The activation of directional stem cell motility by green light-emitting diode irradiation

(1)

The activation of directional stem cell motility by green light-emitting diode

irradiation

Wei-Kee Ong

a

,

1 , How-Foo Chen

b

,

1 , Cheng-Ting Tsai

b

, Yun-Ju Fu

a

, Yi-Shan Wong

a

, Da-Jen Yen

c

,

Tzu-Hao Chang

d

,

e

, Hsien-Da Huang

d

, Oscar Kuang-Sheng Lee

f

, Shu Chien

g

, Jennifer Hui-Chun Ho

a

,

h

,

i

,

*

a_{Center for Stem Cell Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan}

b_{Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan}

c_{Department of Material Science and Engineering, National Tsing Hua University, HsinChu, Taiwan} d_{Institute of Bioinformatics and Systems Biology, National Chiao Tung University, HsinChu, Taiwan} e_{Graduate Institute of Biomedical Informatics, Taipei Medical University, Taiwan}

f_{Institute of Clinical Medicine, National Yang-Ming University, Taiwan}

g_{Departments of Bioengineering and Medicine, Institute of Engineering in Medicine, UC San Diego, La Jolla, CA, USA} h_{Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan}

i_{Department of Ophthalmology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan}

a r t i c l e i n f o

Article history:

Received 12 November 2012 Accepted 29 November 2012 Available online 19 December 2012

Keywords: Cell motility Light-emitting diode Photosensitization Stem cells Wavelength

a b s t r a c t

Light-emitting diode (LED) irradiation is potentially a photostimulator to manipulate cell behavior by opsin-triggered phototransduction and thermal energy supply in living cells. Directional stem cell motility is critical for the efﬁciency and speciﬁcity of stem cells in tissue repair. We explored that green LED (530 nm) irradiation directed the human orbital fat stem cells (OFSCs) to migrate away from the LED light source through activation of extracellular signal-regulated kinases (ERK)/MAP kinase/p38 signaling pathway. ERK inhibitor selectively abrogated light-driven OFSC migration. Phosphorylation of these kinases as well as green LED irradiation-induced cell migration was facilitated by increasing adenosine triphosphate (ATP) production in OFSCs after green LED exposure, and which was thermal stress-independent mechanism. OFSCs, which are multi-potent mesenchymal stem cells isolated from human orbital fat tissue, constitutionally express three opsins, i.e. retinal pigment epithelium-derived rhodopsin homolog (RRH), encephalopsin (OPN3) and short-wave-sensitive opsin 1 (OPN1SW). However, only two non-visual opsins, i.e. RRH and OPN3, served as photoreceptors response to green LED induced OFSC migration. In conclusion, stem cells are sensitive to green LED irradiation-induced directional cell migration through activation of ERK signaling pathway via a wavelength-dependent phototransduction.

1. Introduction

Light provides both illumination and thermal energy. Light

exposure is physiologically responsible for human homeostasis

such as vitamin D synthesis

[1]

, sleep

ewake cycle

[2,3]

, and vision

[4]

. In addition, phototherapy is a standard treatment for skin

disorders

[5,6]

, neovascular retinopathy

[7]

, or musculoskeletal

disorder

[8]

, with the uses of appropriate wavelength, intensity and

duration of light exposure.

Light-emitting diode (LED) is a semiconductor light source

widely used in lighting due to the advantage of high ef

ﬁciency, high

switching rate, and long lifetime

[9]

. Adjuvant phototherapy, using

red or near infra-red (NIR) LED as a light source, has been

demonstrated to confer therapeutic bene

ﬁts on epidermis/dermis

wound healing and arthritis via inhibition of in

ﬂammation

[10

e12]

.

In contrast to incandescent light sources, the multiple colors with

broad spectrum wavelengths make LED light a good

photo-stimulator to manipulate cell behavior by activating

wavelength-speci

ﬁc photosensitizer or providing thermal energy.

Stem cells, with their self-renewal ability, multi-potency, and

paracrine effect, possess a great therapeutic potential for tissue

regeneration during acute injury

[13]

. Homing of stem cells and the

* Corresponding author. Graduate Institute of Clinical Medicine, Taipei Medical University 250 Wu-Hsing Street, Taipei 110, Taiwan. Tel.:þ886 2 29307930x2946; fax:þ886 2 29342285.

E-mail address:[email protected](J. Hui-Chun Ho).

1 _{Wei-Kee Ong and How-Foo Chen contributed equally to this work.}

Contents lists available at

SciVerse ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o m a t e r i a l s

(2)

subsequent inhibition of in

ﬂammation are critical for acute wound

repair

[14

e16]

, and directional stem cell migration leads to an

effective and speci

ﬁc tissue repair

[17]

. Ras homolog gene family,

member A (RhoA) is essential for cell migration to regulate actin

cytoskeleton reorganization and activation of extracellular

signal-regulated kinase (ERK), p38 in stem cells has been reported to

crucial for their migration induced by pro-in

ﬂammatory cytokines,

chemokines or stromal cell-derived factor-1

[16,18

e20]

, all of

which lead to a high speci

ﬁcity and therapeutic efﬁciency of stem

cells in wound repair. Orbital fat stem cells (OFSCs) are multipotent

mesenchymal stem cells (MSCs) isolated from human orbital fat

tissues

[21]

, The anti-in

ﬂammation ability during acute tissue

injury and tolerance in a xenotranplanted model have been

demonstrated in our previous study

[22]

. Therefore, development

of a non-invasive, light-driving method to enhance stem cell

migration ability will be valuable for tissue regeneration in

response to acute injury.

Up to now, only a few studies have investigated the impact of

LED irradiation on stem cell behavior. Recently, red/NIR LED

irra-diation has been found to promote MSCs growth and enhance their

osteogenic differentiation ability

[23

e25]

. It has been

demon-strated that over-expression of channelrhodopsin-2 (ChR2) in

embryonic stem cells successfully differentiates into functional

excitatory neuron under blue LED irradiation

[26,27]

. However, the

mechanism(s) of red/NIR or blue LED irradiation regulating stem

cell behavior is remain elucidative. Moreover, the impact of green

LED irradiation on stem cell biology and the photosensitizers of

green LED are still unclear.

Here we explored the effect as well as mechanism of green LED

irradiation on directional migration of OFSCs. Through the

comparison of migrated cells with non-migrated cells in a transwell

system under green, red LED light exposure or heat, the mechanism

controlling green LED irradiation-induced cell migration and the

role of adenosine triphosphate (ATP) in OFSC migration were

elucidated. The photosensitizers responsible for green LED

irradiation-induced motility in OFSCs were also delineated in this

study.

2. Materials and methods

2.1. Cells Isolation and culture

OFSCs were isolated from human orbital fat tissues with informed consent. Approval from the Institutional Review Board of Wan Fang Hospital, Taipei Medical University was obtained prior to the commencement of the experiments. The procedures of isolating OFSCs were described previously[21]. Briefly, adipose tissues were fragmented, digested, andfiltered. After centrifugation, the cells from the resulting pellet were plated in non-coated tissue cultureflasks (BD Biosciences, San Jose, CA, USA) with MesenPro medium (Invitrogen, Carlsbad, CA, USA). Cells with colony formation ability, MSC surface phenotype profile, and tri-linage differentia-tion capacity were defined as OFSCs. The OFSCs were maintained in MesenPro medium (Invitrogen) under non-contact culture as described previously [28]. Briefly, cells from one flask were detached when their density reached 60e70% of confluence, and were re-seeded into three new flasks. Eighth to tenth passage of OFSCs were used for the experiments.

SV-40 immortalized human corneal epithelial (HCE-T) cells[29]were kindly given by Dr. Araki-Sasaki. The cells were cultured in DMEM/HamF12 (1:1) medium supplemented with 5% fetal bovine serum (HyClone, Logan, UT, USA), 5mg/ml insulin, 0.1 mg/ml cholera toxin (SigmaeAldrich, St. Louis, MO, USA), 10 ng/ml recombinant human epidermal growth factor (hEGF) (BD Biosciences), and 0.5% DMSO, as previous description[30].

2.2. LED photosystem

The LED photosystem is illustrated inFig. 1. OFSCs (7500 cells/transwell) or HCE-T cells (5000 cells/transwell) were seeded on the upper surface of 8mm pore sized transwell culture plate (PI8P01250, Millipore MillicellÒcell culture insert, Millipore, Billerica, MA, USA). The attached cells were covered by undifferentiated medium (MesenPro, Ivitrogen) (Fig. 1B) and exposed to a 530-nm green LED (M530L2, Thorlabs, Inc., Newton, NJ, USA) or a 625-nm red LED (M625L2, Thorlabs) light at a distance of 30 cm away from LED light source (Fig. 1A). The full power density of

LED irradiated on cells was 66.4mW/cm2and the power density could be reduced to 22.8 (ﬁlter 1) or 11.3 (ﬁlter 2)mW/cm2_{by a polariscope at 6 cm from the light. Cells in}

the transwell system under dark at the same time points served as the time-matched control.

2.3. Cell migration assay

The cells in the transwell system were under LED irradiation or dark control with or without 25mM of ERK inhibitor (328005, Merck, Whitehouse Station, NJ, USA) for 24 or 48 h. In this transwell migration system, migrated cells were deﬁned by cells migrating to the bottom surface of transwell plate, while non-migrated cells were deﬁned by cells remaining on the upper surface of transwell plate.

For migrated cell (or non-migrated cell) counting and staining, cells on the upper surface (or on the bottom surface) of transwell culture plate were totally removed by a scraper. Cells migrated to the bottom surface (or remained on the upper surface) wereﬁxed by 3.7% formaldehyde for 20 min, and then stained by TRITC-labeled phalloidin (1:500; SigmaeAldrich, St. Louis, MO, USA) at 37_{C for 1 h,}

followed by 4-,6-diamidino-2-phenylindole (DAPI) for 5 min. Migrated cells on the bottom surface were counted under afluorescence microscopy (Leitz, Germany) with 100 magnification. The migrated cell number in each sample was determined, and the mean value was determined from cell numbers in ten randomfields. More than three independent experiments were performed for each condition.

2.4. Cell proliferation assay

Cells were seeded in a 96-well plate (2000 cells/well) for 4 h before being exposed to LED light or ERK inhibitor. After green LED irradiation for 24, 48, 72 h or incubation with ERK inhibitor (328005, Merck) for 48 h, the culture medium in each well was then replaced by 100ml of serum-free DMEM and 20ml MTS reagent (Promega, Madison, WI, USA). The signal at OD490 was measured by using a microplate reader (Bio-Rad, Hercules, CA, USA) after cells were incubated in the dark at 37C for another 1e4 h.

2.5. Gene expressions

RNAs were extracted using the RNeasy Kit (Qiagan Inc., Valencia, CA, USA) fol-lowed by reversely transcribed to cDNA using an Advantage RT-for-PCR kit (Clon-tech, Palo Alto, CA, USA). cDNA was ampliﬁed using a Mastercycler Gradient 5331 Thermal Cycler (Eppendorf, Germany). For microarray analysis, the differential gene expressions were detected by GeneChipÔ (Affymetrix, Santa Clara, CA, USA) and analyzed by Affymetrix Microarray Suite 5.0. For real-time RT-PCR, gene expression level was represented by monitoring theﬂuorescence signals after each cycle with an ABI 7300 Real-Time PCR system (Applied Biosystems). Primers used for real-time RT-PCR were listed inTable 1.

2.6. Intracellular ATP content

Intracellular ATP level was measured using ATP determination kit (Invitrogen) as per the manufacturer’s instructions. Brieﬂy, cells were trypsinized, resuspended in CelLyticÔ MT mammalian tissue lysis/extraction reagent (100 ul; SigmaeAldrich) to release the intracellular ATP. The supernatant (10ml) was then transferred into a 96-well cell culture cluster (Corning Costar, NY, USA) containing 90ml ATP standard reaction solution and measured by Luminoskan Ascent Luminometer (Thermo Electron Corp., Waltham, MA, USA).

2.7. Intracellular kinase activity

Intracellular kinase activity was determined by using human phospho-kinase array kits (R&D system, Minneapolis, MN, USA) and Western blot analysis accord-ing to the manufacturer’s instructions. Brieﬂy, cells were trypsinized and resus-pended in lysis buffer to obtain the cell lysate. For kinase array, each cell lysate (300 mg) was incubated individually with the antibody-pre-coated membrane overnight. After washing, the membrane was incubated with biotinylated-labeled antibody cocktail for 2 h, and then incubated with streptavidin-HRP for another 30 min.

For Western blot analysis, 30mg of protein was separated on 10% SDS-PAGE and blotted onto PVDF membrane (Amersham Biosciences, Uppsala, Sweden), followed by blocking with 5% skim milk in TBST buffer (50 mm TriseHCl, 150 mm NaCl, 0.1% Tween 20, pH 7.4). The membrane was then blotted with indicated primary antibodies such as ERK (1:1000, Cell signaling, Danvers, MA, USA), p-ERK (1:2000, Cell signaling) anda-tubulin (1:10000, SigmaeAldrich). After 3 times of washes, the membrane was incubated with the HRP-conjugated secondary anti-body (1:5000, Santa Cruz, Santa Cruz, CA, USSA). The signals on each membrane was detected by ECL chemiluminescent reagent (PerkinElmer Life Sciences, Inc.) and their intensities were quantitatively measured by a densitometry (LabWorks, UVP Inc., Upland, CA, USA).

W.-K. Ong et al. / Biomaterials 34 (2013) 1911e1920 1912

(3)

2.8. Temperature measurement

Temperature of medium was measured under dark or LED light exposure using high-sensitivity glass probe thermistor (SP31A, Sensor Scientiﬁc, Inc., Fairﬁeld, NJ, USA). Temperature was recorded every 2 h, and calibration of thermistors was performed before temperature measurement at each time point according to the manufacturer’s instructions.

2.9. Statistical analysis

Statistical analyses were performed using the Statistical Package for Social Science software (v. 16, SPSS Inc., Chicago, IL). Differences in viability with and without ERK inhibitor, kinase activities, temperature or migrated cell numbers between LED irradiation and dark control at the same time point were assessed using the two-tailed, non-paired t-test, and for significance, P-values < 0.05. Differences among the migrated cells numbers and intracellular ATP content at various temperatures, viability or migrated cell numbers with and without LED irradiation at various time points, or data from the gene expression levels (i.e. RhoA, retinal pigment epithelium-derived rhodopsin homolog (RRH), short-wave-sensitive opsin 1 (OPN1SW), and encephalopson (OPN3)), and ATP content in migrated and non-migrated cells with and without LED irradiation were assessed using analysis of variance, Tukey’s post-hoc test, and a 95% confidence level. Different characters represented different levels of significance, and level "ab" indicated statistical level in between level "a" and level "b". Error bars shown in all figures represent standard deviation of mean values.

3. Results

3.1. Effect of green LED irradiation on OFSC migration

The study design is illustrated in

Fig. 1

A and B. The orbital fat

stem cells were initially seeded on the upper surface of the

transwell plate. The cells that have migrated to the bottom

surface of the transwell plate are designated as migrated cells,

Fig. 1. Green light-emitting diode (LED) irradiation enhanced human orbital fat stem cell (OFSC) migration (A) Schema of LED photosystem. (B) Schema of cells in transwell migration system under LED irradiation. Green LED enhanced OFSC migration ability after 48-h irradiation with power density of 66.4 (C), 22.8 (D) and 11.3 (E)mW/cm2_.

Table 1

Primers for real-time reverse transcription-Polymerase Chain reaction. Gene Primer Sequence Product (bp) RRH F: 50-ACCACCAACACTTACATCGG-30 112 R: 50_{-TAGCACCAGTAGGATCTGGG-3}0 OPN1SW F: 50_{-ATGGGCCTCAGTACCACATT-3}0 ₁₃₂ R: 50_{-GCAACTTTTTGTAGCGCAGT-3}0 OPN3 F: 50_{-TCAGTGCACAATGGCTAGAG-3}0 ₁₃₅ R: 50-GCGGTTCCCCGAGTACAT-30 OPN5 F: 50-TTGGGAAGCGGATTTAGTGG-30 125 R: 50_{-TTATTTCAGCGGGTCTCAGC-3}0 ChR2 F: 50_{-CTCACGCGCTGCAATGTCCT-3}0 ₁₁₃ R: 50_{-GCAGCCGAGTGGGGTAATGC-3}0 RhoA F: 50_{-CAGAAAAGTGGACCCCAGAA-3}0 ₁₄₇ R: 50_{-TGCCTTCTTCAGGTTTCACC-3}0 18S rRNA F:50-ATGGCCGTTCTTAGTTGGTG-30 132 R:50-AACGCCACTTGTCCCTCTAA-30

(4)

while non-migrated cells are cells remaining on the upper

surface. Compared to time-matched dark control, 48 h of green

LED irradiation signi

ﬁcantly increased the migrated cell numbers

under the power densities of 11.3 (

Fig. 1

C), 22.8 (

Fig. 1

D) and

66.4 m

W/cm

2

(

Fig. 1

E), but such increases were not seen at 24 h

(

Fig. 1

C

eE), nor at 6 or 12 h (data not shown).

Actin cytoskeleton re-organization is essential for cell migration

[31]

and the formation of actin stress

ﬁbers during cell migration is

regulated by RhoA

[32,33]

. After 48 h of green LED irradiation,

F-actin signals in migrated OFSCs (

Fig. 2

A, right) were stronger than

in migrated cells under dark (

Fig. 2

A, left). Green LED irradiation

signi

ﬁcantly up-regulated RhoA expression in migrated OFSCs, but

did not alter the RhoA expression in non-migrated cells (

Fig. 2

B).

3.2. Effect of green LED irradiation on OFSC proliferation

To determine whether green LED irradiation affected cell

proliferation, the numbers of viable OFSCs under green LED light

exposure at various time points were measured by MTS assay. As

shown in

Fig. 2

C, OFSC number doubled between 48 and 72 h both

in the dark and under green LED exposure; thus, LED irradiation

showed no effect on OFSC proliferation.

To avoid the contribution of cell division to increase in migrated

cell numbers, we performed the following experiments under LED

exposure for up to 48 h at the power density of 66.4

m

W/cm

2

_.

3.3. Effect of green LED irradiation on HCE-T cell migration

To test the cell speci

ﬁcity of the induction of migration by green

LED irradiation, OFSCs were replaced by HCE-T cells, which are

well-differentiated corneal epithelial cells, in the LED photosystem.

HCE-T cell migration was also enhanced under 66.4

m

W/cm

2

of

green LED irradiation for 48 h, but the increase over this time

period was less than 1.5 fold (

Fig. 3

A), as compared with the 3-fold

increase for the OFSCs (

Fig. 1

C). Light-driven F-actin reorganization

in migrated OFSCs (

Fig. 2

A) was also more prominent than in

migrated HCE-T cells (

Fig. 3

B).

3.4. Role of wavelength on LED irradiation-induced cell migration

To further dissect the role of wavelength in such a

photo-induced cell migration, green LED (530 nm) was replaced by red

LED (625 nm), which has no overlap of spectrum wavelength with

the green LED light. Surprisingly, under the same power density to

green LED irradiation (66.4

m

W/cm

2

), red LED irradiation for 48 h

neither affected OFSC (

Fig. 3

C) nor HCE-T cell (

Fig. 3

D) migration in

the photosystem.

3.5. ATP content in green LED irradiated OFSCs

Migration is an energy consumption behavior for a cell. ATP is

essential for phosphorylation of most protein kinases

[34]

and

increases the formation of guanosine triphosphate (GTP) by

transferring phosphate to GDP under light stimulation

[35]

.

Besides, ATP may form cyclic adenosine monophosphate (cAMP) by

conformational change

[34]

. We further investigated the effect of

green LED irradiation on ATP production and OFSCs migration. As

shown in

Fig. 4

A, green LED irradiation markedly increased ATP

production in both migrated and non-migrated OFSCs. Compared

to non-migrated cells, however, the intracellular ATP content in

migrated OFSCs was signi

ﬁcantly lower, suggesting that ATP was

utilized during cell migration.

3.6. Alteration of temperature in green LED irradiated OFSCs

Thermal stress has been reported to increase ATP production

from mitochondria

[36]

. Monitoring the temperature in the culture

medium at various time points under green LED irradiation and

dark control showed that green LED irradiation signi

ﬁcantly

elevated local temperature after 8 h of light exposure. In average,

Fig. 2. Green LED irradiation-induced OFSC migration was associated with F-actin reorganization, and not a consequence of cell division (A) F-actin signal in migrated OFSCs under green LED irradiation (right) was stronger than which in migrated OFSCs under dark control (left). (B) RhoA expression was up-regulated in migrated OFSCs, and green LED irradiation further increased RhoA expression in migrated OFSCs. (C) The doubling time of OFSCs was in between of 48e72 h, and green LED irradiation did not affect OFSC viability.

(5)

the medium temperature under green LED irradiation (37.4

C) was

only 0.1

C higher than dark control (37.3

C) in the

ﬁrst 48 h

(

Fig. 4

B), and which was associated with increase in 100% of ATP

production in OFSCs (

Fig. 4

A).

3.7. Thermal effect on ATP production and cell migration

To determine the role of thermal effect on ATP production and

cell migration, we increased culture medium temperature by heat

instead of LED light for 48 h. As showed in

Fig. 4

C

eF, responses of

ATP production and cell migration to thermal effect could be

observed only in OFSCs (

Fig. 4

C and D), but not HCE-T cells (

Fig. 4

E

and F). Elevation of 1.0

C by heat was associated with a 30%

increase of ATP production in OFSCs (

Fig. 4

C and D), and this

temperature effect on ATP production was not seen in HCE-T cells

(

Fig. 4

E and F). However, elevation of 0.1

C (from 37.3

C to 37.4

C)

by heat did not alter the migration potential of OFSCs (

Fig. 4

G),

indicating that the green LED irradiation-induced ATP production

and cell migration were not attributable to thermal stress.

3.8. Phototransduction for green LED irradiation-induced OFSC

migration

Since green LED irradiation increased ATP production, and ATP

was utilized for OFSC migration (

Fig. 4

A), we looked for the

target(s) of increased ATP during cell migration promoted by green

LED irradiation. Human phosphorylated kinase array was

per-formed to analyze the differential kinase activities in OFSCs

between green LED irradiation and dark control. We found that

eight of the kinases, i.e., p38, ERK 1/2, c-Jun N-terminal kinase

(JNK), MAP kinase kinase (MEK) 1/2, Akt, cAMP response

element-binding (CREB) (

Fig. 5

A), Yes (

Fig. 5

B), and c-Jun (

Fig. 5

C)

were markedly activated after 48-h green LED irradiation, while

three of the kinases, i.e., Lyn, signal transducer and activator of

transcription (STAT) 5 alpha (

Fig. 5

B), and p70S6 kinase (

Fig. 5

C)

were inhibited. Kinases involved in cell cycle, such as p53, p27 and

TOR, were not affected by green LED irradiation (

Fig. 5

D).

To assess the role of ERK signaling pathway in the green LED

irradiation-induced OFSC migration, intracellular ERK activity was

inhibited by using an ERK activation inhibitor peptide II that targets

the phosphorylation of ERK (

Fig. 6

A). OFSC viability was not

affected by the ERK inhibitor (25

m

M) over the 48 h of study

(

Fig. 6

B). The inhibition of ERK activity selectively abrogated the

green LED irradiation-induced OFSC migration, but had no effect on

cell migration in the dark control (

Fig. 6

C). The ERK inhibitor also

decreased F-actin signals in the migrated cells triggered by green

LED irradiation (

Fig. 6

F and G) and did not affect F-actin in the

migrated cells under dark (

Fig. 6

D and E).

3.9. Photosensitizers for green LED irradiation-induced OFSC

migration

To

_{ﬁnd out the putative photosensitive molecules that directed}

the green LED irradiation-induced OFSC migration, microarray

analysis was used for screening the basal expression level of opsins

(OPNs), the photosensitizers in animal cells

[37]

, in OFSCs. As

shown in

Table 2

listing the human opsins have been identi

ﬁed,

gene expressions of RRH, OPN1SW, OPN3 and OPN5 were

detect-able by at least one probe on the microarray chip. OFSCs did not

express rhodopsin (OPN2), long/medium-wave-sensitive opsin 1

(OPN1LW/OPN1MW), ChR2, melanopsin (OPN4), neuropsin (KLK8),

or retinal G protein coupled receptor (RGR). Real-time RT-PCR was

Fig. 3. LED irradiation-induced cell migration was wavelength speciﬁc green LED irradiation enhanced human corneal epithelial (HCE-T) cells migration (A), and F-actin signal in migrated HCE-T cells was not signiﬁcantly increased by green LED irradiation (B). Red LED irradiation neither altered the migration ability in OFSCs (C) nor in HCE-T cells (D).

(6)

Fig. 4. Thermal effect partially contributed in green LED irradiation-induced ATP production for OFSC migration (A) green LED irradiation increased intracellular ATP production by 2 folds in both migrated and non-migrated OFSCs, and ATP consumption was observed during cell migration. (B) Green LED irradiation increased the temperature by 0.1_{C after 8 h of light exposure. (C) Elevation of temperature by heat up to 0.5}_{C and above signiﬁcantly increased ATP} content in OFSCs. (D) Elevation of temperature by heat up to 1C signiﬁcantly increased OFSC migration. Heat-induced thermal effect neither changed the ATP content in HCE-T cells (E) nor HCE-T motility (F). Elevation of temperature from 37.3C to 37.4C by heat did not alter the migration potential in OFSCs (G).

W .-K. Ong et al. / Biomaterials 34 (20 13) 19 11 e 1920 19 1 6

(7)

performed on the photosensitizers detected by microarray under

dark and green LED irradiation for 48 h; the results con

ﬁrmed the

constitutional expression of RRH, OPN1SW and OPN3, but not OPN5

(data not shown). Using transwell migration assay, we found

upregulation of RRH (

Fig. 7

A) and OPN3 (

Fig. 7

B), but not OPN1SW

(

Fig. 7

C), during the

ﬁrst 24 h in migrated OFSCs triggered by green

LED irradiation. In the dark control, the expressions of RRH (

Fig. 7

A)

and OPN1SW (

Fig. 7

C) were not signi

ﬁcantly different between

non-migrated and migrated OFSCs. OPN3 expression was decreased

in migrated OFSCs under dark control in comparison of

non-migrated OFSCs (

Fig. 7

B).

4. Discussion

In this study, we demonstrate that green LED (530 nm)

irradi-ation triggers directional stem cell motility through activirradi-ation of

phototransduction mediated by the ERK signaling pathway. The

enhancement of OFSC migration by green LED irradiation is

wavelength speci

ﬁc, but not limited to OFSCs. OFSCs, stem cells

isolated from orbital fat tissue, are more sensitive to light-induced

migration than the differentiated HCE-T cells. Green LED irradiation

increases ATP production to facilitate ERK/MAPK/p38 kinase

phosphorylation in OFSCs. RRH and OPN3 are photosensitizers in

OFSCs that respond to green LED irradiation, which induced cell

migration away from the light source.

Directional migration of stem cells results in an effective and

speci

ﬁc tissue repair

[17]

. In this study, kinases involving the ERK/

MAPK/p38 signaling pathway were selectively activated by green

LED irradiation (

Fig. 5

), and inhibition of ERK phosphorylation

selectively abrogated the green LED irradiation-induced OFSC

migration (

Fig. 6

), showing that the migration enhancement by LED

irradiation is a consequence of the activation of the ERK/MAPK/p38

signaling pathway. It has been reported that light exposure may

induce activation of ERK/MAPK/p38 signaling pathway in tissue

cells, such as human endothelial cells

[38]

, skin

ﬁbroblast

[39]

and

mouse epidermal cells

[40]

, and it is known that activation of this

signaling pathway is crucial for directional stem cell migration

[16,18

e20]

. Our data indicate that green LED irradiation enhanced

directional cell motility away from the light source through this

signaling pathway axis (

Figs. 1, 2 and 5

). Further studies to evaluate

the therapeutic effect of green LED-irradiated OFSCs on tissue

repair are warranted.

Fig. 5. Green LED irradiation signiﬁcantly activated ERK/MAPK/p38 signaling pathway in OFSCs after 48 h of green LED irradiation, intracellular kinase activities of p38, ERK1/2, JNK, MEK1/2, Akt, CREB (A), Yes (B), and c-Jun (C) were signiﬁcantly increased, while Lyn, STAT 5 alpha (B), and p70S6 kinase (C) were decreased. (D) Kinases involved in cell cycle such as p53, p27 and TOR were not affected.

(8)

Therapeutic effect of red/NIR LED irradiation via thermal

effect-induced ATP production has been reported in the literature.

Elevation of local temperature during red/NIR LED or laser

photo-therapy leads to increase ATP content in the treated animal brain

tissue

[41,42]

, suggesting that thermal effect is one of the

mecha-nism(s) of red/NIR LED phototherapy. In this study, OFSCs were

more sensitive to thermal effect on ATP production and cell motility

than HCE-T cells (

Fig. 4

C

eF). However, green LED irradiation only

minimally increased the local temperature (

Fig. 4

B), and this slight

increase in temperature was not suf

_{ﬁcient to increase the ratio of}

ATP production (

Fig. 4

G), indicating that temperature does not play

a role in green LED irradiation-induced ATP production and cell

migration.

ATP is essential for the phosphorylation of most protein kinases

[34]

. Green LED irradiation increased ATP production in OFSCs

(

Fig. 4

A) accompanied by activation of ERK/MAPK/p38 kinase

activities (

Fig. 5

) and migration enhancement (

Fig. 1

), suggesting

that the increase in ATP production may facilitate ERK 1/2, MEK,

p38, JNK, and c-Jun phosphorylation for OFSC migration. However,

the kinase phosphorylation selectivity by ATP requires appropriate

photosensitizer-triggered phototransduction.

Opsins are photoreceptors in animal cells and there are more

than sixty opsins have been identi

ﬁed. Most opsins are

chromo-phore containing trans-membranous G-protein coupled receptors,

and cellular signal delivery follows speci

ﬁc opsin activation by light

so that cell behavior may altered by photoirradiation

[37]

. Among

human opsins, it can be divided into visual opsins and non-visual

opsins. Visual opsins are photorsensitizers expressing in

photore-ceptor cells, i.e. OPN1LW, OPN1MW and OPN1SW to mediate

wavelength-speci

ﬁc phototransduction for color vision in cone

cells, and OPN2 in rod cells for night vision

[43

e45]

. Non-visual

opsins are phosensitizers such as OPN3, OPN4, OPN5, RRH, KLK8

and RGR. OPN3 (encephalopsin or panopsin) is strongly expressed

in brain and testes

[46,47]

, but functional unclear. Recently, OPN3

expression in lung bronchial epithelia and immune cells has been

reported to associate with asthma and modulation of T-cell

response

[48]

. OPN4 are located in retinal ganglion cells and retinal

pigment epithelial cells for pupillary light response, light

entrain-ment of the circadian rhythm, and photopigentrain-ment regeneration

[49

e51]

. RRH, OPN5 and RGR are opsins expressing in retina, and

may encode a protein with photoisomerase activity

[52

e54]

.

Up to now, what kinds of opsins are expressed in stem cells, as

well as their functions in stem cells, has not been reported in the

literature. Recently, OPN1SW, OPN2, OPN3, OPN4, OPN5, and RRH

expressions are found in spontaneously immortalized human

Müller cell lines exhibiting retinal progenitor characteristics

[55]

,

but the function of those opsins in retinal progenitor cells needs be

de

ﬁned. It is well accepted that animal vision starts with cAMP

signaling mediated by opsin-G protein cascade

[56]

. In

hippo-campus, light/dark cycle with oscillation reactivates ERK1/2, MAPK

and cAMP/CREB signaling pathway is critical for persistent

Fig. 6. Inhibition of ERK activity abrogated green LED irradiation-induced OFSC migration (A) Twenty-fivemM ERK inhibitor blocked ERK phosphorylation in OFSCs within 2 h. Persistent treatment of 25mM ERK inhibitor did not affect OFSC viability (B), but totally abrogated green LED irradiation-induced OFSC migration in thefirst 48 h (C). (D, E) Under dark control, inhibition of ERK activity did not change actin organization in migrated OFSCs. (F, G) Under green LED irradiation, inhibition of ERK activity significantly affected F-actin re-organization in migrated OFSCs.

Table 2

Gene expression of photosensitizers in OFSCs.

Gene symbol Gene Title Expression in OFSCs RRH retinal pigment epithelium-derived

rhodopsin homolog

þ-RHO rhodopsin (opsin 2, rod pigment) -OPN1SW short-wave-sensitive opsin 1

(cone pigments) þ-OPN1LW/ OPN1MW long/medium-wave-sensitive opsin 1 (cone pigments) -ChR2 channelrhodopsin-2 -OPN3 opsin 3 (encephalopsin, panopsin) þþ OPN4 opsin 4 (melanopsin)

-OPN5 opsin 5

þ-KLK8 kallikrein 8 (neuropsin/ovasin) -RGR retinal G protein coupled receptor -þþ: gene expression can be detected by two independent probes. þ: gene expression can be detected by one of two independent probes. -: negative gene expression.

(9)

of long-term memory

[57]

. In this study, green LED

irradiation-activated CREB, ERK1/2 and MAPK (

Fig. 5

A) supported that

activation of ERK/MAPK/p38 pathway may a consequence of

light-induced opsin-G protein cascade. OFSCs constitutionally express

OPN1SW, RRH and OPN3 (

Table 2

). OPN1SW is a visual opsin, while

the other two are non-visual opsins. Hence, RRH and OPN3

expression were selectively upregulated in migrated cells, but not

in non-migrated cells (

Fig. 7

A and B), suggesting that only RRH and

OPN3 are responsible for green LED irradiation-induced OFSC

migration. RRH and OPN3, the two non-visual opsins in OFSCs,

serve as the photoreceptors of green LED irradiation for the

acti-vation of ERK/MAPK/p38 signaling pathway during OFSC migration.

5. Conclusion

Green LED irradiation enhanced directional OFSC migration

away from light source through activation of ERK signaling

pathway. Increased ATP production facilitated kinase

phosphory-lation and the selectivity of phosphorylating on target kinases was

governed by phototransduction mediated by RRH and OPN3.

Pretreatment of OFSCs with green LED irradiation may serve

a useful platform for future studies regarding wound repair using

OFSCs.

Acknowledgments

The authors acknowledge the support of research grants from

the National Science Council (NSC 101-2314-B-038-022-MY3 to

JHH and OKL; NSC 98-2314-B-038-010-MY3,

NSC101-2120-M-010-002 to JHH, HFC and OKL; NSC 100-2911-I-010-503 to SC, OKL and

JHH; NSC100-2314-B-010-030-MY3, NSC101-2321-B-010-009, NSC

101-2911-I-010-503, and NSC 99-3114-B-002-005 to OKL). This

work was also supported in part by the UST-UCSD International

Center of Excellence in Advanced Bio-engineering sponsored by the

Taiwan National Science Council I-RiCE Program under Grant

Number: NSC100-2911-I-009-101. The authors also acknowledge

the

ﬁnancial support from Wan Fang Hospital, Taipei Medical

University (101swf02 to JHH and OKL).

References

[1] Mason RS, Sequeira VB, Gordon-Thomson C. Vitamin D: the light side of sunshine. Eur J Clin Nutr 2011;65:986e93.

[2] Chellappa SL, Gordijn MC, Cajochen C. Can light make us bright? Effects of light on cognition and sleep. Prog Brain Res 2011;190:119e33.

[3] Schmoll C, Lascaratos G, Dhillon B, Skene D, Riha RL. The role of retinal regulation of sleep in health and disease. Sleep Med Rev 2011;15:107e13. [4] Sung CH, Chuang JZ. The cell biology of vision. J Cell Biol 2010;190:953e63. [5] Zandi S, Kalia S, Lui H. UVA1 phototherapy: a concise and practical review.

Skin Ther Lett 2012;17:1e4.

[6] Walker D, Jacobe H. Phototherapy in the age of biologics. Semin Cutan Med Surg 2011;30:190e8.

[7] Schmidt-Erfurth U, Kiss C, Sacu S. The role of choroidal hypoperfusion asso-ciated with photodynamic therapy in neovascular age-related macular degeneration and the consequences for combination strategies. Prog Retin Eye Res 2009;28:145e54.

[8] Baratto L, Calza L, Capra R, Gallamini M, Giardino L, Giuliani A, et al. Ultra-low-level laser therapy. Lasers Med Sci 2011;26:103e12.

[9] Mills E. Environment. The specter of fuel-based lighting. Science 2005;308: 1263e4.

[10] Whelan HT, Smits Jr RL, Buchman EV, Whelan NT, Turner SG, Margolis DA, et al. Effect of NASA light-emitting diode irradiation on wound healing. J Clin Laser Med Surg 2001;19:305e14.

[11] de Morais NC, Barbosa AM, Vale ML, Villaverde AB, de Lima CJ, Cogo JC, et al. Anti-inﬂammatory effect of low-level laser and light-emitting diode in zymosan-induced arthritis. Photomed Laser Surg 2010;28:227e32. Fig. 7. Upregulation of RRH and OPN3 in OFSCs accompanied green LED irradiation induced cell motility OFSCs expressed three photosensitizers, which were RRH (A), OPN3 (B) and OPN1SW (C). After green LED irradiation, gene expressions of RRH (A) and OPN3 (B) were upregulated in migrated OFSCs, but not in non-migrated OFSCs. (C) OPN1SW was not sensitive to green LED irradiation.

(10)

[12] Barolet D. Light-emitting diodes (LEDs) in dermatology. Semin Cutan Med Surg 2008;27:227e38.

[13] He S, Nakada D, Morrison SJ. Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol 2009;25:377e406.

[14] Liang X, Bhattacharya S, Bajaj G, Guha G, Wang Z, Jang HS, et al. Delayed cutaneous wound healing and aberrant expression of hair follicle stem cell markers in mice selectively lacking Ctip2 in epidermis. PLoS One 2012;7: e29999.

[15] Wu Y, Wang J, Scott PG, Tredget EE. Bone marrow-derived stem cells in wound healing: a review. Wound Repair Regen 2007;15(Suppl 1):S18e26. [16] Fu X, Han B, Cai S, Lei Y, Sun T, Sheng Z. Migration of bone marrow-derived

mesenchymal stem cells induced by tumor necrosis factor-alpha and its possible role in wound healing. Wound Repair Regen 2009;17:185e91. [17] Huang C, Jacobson K, Schaller MD. MAP kinases and cell migration. J Cell Sci

2004;117:4619e28.

[18] Ryu CH, Park SA, Kim SM, Lim JY, Jeong CH, Jun JA, et al. Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction path-ways. Biochem Biophys Res Commun 2010;398:105e10.

[19] Liu W, Feng Y, Shang X, Zheng Y. Rho GTPases in hematopoietic stem/ progenitor cell migration. Methods Mol Biol 2011;750:307e19.

[20] Wang L, Yang L, Filippi MD, Williams DA, Zheng Y. Genetic deletion of Cdc42GAP reveals a role of Cdc42 in erythropoiesis and hematopoietic stem/ progenitor cell survival, adhesion, and engraftment. Blood 2006;107:98e105. [21] Ho JH, Ma WH, Tseng TC, Chen YF, Chen MH, Lee OK. Isolation and charac-terization of multi-potent stem cells from human orbital fat tissues. Tissue Eng Part A 2011;17:255e66.

[22] Chien MH, Bien MY, Ku CC, Chang YC, Pao HY, Yang YL, et al. Systemic human orbital fat-derived stem/stromal cell transplantation ameliorates acute inﬂammation in lipopolysaccharide-induced acute lung injury. Crit Care Med 2012;40:1245e53.

[23] Kim HK, Kim JH, Abbas AA, Kim DO, Park SJ, Chung JY, et al. Red light of 647 nm enhances osteogenic differentiation in mesenchymal stem cells. Lasers Med Sci 2009;24:214e22.

[24] Li WT, Leu YC, Wu JL. Red-light light-emitting diode irradiation increases the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells. Photomed Laser Surg 2010;28(Suppl 1):S157e65.

[25] Peng F, Wu H, Zheng Y, Xu X, Yu J. The effect of noncoherent red light irra-diation on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Lasers Med Sci 2012;27:645e53.

[26] Weick JP, Johnson MA, Skroch SP, Williams JC, Deisseroth K, Zhang SC. Functional control of transplantable human ESC-derived neurons via opto-genetic targeting. Stem Cells 2010;28:2008e16.

[27] Stroh A, Tsai HC, Wang LP, Zhang F, Kressel J, Aravanis A, et al. Tracking stem cell differentiation in the setting of automated optogenetic stimulation. Stem Cells 2011;29:78e88.

[28] Ho JH, Chen YF, Ma WH, Tseng TC, Chen MH, Lee OK. Cell contact accelerates replicative senescence of human mesenchymal stem cells independent of telomere shortening and p53 activation: roles of Ras and oxidative stress. Cell Transplant 2011;20:1209e20.

[29] Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, et al. An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci 1995;36:614e21.

[30] Ho JH, Chuang CH, Ho CY, Shih YR, Lee OK, Su Y. Internalization is essential for the antiapoptotic effects of exogenous thymosin beta-4 on human corneal epithelial cells. Invest Ophthalmol Vis Sci 2007;48:27e33.

[31] Papakonstanti EA, Stournaras C. Cell responses regulated by early reorgani-zation of actin cytoskeleton. FEBS Lett 2008;582:2120e7.

[32] Fukata M, Nakagawa M, Kaibuchi K. Roles of Rho-family GTPases in cell polarisation and directional migration. Curr Opin Cell Biol 2003;15:590e7. [33] Evers EE, Zondag GC, Malliri A, Price LS, ten Klooster JP, van der Kammen RA,

et al. Rho family proteins in cell adhesion and cell migration. Eur J Cancer 2000;36:1269e74.

[34] Schwartz PA, Murray BW. Protein kinase biochemistry and drug discovery. Bioorg Chem 2011;39:192e210.

[35] Tanaka N, Ogura T, Noguchi T, Hirano H, Yabe N, Hasunuma K. Phytochrome-mediated light signals are transduced to nucleoside diphosphate kinase in Pisum sativum L. cv. Alaska J Photochem Photobiol B 1998;45:113e21. [36] Rivera RM, Dahlgren GM, De Castro EPLA, Kennedy RT, Hansen PJ. Actions of

thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development. Reproduction 2004; 128:33e42.

[37] Terakita A. The opsins. Genome Biol 2005;6:213.

[38] Rau CS, Yang JC, Jeng SF, Chen YC, Lin CJ, Wu CJ, et al. Far-infrared radiation promotes angiogenesis in human microvascular endothelial cells via extracel-lular signal-regulated kinase activation. Photochem Photobiol 2011;87:441e6. [39] Shim JS, Kwon YY, Han YS, Hwang JK. Inhibitory effect of panduratin A on UV-induced activation of mitogen-activated protein kinases (MAPKs) in dermal ﬁbroblast cells. Planta Med 2008;74:1446e50.

[40] Nomura M, Kaji A, Ma WY, Zhong S, Liu G, Bowden GT, et al. Mitogen- and stress-activated protein kinase 1 mediates activation of Akt by ultraviolet B irradiation. J Biol Chem 2001;276:25558e67.

[41] Mochizuki-Oda N, Kataoka Y, Cui Y, Yamada H, Heya M, Awazu K. Effects of near-infra-red laser irradiation on adenosine triphosphate and adenosine diphosphate contents of rat brain tissue. Neurosci Lett 2002;323:207e10. [42] Lapchak PA, De Taboada L. Transcranial near infrared laser treatment (NILT)

increases cortical adenosine-5’-triphosphate (ATP) content following embolic strokes in rabbits. Brain Res 2010;1306:100e5.

[43] Chen J, Tucker CL, Woodford B, Szel A, Lem J, Gianella-Borradori A, et al. The human blue opsin promoter directs transgene expression in short-wave cones and bipolar cells in the mouse retina. Proc Natl Acad Sci U S A 1994;91:2611e5. [44] Nickle B, Robinson PR. The opsins of the vertebrate retina: insights from structural, biochemical, and evolutionary studies. Cell Mol Life Sci 2007;64: 2917e32.

[45] Kefalov VJ. Rod and cone visual pigments and phototransduction through pharmacological, genetic, and physiological approaches. J Biol Chem 2012; 287:1635e41.

[46] Blackshaw S, Snyder SH. Encephalopsin: a novel mammalian extraretinal opsin discretely localized in the brain. J Neurosci 1999;19:3681e90. [47] Halford S, Freedman MS, Bellingham J, Inglis SL, Poopalasundaram S, Soni BG,

et al. Characterization of a novel human opsin gene with wide tissue expression and identiﬁcation of embedded and ﬂanking genes on chromo-some 1q43. Genomics 2001;72:203e8.

[48] White JH, Chiano M, Wigglesworth M, Geske R, Riley J, White N, et al. Iden-tiﬁcation of a novel asthma susceptibility gene on chromosome 1qter and its functional evaluation. Hum Mol Genet 2008;17:1890e903.

[49] Lucas RJ, Hattar S, Takao M, Berson DM, Foster RG, Yau KW. Diminished pupillary light reﬂex at high irradiances in melanopsin-knockout mice. Science 2003;299:245e7.

[50] Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, Hankins MW, et al. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 2003;424:76e81.

[51] Fu Y, Zhong H, Wang MH, Luo DG, Liao HW, Maeda H, et al. Intrinsically photosensitive retinal ganglion cells detect light with a vitamin A-based photopigment, melanopsin. Proc Natl Acad Sci U S A 2005;102:10339e44. [52] Hara T, Hara R. Regeneration of squid retinochrome. Nature 1968;219:450e4. [53] Sun H, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J. Peropsin, a novel visual pigment-like protein located in the apical microvilli of the retinal pigment epithelium. Proc Natl Acad Sci U S A 1997;94:9893e8.

[54] Hao W, Fong HK. The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium. J Biol Chem 1999;274:6085e90. [55] Hollborn M, Ulbricht E, Rillich K, Dukic-Stefanovic S, Wurm A, Wagner L, et al. The human Muller cell line MIO-M1 expresses opsins. Mol Vis 2011; 17:2738e50.

[56] Koyanagi M, Takano K, Tsukamoto H, Ohtsu K, Tokunaga F, Terakita A. Jellyﬁsh vision starts with cAMP signaling mediated by opsin-G(s) cascade. Proc Natl Acad Sci U S A 2008;105:15576e80.

[57] Eckel-Mahan KL, Phan T, Han S, Wang H, Chan GC, Scheiner ZS, et al. Circadian oscillation of hippocampal MAPK activity and cAmp: implications for memory persistence. Nat Neurosci 2008;11:1074e82.