Acta Biomaterialia 87 (2019) 256–264

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Patterning of human epidermal stem cells on undulating elastomer

substrates reflects differences in cell stiffness
Seyedeh Atefeh Mobasseri a, Sebastiaan Zijl a, Vasiliki Salameti a, Gernot Walko a,b, Andrew Stannard c,
Sergi Garcia-Manyes c, Fiona M. Watt a,⇑
a
Centre for Stem Cells and Regenerative Medicine, King’s College London, 28th Floor, Tower Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, United Kingdom
b
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
c
Department of Physics and Randall Institute of Cell and Molecular Biophysics, King’s College London, WC2R 2LS, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: In human skin the junction between epidermis and dermis undulates, the width and depth of the undu-
Received 11 September 2018 lations varying with age and disease. When primary human epidermal keratinocytes are seeded on
Received in revised form 15 January 2019 collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the epidermal-dermal
Accepted 29 January 2019
interface, the stem cells become patterned by 24 h, resembling their organisation in living skin. We found
Available online 30 January 2019
that cell density and nuclear height were higher at the base than the tips of the PDMS features. Cells on
the tips not only expressed higher levels of the stem cell marker b1 integrin but also had elevated E-
Keywords:
cadherin, Desmoglein 3 and F-actin than cells at the base. In contrast, levels of the transcriptional cofactor
Epidermal stem cells
Topographical substrates
MAL were higher at the base. AFM measurements established that the Young’s modulus of cells on the
AFM tips was lower than on the base or cells on flat substrates. The differences in cell stiffness were dependent
Cell-cell junctions on Rho kinase activity and intercellular adhesion. On flat substrates the Young’s modulus of calcium-
dependent intercellular junctions was higher than that of the cell body, again dependent on Rho kinase.
Cell patterning was influenced by the angle of the slope on undulating substrates. Our observations are
consistent with the concept that epidermal stem cell patterning is dependent on mechanical forces
exerted at intercellular junctions in response to undulations in the epidermal-dermal interface.

Statement of significance

In human skin the epidermal-dermal junction undulates and epidermal stem cells are patterned accord-
ing to their position. We previously created collagen-coated polydimethylsiloxane (PDMS) elastomer
substrates that mimic the undulations and provide sufficient topographical information for stem cells
to cluster on the tips. Here we show that the stiffness of cells on the tips is lower than cells on the base.
The differences in cell stiffness depend on Rho kinase activity and intercellular adhesion. We propose that
epidermal stem cell patterning is determined by mechanical forces exerted at intercellular junctions in
response to the slope of the undulations.
Ó 2019 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

1. Introduction attached to an underlying basement membrane [2] and cells

Mammalian skin is built from two histologically and physiolog-
ically distinct tissue compartments: an epithelial layer called the
epidermis and an underlying connective tissue layer called the der-
mis. In humans, the interface between the epidermis and dermis is
not flat but undulates [1]. The interfollicular epidermis (IFE) com-
prises multiple cell layers, with the stem cell compartment
⇑ Corresponding author.
E-mail address: fiona.watt@kcl.ac.uk (F.M. Watt).
undergo terminal differentiation as they move through the supra-
basal layers [3]. Extrinsic signals such as interactions with neigh-
boring cells, extracellular matrix (ECM) adhesion, tissue stiffness
and secreted factors are known to regulate the behavior of stem
cells [2]. Physical forces such as cell shape, shear forces and sub-
strate stiffness all affect the balance between stem cell prolifera-
tion and differentiation [4]. Internal and external mechanical
loading affects the biology of both epidermis and dermis and is
mediated through mechanochemical transduction processes that
involve both cell-cell and cell-ECM adhesion [5].

https://doi.org/10.1016/j.actbio.2019.01.063
1742-7061/Ó 2019 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264
The importance of physical parameters has been explored by
seeding individual epidermal cells (keratinocytes) on ECM-coated
micro-patterned islands. Restricting keratinocyte spreading on
20 lm diameter circular islands triggers terminal differentiation
whereas cells on 50 lm diameter islands remain spread and do
not differentiate [6,7]. On larger islands, that can accommodate
approximately 10 cells, keratinocytes form a stratified ‘micro-
epidermis’ with stem cells in the basal layer and differentiated cells
(which express markers such as involucrin and transglutaminase
1) in the suprabasal layer. Actin polymerisation, desmosomes
and adherens junctions are key mediators of micro-epidermis
assembly [7].
Several of the signal transduction pathways that regulate ker-
atinocyte differentiation in response to physical cues have been
identified [8]. One of the key mechanotransduction mechanisms
is YAP/TAZ signalling. The subcellular localisation of YAP and TAZ
is controlled by surface topography, ECM stiffness and cell shape.
YAP and TAZ translocate between nucleus and cytoplasm in
response to mechanical cues [9]. Another key pathway is mediated
by the SRF (serum-response factor) transcription factor, which is
regulated by RhoA, actin polymerisation and the transcriptional
cofactor MRTF-A (MAL). Actin polymerisation controls transloca-
tion of MAL into the nucleus in response to cell-ECM and cell-cell
adhesion [10]. MAL and SRF mediate shape induced terminal dif-
ferentiation of individual keratinocytes [11], while YAP/TAZ sig-
nalling in keratinocytes is regulated by intercellular adhesion [12].
In human epidermis the cells in the basal layer are patterned,
with stem cells expressing highest levels of b1 integrin clustered
where the basal layer comes closest to the skin surface. We are able
to mimic the undulations by creating collagen-coated PDMS sub-
strates and have shown that the topography that most closely
resembles healthy human skin induces stem cell clustering, with
nuclear YAP, on the tips [13,12]. Patterning of stem cells and
nuclear YAP can be disrupted by treating cells with a Rho kinase
inhibitor (Y-27632), a Non-muscle Myosin 2 inhibitor (Blebbis-
tatin) or by preventing the assembly of desmosomes and adherens
junctions by reducing the extracellular calcium ion concentration
[12].
Although ECM adhesion is known to regulate both the spatial
location and the differentiation of keratinocytes in vivo and
in vitro [1,2], it is hard to envisage how a uniform collagen coating
could determine the patterning of stem cells on undulating
topographies. An alternative explanation, and one that is attractive
given the response of the YAP pathway to the topographies [12],
would be that patterning is controlled by the forces exerted by
neighbouring cells, which could, in turn, depend on the slope of
the undulations. In the present study, we therefore used the undu-
lating collagen-coated PDMS substrates to explore the potential
physical and cell biological mechanisms by which stem cells clus-
ter on the tips of features. Our study reveals marked differences in
cell stiffness in different locations and suggests that forces medi-
ated by cell-cell adhesion in response to the slopes of the undula-
tions determine stem cell patterning.
2. Materials and methods
2.1. Substrate preparation
Patterned and flat polydimethylsiloxane (PDMS) substrates
were prepared as previously described [13,14]. Briefly, the molding
precursor, made of polymerised hydroxyethyl methacrylate
(PHEMA), was cast on a methacrylated glass slide. By using a pho-
tomask and UV light, the PHEMA films were crosslinked and the
negative pattern was created. The PDMS precursor was then mixed
257
with the curing agent (10:1 ratio by weight, Sylgard 184s Silicone
Elastomer Kit, Dow Corning) and cast on the mold. The PDMS was
cured in an oven at 80 °C and then peeled off from the mold. Prior
to cell culture, the patterned PDMS substrates were sterilised using
70% ethanol and washed with PBS, followed by collagen coating
(100 lg mL 1 rat tail type 1 collagen, from Becton Dickinson) by
adsorption for 2 h at 37 °C.
2.2. Keratinocyte culture
Primary human keratinocytes (strain km) were cultured on
mitotically inactivated J2 3T3 cells in complete FAD medium
(consisting of one part Ham’s F12 medium, three parts Dulbecco’s
modified Eagle’s medium (DMEM), 1.8  10 4 M adenine, 10%
fetal calf serum (FCS), 0.5 lg mL 1 hydrocortisone, 5 lg mL 1
insulin, 10 10 M cholera toxin and 10 ng mL 1 epidermal growth
factor). Low calcium complete FAD contained calcium-free F12
and DMEM supplemented with Chelex 100 (BioRad)-treated FCS
[15].
Collagen-coated patterned and flat PDMS substrates were used
to culture keratinocytes at a seeding density of 150,000 per cm2.
After 45 min at 37 °C non-adherent cells were removed and the
substrates were transferred to 12-well plates containing J2 3T3
feeders at a density of 20,000 cells per cm2. In some experiments
cells were treated with Rho kinase (ROCK) pathway inhibitor Y-
27632 (Life Science Enzo) at a final concentration of 10 lM.
2.3. Scanning electron microscopy (SEM)
PDMS substrates seeded with keratinocytes were fixed with
2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h at room tem-
perature. Samples were washed for 2  10 min in phosphate buf-
fer. The samples were dehydrated with a gradient of 30%, 50%,
70%, 90% and 3  100% ethanol for 15 min at each step. Samples
were then dried using hexamethyldisilazane:ethanol (1:1) for
5 min, mounted on an aluminum stub and sputter gold coated
(4 nm thickness). Image acquisition was performed using a desk-
top SEM (JEOL NeoScope JCM 6000Plus).
2.4. Immunofluorescence microscopy
Cells seeded on PDMS substrates were fixed in 4%PFA for 15 min
followed by three washes in PBS. Permeabilisation and blocking
(PB) buffer (0.5% skimmed milk powder, 0.25% fish skin gelatin,
0.5% TritonX100, 1 HEPES-buffered saline) was added and incu-
bated at room temperature for 1 h. Substrates were incubated in
primary antibodies for 1 h at room temperature. Samples were
washed three times in PBS and incubated with secondary antibod-
ies at room temperature for 1 h. Samples were washed three times
in PBS and then mounted on glass slides in Prolong antifade
mounting reagent (Life technologies) containing DAPI. Samples
were imaged using a Nikon A1 upright confocal microscope with
a 20 objective. Images are presented as maximum intensity
projections.
Primary antibodies used were as follows: anti-b1 integrin
mouse monoclonal (clone P5D2; 1:500), anti-involucrin rabbit
polyclonal (DH1; 1:200), anti-E-Cadherin mouse monoclonal
(clone HECD1; 1:200), anti-Desmoglein 3 mouse monoclonal
(Abcam; 1:25), anti-MAL rabbit polyclonal (Abcam; 1:200),
anti-Ki67 rabbit polyclonal (Abcam; 1:200), and anti-YAP mouse
monoclonal (Santa Cruz; 1:500). Alexa FluorTM 488 Phalloidin
(ThermoFisher Scientific; 1:1000) was also used. Secondary anti-
bodies of the appropriate species were conjugated to Alexa FluorTM
488, 555 and 647 (ThermoFisher Scientific).
258 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264

Fig. 1. SEM of keratinocytes cultured on PDMS substrates. (A, B) Side views of (A) patterned and (B) flat PDMS substrates prior to collagen coating. (C) top view of patterned
PDMS substrate prior to collagen coating. (D-G) Keratinocytes on (D) flat and (E, F, G) undulating PDMS substrates. (F) is higher magnification view of boxed region in (E).
Scale bars: 100 lm (A, B, C, G), 50 lm (D, E).

2.5. Atomic force microscopy (AFM) of the cells. Keratinocytes were seeded on substrates 24 h prior
to analysis and measurements were conducted at 37 °C in com-
AFM synchronised with optical microscopy (BioScope ResolveTM plete FAD medium. Optical microscopy was used to localise the
BioAFM, Bruker) was used to assess the biomechanical properties AFM tip on the top or base of PDMS topographical features. Silicon

Fig. 2. Effect of undulating topography on keratinocyte proliferation, differentiation and morphology. (A, B, D) Representative images (maximum intensity projections) of
cultured keratinocytes labelled for the markers indicated on (A, D) patterned and (B) flat PDMS for the length of time shown. Scale bars: 100 lm. (C, F) Quantification of
percentage of involucrin positive cells (% Inv+) (C), Ki67+ cells (F) cell density (F). The total % involucrin-positive cells per topography regardless of location is shown in the
middle panel of (C). (E) Quantitation of nucleus volume (nucl. Vol.). *P < 0.05, *** P < 0.001, ****P < 0.0001. (n = 3, 9 images (each corresponding in area to 0.40 mm2) per group
were analysed).
 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264 259

nitride MLCT cantilevers (Bruker) with nominal spring constants of
0.02 N/m (MLCT-B, for undulating substrates) and 0.6 N/m (MLCT-
F, for flat substrates) were used. Both cantilever types have pyrami-
dal tips with effective semi-included angles of 18° and a nominal
radius of curvature of 20 nm. The size for each force-volume scan
was selected in the range of 15–50 lm2 with 256 (=16  16) or
1024 (=32  32) force-separation measurement acquired. Each
force-separation measurement was acquired with a maximum
ramp size of 6 mm and ramp rate 0.5 Hz, up to a maximum force
of 2 nN. Analysis of force-volume scans to obtain the Young’s mod-
ulus maps was performed with Nanoscope Analysis software (Bru-
ker) using the linearized Sneddon conical model over the force
range 0.4–1.6 nN and assuming a Poisson’s ratio of 0.5.
2.6. Live cell imaging
Keratinocytes were seeded on collagen-coated PDMS substrates
24 h prior to live imaging. In some experiments, keratinocytes
were infected with lentiviral vectors encoding GFP (pLenti-CMV
GFP puro (658-5), gift from Eric Campeau, Addgene plasmid #
17448,) and mCherry (pLV-mCherry, gift from Pantelis Tsoulfas,
Addgene plasmid # 36084). Keratinocytes were transduced with
lentiviral plasmids carrying GFP and mCherry reporters 2 days
prior to live imaging. A JuLITM Stage Real-Time Cell History Recorder
device was used to acquire images from live keratinocytes. Images
were captured with an interval time of 1–2 h and videos were cre-
ated using JuLITM Stage edit software.
2.7. Statistical analysis and image quantification
Prism7 GraphPad software was used to generate graphs and
perform statistical analysis. All data were analysed by unpaired
or paired ANOVA or paired t-test and presented as the
mean ± standard deviation. The number of replicate experiments
is reported in the figure legends.
The immunofluorescence labeled images were processed by
imageJ and presented as maximum intensity projections. Using
imageJ, cells labelled with DAPI, Ki67, involucrin or other markers
were quantified and presented as a percentage of positive cells.

Fig. 3. Immunofluorescence labelling of keratinocytes on patterned substrates 24 h post seeding. Representative images (maximum intensity projections) showing
expression of F-actin (A), E-cadherin (B), Desmoglein 3 (C) and MAL (D) with DAPI counterstain (blue). Scale bars: 100 lm. (E) Quantitation of immunofluorescence images.
**P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 independent experiments, 9 images (each corresponding in area to 0.40 mm2) per experiment. one-way ANOVA for E-cadherin,
F-actin, Desmoglein 3 and paired t-test for MAL.
260 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264

Nuclear height and volume were measured using the 3D object

counter plugin.

3. Results

3.1. Seeding keratinocytes on undulating topographies

We previously generated a set of 9 PDMS substrates (including

one flat control substrate) by using three different photomasks and
three different UV exposure times [13]. Differences in topography
wavelength were determined by the photomask used, while the
amplitude was controlled by UV exposure. The topography that
best mimicked the undulations of healthy human skin (S1 in
[13]) had centre-to-centre (l) and circle diameter (d) equal to
300 and 150 lm (Fig. 1A and C). Although PDMS is hydrophobic,
we have previously shown that it can be coated with collagen by
adsorption [13]. This topography and a flat PDMS control (S6 in
[13]) (Fig. 1B) were coated with collagen and keratinocytes were
allowed to attach for 45 min, ensuring enrichment for stem cells.
After 24 h SEM revealed that both substrates were covered by a
confluent multilayered sheet of keratinocytes (Fig. 1D and E).
However, whereas cell morphology was rather uniform on the flat
substrate (Fig. 1D), cell morphology was more heterogeneous on
the undulating substrate (Fig. 1E–G).

3.2. Topographical features dictate the location of stem cells and

differentiated cells, and influence cell movements

To examine the evolution of cell patterning on the undulating

substrates, keratinocytes were cultured on the collagen-coated
patterned and flat PDMS substrates for 1 h, 4 h, 24 h and 4 days
(Fig. 2). b1 integrin bright cells clustered on tips of the undulating
topography as early as 1 h and the pattern was pronounced from
4 h to 4 days (Fig. 2A; Suppl. Fig. 1). The percentage of
involucrin-positive differentiated cells was 5.5% and 6.7% at 1 h
and 4 h time points, respectively (Fig. 2C), confirming that the
majority of adherent cells were undifferentiated. The number of
differentiated cells was significantly higher at the base and slopes
of the features than the tips at both time points (Fig. 2C), consistent
with earlier observations [13]. When we compared the base and
slopes separately there were more involucrin-positive cells on
the slopes than the base at 24 h (Suppl. Fig. 1B).
Keratinocyte motility has been reported to distinguish stem
cells from differentiated cells [16], with stem cells having the high-
est rotational speed. It has also been suggested that rotational
motion of individual keratinocytes results in collective motion of
stem cells [17]. To determine whether on undulating substrates
cells differentiated according to their location or whether there Fig. 4. Biomechanical properties of keratinocytes and the effect of Rho kinase
was significant cell movement, we collected time-lapse images. inhibition. (A) AFM measurements of keratinocytes were carried out at different
We mixed cells that had been labelled with GFP or mCherry len- positions on undulating substrates by positioning the cantilever tip at the tips or
base of substrates using an optical microscope. (B) Representative image of depth-
tiviral vectors to facilitate observation of cell-cell interactions.
coding of Z stack and cross section of DAPI channel showing cultured keratinocytes
The cultures also contained unlabeled cells (Movie 1). on patterned PDMS substrate. (C) Representative images of height and Young’s
Initially cells were observed moving on the base and tips of the modulus maps. Each square corresponds to one force displacement curve. (D)
substrates; however, when cells reached confluence movement Quantification of Young’s modulus measurements of keratinocytes on patterned
largely ceased. Prior to confluence GFP and mCherry labeled ker- (tip, base) and flat PDMS substrates 24 h post seeding in the presence or absence of
ROCKi. ** P < 0.01, *** P < 0.001. (n = 3, 9 random (15  15 lm) areas per group, 20
atinocytes were observed not only moving relative to one another measurements per area) (E) Representative images (maximum intensity projec-
but also sliding over one another, indicating that once cells have tions) of keratinocytes 24 h post seeding in the presence of ROCKi labelled for the
differentiated and left the basal layer they can translocate from markers shown. Scale bars: 100 lm (n = 3, 9 images per group were analysed).
their original position (Movie 1). By lower power time-lapse imag-
ing of unlabeled cells, we could detect more movement on the
bases than the tips, with movement resembling a push and pulling These results suggest that patterning is achieved by cues pro-
pattern (Movie 2). No apoptotic cells were observed by time-lapse vided by the topography of the culture substrate rather than by cell
imaging. sorting due to cell migration.
 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264 261

3.3. Topographical features dictate cell density and nuclear
morphology
Mechanical forces experienced by cells can lead to nuclear
deformation and flattening, and mechanosensing pathways are
regulated by active and passive transport of proteins through
nuclear pores [18]. To determine whether, like cell shape (Fig. 1),
nuclear morphology was affected by substrate topography, we
measured nuclear height and volume in cells at the tips and base
1 h and 24 h after seeding (Fig. 2E). We compared cells on pat-
terned and flat topographies. Overall, there was no difference in
nuclear volume at either time point and in any location, the excep-
tion being a reduction in volume of cell nuclei at the base of fea-
tures between 1 h and 24 h. Nuclear height was reduced after
24 h, regardless of cell location. In addition nucleus height
reflected cell position, being highest in cells on flat surfaces, and
lowest in cells on the tips of undulating substrates (Fig. 2E). The
nuclear volume of attached cells on the tips and base was compa-
rable (Fig. 2E).
At 1 h and 4 h there was no difference in cell density between
tips and bases, but from 24 h onwards cells were significantly more
crowded on the bases (Fig. 2F). Quantification of cell proliferation
as the percentage of Ki67 positive cells confirmed that, as reported
previously [12], there were no differences in proliferation between
cells at tips and bases (Fig. 2D). The differences in cell density and
nuclear morphology are thus more likely to reflect differences in
cell shape (Fig. 1).
3.4. Influence of surface topography on the actin cytoskeleton,
intercellular adhesion and MAL
The differences in the shape and motility of cells on tips and
base of the topographies suggested there would be corresponding
differences in the cytoskeleton and intercellular junctions. At 24 h
we observed higher F-actin levels in cells on the tips than on the
bases, correlating with the location of b1 integrin bright cells
(Fig. 3A and E). E-cadherin, a marker of adherens junctions, and
Desmoglein 3, a desmosome marker, were also expressed at higher
levels tips (Fig. 3B, C, and E). In contrast, keratinocytes attached on
the tips expressed lower levels of MAL compared to cells at the
base (Fig. 3D and E).
3.5. Influence of surface topography on cell stiffness
Since physical forces are transmitted between epithelial cells
via intercellular junctions [5] and keratins provide mechanical
strength against cellular deformation [19], we hypothesised that
cell stiffness might vary according to cell position on the patterned
topographies. We therefore used AFM to measure the Young’s
modulus of keratinocytes in different regions of the substrate
(Fig. 4A–C). The nature of the force-volume AFM data precluded
obtaining stiffness measurements with subcellular resolution
(Fig. 4A) and therefore the measurements represent the average
for the entire cell (nucleus, cytoplasm and intercellular junctions)
in each location on the topography. We could only make measure-
ments of cells on the tips and base, not on the slopes. We did not
find any difference in the stiffness of PDMS in different regions of
the topography (Suppl. Fig. 2).
Quantification of Young’s modulus revealed that the stiffness of
cells on the tips (3.9 ± 5.2 kPa) was significantly lower than that of
cells on the base (18.0 ± 12.3 kPa) (Fig. 4D). The stiffness of cells on
the flat substrates (15.9 ± 8.2 kPa) was comparable to cells on the
base of patterned substrates. Treatment with a Rho kinase inhibitor
(ROCKi) reduced the Young’s modulus of cells in all experimental
groups. Furthermore, in the presence of the inhibitor, the Young’s
modulus of cells on tips (7.1 ± 3.4 kPa) and base (6.5 ± 4.4 KPa)
was comparable (Fig. 4D).

Fig. 5. Biomechanical properties of the cell body and cell-cell junctions of keratinocytes cultured on flat substrates. (A) Representative images of immunolabled keratinocytes
following culture in low or high calcium FAD, or high calcium FAD containing ROCKi. Scale bars: 100 lm. (B) Representative images of height and Young’s modulus map. (C)
Quantitation of mean Young’s modulus of cell-cell borders and cell body. **P < 0.01, ***P < 0.001, ****P < 0.0001. (n = 3, 9 random (50  50 lm) areas per group, 20
measurements per area).
262 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264

Cell shape and ROCK regulate differentiation of epidermal stem
cells [6]. Inhibition of Rho GTPase signaling disrupts adherens
junction and stimulates keratinocyte colony expansion in culture
[12] (Fig. 4E). In the presence of ROCKi, clusters of integrin bright
cells remained at the tips, which expressed high levels of F-Actin
and low levels of MAL. However, E-cadherin staining was dis-
rupted, confirming the requirement for Rho kinase activity to
maintain adherens junctions.
3.6. Differential stiffness of cell-cell junctions and the cell body
To examine the influence of cell-cell junctions on biomechani-
cal properties, keratinocytes were cultured on collagen-coated
glass in low calcium FAD medium for 48 h to impair the formation
of E-cadherin mediated adherens junctions. Following AFM mea-
surements, the medium was changed standard (high calcium)
FAD. In a separate set of samples, ROCKi was added to standard
FAD. Cells were incubated overnight and the AFM measurements
were repeated. High levels of nuclear YAP and low levels of mem-
brane E-cadherin expression were observed in low calcium FAD.
After increasing the calcium concentration, YAP expression loca-
lised in the cytoplasm and E-cadherin expression increased signif-
icantly (Fig. 5A).
The AFM measurements indicated that the cell body of ker-
atinocytes cultured in low calcium FAD displayed a lower Young’s
modulus (2.0 ± 1.6 kPa) than keratinocytes cultured in standard
medium. Switching cells from low calcium to standard medium
led to a significant increase in cell body stiffness (4.1 ± 3.4 kPa),

Fig. 6. Effect of culturing keratinocytes on topographies with different slopes. (A) SEM images of uncoated patterned PDMS with steep (A) and shallower (B) slopes. (C-F)
Representative images (maximum intensity projections) of keratinocytes immunolabelled for b1 integrin (red) with DAPI counterstain (blue). Cells were cultured for the
number of days shown in the presence or absence of ROCKi. (C, E) steep, (D, F) shallow slopped topographies. Scale bars: 200 lm (A, B), 100 lm (C–F). (n = 3, 9 images per
group (each corresponding in area to 0.40 mm2) were analysed).
 S.A. Mobasseri et al. / Acta Biomaterialia 87 (2019) 256–264
which could be prevented by ROCKi (1.4 ± 0.7 kPa) (Fig. 5C). Under
all experimental conditions, the stiffness of the cell body was sig-
nificantly lower than that of cell-cell borders (Fig. 5C).
3.7. Effect of surface topography on cell-cell junctions
We hypothesised that stem cell patterning is controlled by
forces exerted by cells on the slopes of the topographical features,
rather than the tips or bases. To evaluate this, two additional
topographies [12] were used. Keratinocytes seeded on a substrate
with steep features (Fig. 6A; S5 in [12]) formed an intact sheet at
day 1, but the cells subsequently retracted from the tips and by
day 4 there were no longer any cells on the tips (Fig. 6, C). This
effect – i.e. extent of retraction - was less substantial for a substrate
with shallower slopes (Fig. 6B, D; S9 in [12]). By disrupting cell-cell
junctions with ROCKi, keratinocytes failed to form a confluent
sheet covering the tips, even on the substrate with the shallower
slope (Fig. 6E and F).
4. Discussion
When primary human keratinocytes are seeded on a collagen-
coated undulating polydimethylsiloxane (PDMS) elastomer
substrate that mimics the topography of the epidermal-dermal
junction in healthy, young skin, the cells become patterned such
that the b1 integrin bright stem cells localise to the tips of the fea-
tures [13]. Here we show that the cells at the tips of the features
are softer than those at the base, correlating with enhanced accu-
mulation of E-cadherin, Desmoglein 3 and F-actin at cell-cell bor-
ders. The differential stiffness of the cells was dependent of Rho
kinase activity. Our observations lead us to propose that epidermal
stem cell patterning is achieved through forces exerted on stem
cells by cells on the slopes, mediated by intercellular adhesion. This
would be consistent with earlier work showing that ker-
atinocytes are stiffer than other cell types because of their keratin
intermediate filament network [20], and that mutations or anti-
body treatments that impair desmosomes alter the viscoelastic
properties of keratinocytes [21–23].
While the dimensions of the PDMS substrates precluded com-
paring forces at intercellular junctions, we were able to measure
them on flat substrates. We observed that intercellular junctions
were stiffer than cell bodies and that their stiffness depended on
Rho kinase. In future it would be interesting to discover whether
forces exerted via intercellular junctions differ according to the
slope of the undulations.
We have previously reported that tip cells have nuclear YAP, a
localisation that is dependent on Rho kinase and intercellular
adhesion [12]. We now report that cells at the base of the topogra-
phies have higher levels of MAL, again dependent on Rho kinase.
Given that YAP and MAL/SRF differentially regulate differentiation
[12,6], the localisation of MAL is consistent with the higher fre-
quency of differentiation at the base of the substrates. Neverthe-
less, the fact that basal and suprabasal keratinocytes were able to
move relative to one another indicates that the accumulation of
involucrin-positive cells at the base of the features [13] could be
due to a combination of MAL/SRF signalling in the basal layer
and movement of suprabasal cells relative to the underlying basal
cells.
We have recently created a dynamic rig and shown that topo-
graphical features can impose patterning on a flat sheet of cells
within 48 h [24]. On the dynamic substrates stem cell clustering
can be induced independent of the location of differentiating,
involucrin-positive cells. This is consistent with the finding that
differentiating cells can move relative to underlying basal cells,
both in culture and during wound healing [25,26]. In addition,
263
studies of keratinocytes growing on the dynamic rigs lead us to
speculate that it is the undulations rather than whether cells are
on tips or base that is important in determining stem cell pattern-
ing. We provide further indirect support for this with the finding
that the ability of integrin-bright stem cells to localise to the tips
of PDMS substrates depends on the angle of the slope.
Crowding in the epidermal basal layer, which we observed on
the base of the topographies, is known to affect cell shape and
can trigger movement into the suprabasal layer through a decrease
in cortical tension and increased cell-cell adhesion [27,28]. We
speculate that the crowding of cells on the base of the topographies
is due to cells being pulled from the tips. It is possible that the dif-
ferences in cell stiffness between tips and base reflect differences
in cell crowding. It is important to emphasise that in our experi-
mental model it is the stiffness of the cells not the substrate that
is patterned.
Our finding that topography affects cell stiffness has implica-
tions for the changes in epidermal-dermal topography that occur
in human skin with age and inflammation. The undulations
decrease with age [29] and increase in psoriatic lesions [30,31].
While previous studies have focused on changes in the height of
the undulations, our studies suggest that changes in the slope
may also impact on the disruption of epidermal homeostasis that
occurs in psoriatic lesions. In the case of corneal injury biomechan-
ical changes in stromal cells are linked to accumulation of inflam-
matory cells [32] and it is therefore tempting to speculate that
differences in the stiffness of epidermal cells and their junctions
could lead to an altered immune infiltrate. It is also possible that
materials that mimic the undulating topography of the
epidermal-dermal junction, for example by casting topographies
out of collagen gels, could enhance wound healing or reduce
scarring.
Acknowledgements
We gratefully acknowledge the helpful input of our colleagues
Simon Broad, Bénédicte Oulès, Priya Viswanathan, Toru Hiratsuka
and Elvira Infante for technical assistance and discussion of AFM
measurements of keratinocytes. This work was supported by the
UK Medical Research Council (MR/PO18823/1), Biotechnology
and Biological Sciences Research Council (BB/M007219/1) and
the Wellcome Trust (206439/Z/17/Z). This work was also sup-
ported by the European Commission (Mechanocontrol, grant
agreement SEP-210342844), an EPSRC Fellowship K00641X/1,
EPSRC Strategic Equipment Grant (EP/M022536/1), and the Lever-
hulme Trust Research Leadership Award (RL-2016-015) to S.G-M.
We also acknowledge funding from the Department of Health via
the National Institute for Health Research comprehensive Biomed-
ical Research Centre award to Guy’s & St Thomas’ National Health
Service Foundation Trust in partnership with King’s College London
and King’s College Hospital NHS Foundation Trust. FMW is cur-
rently on secondment as Executive Chair of the Medical Research
Council.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.actbio.2019.01.063.
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