves and the
OP65 nanopillar surface. Genes including MHC,GATA6 and
NKX2.5 for mESCs were not expressed (i.e. Ct 438) as shown in
Fig. S2 (ESI†).
Heterogeneity of stem cells on nanotopographies
Multiple lineage genes were up-regulated on the nanotopo-
graphies especially on OP65 (Fig. 6A). Co-expression of OCN
and NKX2.5 proteins within one colony was found on OP65 and
the flat control, which was consistent with the gene expression
Table 1 Characterisation of nanotopographies. Value = mean STDEV (n=5)
OP65 HP50 MP20 LP10 SG40 DG400
Pillar density (% area) 65.5 1.5 50.2 1.7 21.8 0.6 10.3 0.5 — —
Pillar diameter (nm) 542.1 17.9 482.6 32.8 447.7 54.8 478.6 34.3 — —
Height (nm) 445.6 23.0 463.7 21.6 761.3 31.7 732.9 12.8 48.5 0.1 389.8 0.6
Roughness (nm) 72.9 208 273 217 22.7 181
Groove width (nm) — — — — 802.7 66.9 924.2 23.6
Ridge width (nm) — — — — 649.9 38.7 516.7 23.3
Journal of Materials Chemistry B Paper
7932 |J. Mater. Chem. B, 2017, 5, 7927--7938 This journal is ©The Royal Society of Chemistry 20 17
Fig. 2 Cell morphology of (A) hAM-MSCs and (B) mEBs after 3 days. The arrows in G400 indicate the direction of the nanogrooves. Full data are shown
in the ESI†(Fig. S1). BF: bright field; fluore: DAPI and phalloidin–TRITC staining. Nanotopography-induced morphological changes of (C) hAM-MSCs and
(D) mEBs after 3 days. All measurements were taken from fluorescence microscope images at day 3 of cell culture. The alignment (degrees) and elongation
(length-to-width) of single hAM-MSCs and mEBs were analysed. Value = mean SEM (n=74–238).*,po0.05; **, po0.01; ***, po0.001 vs. flat.
(E) Growth rate of hAM-MSCs and colony size change of mEBs after 3 days. For hAM-MSCs, the cell number at day 3 was compared to that at day 1.
For mEBs, the colony size at day 3 was compared to that at day 1. Value = mean SEM (n=10–12).*,po0.05; **, po0.01; ***, po0.001 vs. flat.
Paper Journal of Materials Chemistry B
This journal is ©The Royal Society of Chemistry 201 7 J. Mater. Chem. B, 2017, 5, 7927--7938 | 7933
data (Fig. 6B). A small portion of mEB-derived beating cells
(GFP positive cells, Video S1B and D, ESI†) were found within
one colony on the surfaces (e.g. OP65 and DG400 Fig. 6C and
Video S1A–D, ESI†) indicating that the cell population was
diverse. Specific nanotopographies, for example OP65, can thus
induce stem cell differentiation compared to the flat control,
but may not be strong enough to guide mESCs into one specific
lineage.
Discussion
Surface nanotopography-induced morphological changes and
differentiation of stem cells have previously been studied.
1
However, the majority of the work has focused on one type of
surface structure, one type of stem cell and/or one downstream
differentiated cell type. Various cell types, different cell culture
protocols, and the diverse types of surfaces/materials used lead
to difficulties in drawing clear conclusions about the specific
role played by nanotopography. For example, Pan et al. reported
that 350 nm-width nanogrooves enhanced neurogenesis
(i.e. increase of neuronal markers) for hiPSCs (i.e. human
induced pluripotent stem cells) compared to microgrooves and
flat controls.
22
However, analysis of other genes was missing,
although the cell morphology was neuron-like. Another study
Fig. 3 SEM images of cell–nanotopography interactions. The images
show the interaction of (A) hAM-MSCs and (B) mEBs with the nanotopo-
graphies. Double-headed arrow indicates groove direction. Arrowheads
indicate the detailed location between the cells (i.e. filopodia and/or cell
extensions) and the nanoscale features.
Fig. 4 Confocal images of stem cells on nanotopographies. (A) AM-MSCs
and (B) mESCs were cultured on DG400, OP65, and the flat control for
24 h. Cell nucleus, F-actin, and vinculin were stained using DAPI (blue),
TRITC (red), and FITC (green). White arrows indicate the clear F-actin and
vinculin overlapping.
Journal of Materials Chemistry B Paper
7934 |J. Mater. Chem. B, 2017, 5, 7927--7938 This journal is ©The Royal Society of Chemistry 201 7
showed that 250 nm-diameter nanopillars enhanced chondro-
genic differentiation of hMSCs. Again, the study did not analyse
other mesoderm genes such as adipocytes.
23
To address this, in
this systematic study we investigated the effects of two types of
surface nanostructures made by a combination of approaches
and examined expression of genes associated with various
lineages of the two stem cell types. The aim was to understand
whether a specific nanotopography can stimulate stem cells into
one specific cell type in early stages of growth without chemical
induction.
Colloidal lithography is a cost-effective approach which uses
particle monolayers as masks to generate nanostructures after
etching the substrate.
18,24
Surprisingly, very few studies have
used colloidal lithography to fabricate nanostructures for cell
culture. One possible reason is that the ability of controlling
colloidal self-assembly for the fabrication of either close packed
or randomly distributed particle masks has been lacking.
In this study, we used an exquisite approach to assemble
submicron polystyrene particles into a monolayer at the air–
water interface, and then deposited the monolayer on a silicon
wafer. Other masks with decreasing particle densities were
prepared using spin coating. Then, reactive ion etching and
soft lithography were used for the production of surface nano-
topographies. To the best of our knowledge, this is the first
time that various nanopillar densities generated in a large area
(2 2cm
2
) for stem cell culture have been made. This
combination of approaches and the types of nanostructures
produced are very useful in biomedical engineering as well as
other fields such as photonics and solar cells.
25
We previously demonstrated that cell alignment on nano-
grooves was mainly depth dependent (i.e. C2C12 myoblasts,
19,26,27
rat BM-MSCs,
17
mouse cardiomyocytes,
8
and porcine anterior
cruciate ligament cells
20
). In this study, the same phenomenon
was observed again on hAM-MSCs and mEBs. The morphology of
Fig. 5 Gene expression of different lineage markers of (A) hAM-MSCs and (B) mEBs on the different nanotopographies. hAM-MSCs were cultured on
nanopatterns for 14 days, while mEBs were cultured for 7 days in normal culture medium. All gene expression was normalized to TCPS (value = 1). The
gene expression of each gene on different surfaces was compared to that on the flat control, and the fold changes are indicated using red boxes. A gene
expression greater than 2-fold vs. flat was considered significant. *, 42-fold; **, 43-fold; ***, 44-fold. Value = mean STDEV (n= 3). Genes from bone
(COL1,ALP,RUNX2RUNX2), cartilage (SOX9,ACAN), and fat (PPARg) were selected for hAM-MSCs. Genes from bone (COL1,OCN), cartilage (COL2),
skeletal muscle (MYOG), heart (GATA4), nerve (NESTIN), mesodermal (PDGFR), liver (ALB,AFP) and pluripotency (KLF4,OCT4) were selected for mEBs.
Paper Journal of Materials Chemistry B
This journal is ©The Royal Society of Chemistry 201 7 J. Mater. Chem. B, 2017, 5, 7927--7938 | 7935
hAM-MSCs and spheroidal mEBs was elongated on DG400
(800 nm width/400 nm depth) indicating that the submicron
nanogrooves of this specific size are an effective modulator of
contact guidance. On the other hand, nanopillars do not provide
any specific guidance on cell direction. hAM-MSCs on nanopillars
have smaller cell sizes, especially on the HP50 and LP10 surfaces,
while mEBs on nanopillars seem to have more 3D structure,
especially on OP65 and LP10, indicating that nanopillars inhibit
the spreading of both types of stem cells. This phenomenon was
reported previously on different materials with nanopillar
28
and
nanoprotrusion
18,24
structures.
This inhibition changes subsequent cell proliferation, espe-
cially for mEBs. The elongation and alignment of hAM-MSCs
on DG400 decreases the growth rate after 3 days. On the
nanopillars, the growth rate of hAM-MSCs did not significantly
change compared to the flat control. On the other hand, the
growth rate, or more specifically the colony size, of mEBs,
increased on DG400 and HP50, but decreased on the OP65
and LP10 surfaces. There is also a correlation between cell
morphology and growth rate. On the DG400 and HP50 surfaces,
colonies appear flat, while on the OP65 and LP10 surfaces they
take on a more 3D-like structure. Decreasing the pillar density
from HP to LP seems to inhibit cell growth. However, the cell
growth rate on the OP65 surface is low and it seems that this is
another factor that interferes with cell growth such as the pillar
size. The pillar size between surfaces is different and the
symmetry of pillars could be another factor causing growth
inhibition.
10,29
Since we do not have a nanopattern having the
same pillar density but different ordering (due to fabrication
difficulties), the effect of symmetry needs to be studied in the
future if these types of patterns can be made. Nevertheless,
we prove here that nanotopography can modulate stem cell
morphology, which in turn changes the cell proliferation in a
cell type- and pattern-dependent manner.
Our gene expression results are interesting and show that
these nanotopographies do not improve hAM-MSC differentiation.
Although ALP,RUNX2,andPPARgare slightly higher and SOX9 and
ACAN are slightly lower on some patterns compared to the flat
control, none of them exhibit more than 2-fold changes compared
to the flat control. Previous studies reported that nanogrooves
suppress osteogenesis of osteoprogenitors.
30
Similarly, specific
pillar structures enhance osteogenesis of human mesenchymal
dental pulp-derived stem cells.
31
Compared to these previous
studies, we did not find the same outcome in this study. This is
possible due to the different cell types and pattern sizes used in
this study compared to previous ones. Overall, the nanogrooves
and nanopillars used in this study did not improve the differentia-
tion of hAM-MSCs toward any of the cell types of the mesenchymal
lineage.
On the other hand, mEBs exhibited higher sensitivity to
the different nanotopographies. Nanogrooves enhanced OCN,
NESTIN,PDGFR,MYOG and ALB expression, which are at least
2-fold greater compared to the flat control. Nanopillars, especially
OP65, enhanced COL1,OCN,MYOG,COL2,NESTIN,PDGFR,and
ALB expression, but inhibited GATA4 expression, compared to flat
controls in a surface dependent manner. Interestingly, three
cardiac genes, i.e. NKX2.5,GATA6,andMHC have no expression
on the nanopatterns, indicating that the selected nanotopo-
graphies were unable to stimulate cardiac differentiation of
mESCs. It is important to note that the OP65 surface can
stimulate mEBs to express multiple lineage markers without
biochemical stimulation. This outcome implies that biophysical
stimulation alone may not be strong enough for driving stem cell
fate specifically although the fate of stem cells was modulated.
Pluripotent stem cells such as mESCs routinely display hetero-
geneous gene expression in in vitro culture.
32
Aspects of
this heterogeneity are linked to differences in the propensity
of individual cells to either self-renew or commit towards
differentiation. On our nanotopographies, especially the OP65
and SG40 surfaces, the expressions of various lineage genes
were upregulated. This indicates that these two nanotopo-
graphies may induce stem cell differentiation but may not be
Fig. 6 Correlation of gene and protein expressions of mEBs. (A) Gene expression on OP65 normalized to TCPS and the flat control. (B) Immunostaining
of OCN (red) on OP65 and the flat control. Cell nuclei were stained using DAPI (blue) and NKX2.5 positive cells expressed GFP (green). White arrows
indicate strong GFP positive cells. (C) Video images show that a small portion of cells were cardiac-like beating cells, while other cells were non-beating
cells. Double-headed arrow indicates groove direction. Bright-field and GFP videos are available in the ESI†(Video S1A–D).
Journal of Materials Chemistry B Paper
7936 |J. Mater. Chem. B, 2017, 5, 7927--7938 This journal is ©The Royal Society of Chemistry 2017
able to direct the differentiation into a specific lineage. It is
possible that either each mEB contains a heterogeneous cell
population or diverse mEBs are formed on the surface. To
confirm this, further studies using single colony or single cell
analysis such as fluorescence activated cell sorting (FACS) and
other emerging advanced technologies
33
are needed.
Previous studies have shown that extracellular matrix (ECM)
proteins can guide human bone marrow MSCs into different
lineages.
34
For example, collagen participates in the formation of
fibrils guiding the differentiation of cardiomyocytes, adipocytes,
and osteoblasts, while fibronectin does not significantly affect
stem cell differentiation.
34
Cells or stem cells can sense
both biochemical and biophysical signals surrounding them.
In the same way collagen can form fibrils to induce stem cell
differentiation into different cell types, nanogrooves and nano-
pillars are also present in the microenvironment of various
tissues. Thus, it is possible that one type of nanopattern
can induce multiple lineage differentiation of stem cells even
without the need for additional growth factors.
We propose a mechanism by which nanotopography can
affect stem cell function most likely indirectly through the
changes in the cytoskeleton and nucleus shape. In general,
biophysical cues are not as efficient as biochemical cues.
Biochemical cues such as small molecules or growth factors
can either bind to cell surface receptors or permeabilize the
cellular membrane to directly activate intracellular signalling
pathways. On the other hand, biophysical cues normally pre- or
post-exert stress and/or stretching on cells, which leads to
changes in focal adhesions, the cytoskeleton (i.e. cell morphology)
and/or the shape of the cell nucleus. These changes in turn can
affect intracellular molecular assembly and then cell function.
In this study, we found that the population of differentiated mESCs
was heterogeneous even with nanotopographical modulation. This
could be due to the fact that, in EBs, cell–cell contact has a
stronger effect on cell function than cell–surface interactions,
resulting in only a small portion of cells being influenced by
surface nanotopographies. Another possibility is that the dimen-
sions of selected nanostructures were not optimized. Since the
substrate solely affects the cell–substrate interface to indirectly
alter cell function, this effect may be minimized when cells do
not exist as a monolayer culture. Nevertheless, our results show
that the OP65 nanotopography stimulates early differentiation of
mESCs especially into the mesodermal lineage (i.e. bone and
muscle) compared to the flat control. Future studies that assess
the long-term effect of nanotopographies on stem cells would
allow us to further understand the effect of early stimulation of
gene and protein expression on the function of stem cells.
Conclusion
Substrate nanotopographies have a tremendous influence on the
morphology, migration, and proliferation of stem cells. However,
downstream events such as nanotopography-induced differen-
tiation remain unclear. Although some specific nanotopographies
such as nanogrooves have been suggested to be able to trigger
stem cell differentiation into, for example, neurons, it is unclear
to what extent nanotopographies contribute and whether or not
the cell population is homogenous. In this systematic study, we
demonstrated that nanogrooves and nanopillars can affect the
growth and proliferation of mESCs and hAM-MSCs with different
levels of cellular response occurring depending on the specific
nanogrooves and nanopillars. In terms of the cell populations,
nanotopography induced or inhibited mEB differentiation was
found to be dependent on the structure and size of the features.
We observed an increase of various lineage genes and markers
on nanotopographies, especially on OP65, indicating that the
cell population is heterogeneous. This study uses accessible
approaches for the fabrication of commonly nanostructures
found in the native extracellular matrix, and demonstrates that
hAM-MSCs are not sensitive to these nanotopographies, while
mEBs can be affected by nanotopographies but most likely
other factors will be needed such as biochemical factors to
precisely control the stem cell fate and homogeneity.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
This work was supported by the Biomedical Research Victoria
for the Undergraduate Research Opportunities Program (UROP).
The Australian Research Council (ARC) is acknowledged for
providing the Discovery Early Career Researcher Award (DECRA:
DE150101755) to P.-Y. Wang. Taipei Medical University and MOST,
Taiwan are also acknowledged for providing funding support
to P.-Y. Wang (TMU105-AE1-B13 and 105-2314-B-038-088-MY2).
R. Wong was supported by the Medical Advances Without Animal
Fellowship and the Louisa Jean de Bretteville Bequest. We espe-
cially thank Prof. Wei-Bor Tsai for providing the nanogrooved
patterns. This work was performed at both the Biointerface
Engineering Hub at Swinburne University and MCN as part of
the Victorian Node of the Australian National Fabrication Facility,
a company established under the National Collaborative Research
Infrastructure Strategy to provide nano- and microfabrication
facilities to Australia’s researchers. The Centre for Eye Research
Australia receives operational infrastructure support from the
Victorian Government.
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