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HJ Lee, et al
584 Ann Dermatol
Received August 29, 2013, Revised October 30, 2013, Accepted for
publication November 6, 2013
Corresponding author: Dong Hyun Kim, Department of Dermatology,
CHA Bundang Medical Center, CHA University, 59 Yatap-ro,
Bundang-gu, Seongnam 463-712, Korea. Tel: 82-31-780-5240, Fax:
82-31-780-5247, E-mail: terios92@cha.ac.kr
T
his is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http:/
/
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Ann Dermatol Vol. 26, No. 5, 2014 http://dx.doi.org/10.5021/ad.2014.26.5.584
ORIGINAL ARTICLE
Efficacy of Microneedling Plus Human Stem Cell
Conditioned Medium for Skin Rejuvenation:
A Randomized, Controlled, Blinded Split-Face Study
Hee Jung Lee, Eo Gin Lee, Sangjin Kang
1
, Jong-Hyuk Sung
1,2
, Hyung-Min Chung
3,4
, Dong Hyun Kim
Department of Dermatology, CHA Bundang Medical Center, CHA University, Seongnam,
1
Department of Applied Bioscience, CHA University, Pocheon,
2
College of Pharmacy, Yonsei University, Incheon,
3
Stem Cell Research Laboratory, Department of Biomedical Science, CHA Stem Cell Institute, CHA University,
4
Department of Stem Cell Biology, Konkuk University School of Medicine, Seoul, Korea
Background: The use of growth factors in skin rejuvenation
is emerging as a novel anti-aging treatment. While the role of
growth factors in wound healing is well established, their use
in skin rejuvenation has only recently been to be studied and
no controlled trials have been performed. Objective: We
evaluated the anti-aging effects of secretory factors of
endothelial precursor cells differentiated from human em-
bryonic stem cells (hESC-EPC) in Asian skin. Methods: A total
of 25 women were included in this randomized, controlled
split-face study. The right and left sides of each participant’s
face were randomly allocated to hESC-EPC conditioned
medium (CM) or saline. To enhance epidermal penetration,
a 0.25-mm microneedle roller was used. Five treatment
sessions were repeated at 2-week intervals. Results: Phy-
sician’s global assessment of pigmentation and wrinkles after
treatment revealed statistically significant effects of micro-
needling plus hESC-EPC CM compared to microneedling
alone (p＜0.05). Skin measurements by Mexameter and
Visiometer also revealed statistically significant effects of
microneedling plus hESC-EPC CM on both pigmentation and
wrinkles (p＜0.05). The only minimal adverse event was
mild desquamation in one participant. Conclusion: Secre-
tory factors of hESC-EPC improve the signs of skin aging and
could be a potential option for skin rejuvenation. (Ann
Dermatol 26(5) 584∼591, 2014)
-Keywords-
Aging, Conditioned culture media, Embryonic stem cell,
Microneedling, Rejuvenation, Skin aging
INTRODUCTION
Skin aging includes pigmentary alterations, wrinkling,
thinning, and loss of elasticity owing to both genetic and
environmental factors. Various medical treatments and
topical cosmeceuticals are used to treat symptoms of
aging. However, the results have been unsatisfactory thus
far.
Endothelial precursor cells (EPCs) differentiated from
human embryonic stem cells (hESCs) demonstrated impro-
vement of blood perfusion in damaged tissues secreting
high levels of growth factors and cytokines
1,2
. Conditioned
medium (CM) of hESC-derived EPCs (hESC-EPCs), which
comprises several growth factors and cytokines, signi-
ficantly improved the proliferation and migration of der-
mal fibroblasts and epidermal keratinocytes as well as in-
creased collagen synthesis in fibroblasts
2
. In this respect,
growth factors may be beneficial for reducing signs of skin
aging
3
. Some growth factors also exhibit a whitening effect
by inhibiting melanogenesis
4
. However, the beneficial
role of growth factors for skin rejuvenation has only
recently been to be studied
5-7
, and no controlled clinical
Microneedling Plus hESC-EPC CM for Rejuvenation
Vol. 26, No. 5, 2014
585
Table 1. Multiplex cytokine analysis of secreted factors obtained
from conditioned medium of hESC-EPCs
hESC-EPC (pg/ml) EGM-2 (pg/ml)
EGF 10,168 9
FGF-2 421 61
Fractakine 1,372 133
GM-CSF 689 313
IL-6 4,294 2,463
IL-8 193,789 7
IL-9 302 9
IP-10 491 511
PDGF-AA 7,379 41
VEGF 4,092 42
hESC-EPC: human embryonic stem cell-derived endothelial pre-
cursor cell, EGM-2: endothelial growth medium-2, EGF: epider
-
mal growth factor, FGF-2: basic fibroblast growth factor, GM-
CSF: granulocyte macrophage colony stimulating factor, IL: inter
-
leukin, IP-10: interferon-inducible protein-10, PDGF: platelet
derived growth factor, VEGF: vascular endothelial growth factor.
EGM-2 means serum-free endothelial culture medium which is
used in culture of hESC-EPC, and was used as vehicle control
medium in Milliplex analysis.
trials have been performed.
Hydrophilic molecules larger than 500 Da have poor
penetration through the stratum corneum
8,9
. Most growth
factors are large hydrophilic molecules greater than 20
kDa; therefore, they are unlikely to penetrate the epidermis
in measurable quantities to produce pharmacologic effects.
This 12-week double-blinded randomized split-face study
was performed to investigate the effects of the secretory
factors of hESC-EPC on aged skin in Asians. Microneedling
was used to enhance the skin penetration of hESC-EPC
CM. An in vitro experiment to confirm the cutaneous
absorption of hESC-EPC CM after microneedling was also
performed. In particular, this study used diverse nonin-
vasive skin-measuring devices to objectively assess
changes in the biophysical properties of the skin following
hESC-EPC CM treatment.
MATERIALS AND METHODS
Preparation of conditioned medium and multiplex
cytokine assay
We used commercially available CM of hESC-EPCs, which
were generated as described previously
10
. The multiplex
cytokine array was performed using the Milliplex and
Luminex systems (Millipore; Luminex Corp., Austin, TX,
USA) with concentrated CM; this system can be used to
analyze all or any combination of cytokines and
chemokines in tissue/cell lysate and culture supernatant
samples by using a microbead-based tagging system
2
.
Endothelial growth medium-2 (EGM-2; Lonza, Walker-
sville, ML, USA) was used as control medium. The mul-
tiplex cytokine analysis of CM revealed that hESC-EPCs
strongly expressed several growth factors including epi-
dermal growth factor (EGF), fibroblast growth factor-2
(FGF-2), fractalkine, granulocyte macrophage colony-
stimulating factor (GM-CSF), interleukin-6 (IL-6), platelet-
derived growth factor-AA (PDGF-AA), and vascular
endothelial growth factor (VEGF) (Table 1).
Cutaneous absorption experiment
To confirm transdermal absorption, proteins in hESC-EPC
CM were labeled by using the Alexa Fluor 488 Protein
Labeling Kit (Invitrogen Co., Carlsbad, CA, USA) accor-
ding to the manufacturer’s protocol. Briefly, Alexa Fluor
488 reactive dye was mixed with 1 mg protein in 1.5 ml
of 0.1 M sodium bicarbonate buffer. After 1 hour, the
unreacted dye was separated by using a purification resin
column. Female miniature pig skins (Medi Kinetics Micro-
pigs; Medi Kinetics Co., Ltd, Busan, Korea) were micro-
needled using 0.25-mm-long microneedles. Fluorescence
dye-protein conjugates were subsequently applied to the
skin and incubated for 1 hour. Tissues were immediately
embedded in frozen sectioning compound (Leica Micro-
systems GmbH, Wetzlar, Germany) in liquid nitrogen.
The sections were approximately 16 μ thick and were
dried overnight at room temperature. The slides were
washed several times with PBS and incubated with Flu-
oroshield Mounting Medium with DAPI (ImmunoBio-
Science Co., Mukilteo, WA, USA). Images were captured
by a Nikon fluorescence microscope (Nikon, Tokyo,
Japan) at excitation wavelengths of 405 nm and 488 nm
and merged.
Study design and participants
Twenty-five participants were recruited for this prospective
randomized controlled observer-blinded split-face study.
Participants were 41∼64 years old (mean, 51.6 years) and
had Fitzpatrick Skin Type III or IV. The exclusion criteria
were as follows: use of bleaching creams, history of any
skin rejuvenation treatment within 6 months, history of
keloids, and active eczema. The study protocol and
informed consent form were submitted to and approved
by the CHA University institutional review board (PBC10-
062).The participants were informed of the benefits, risks,
and possible complications of the treatment before en-
rollment; all provided informed consent prior to parti-
cipation.
The left and right sides of the face of each participant were
randomly assigned to treatment with microneedling alone
(control) or microneedling plus hESC-EPC CM. The ran-
HJ Lee, et al
586 Ann Dermatol
domization procedure involved sealed envelopes numbered
1∼25 in which the allocation was indicated. The ran-
domization was based on a computer generated random
list (GraphPad Software Inc., La Jolla, CA, USA) created by
an independent cooperator, and envelopes were opened
in ascending order. All participants and 2 dermatologists
assessing outcomes were blinded until all the participants
finished final assessments.
Participants received 5 treatments at 2-week intervals.
First, the face was anesthetized by topical 4% lidocaine
cream (LMX4; Ferndale Laboratories Inc., Ferndale, MI,
USA) approximately 30 minutes before the procedure.
The face was cleansed with a mild soap and 70% alcohol.
For the microneedling alone treatment, 1.5 ml of normal
saline was painted on the skin, and 2 passes of micro-
needling with dermarollers (0.25-mm DTS roller; TCellBio,
Seoul, Korea) were performed. The endpoint of treatment
was the presence of uniform erythema over the face. For
the microneedling plus hESC-EPC CM treatment, 1.5 ml of
hESC-EPC CM was painted on the face and microneedling
was performed in the same manner. An epidermal cooling
device (CARESYS; Danil SMC, Seoul, Korea) was used to
relieve pain and erythema after microneedling therapy.
Clinical assessments
Participants were assessed at baseline and 2 weeks after
final treatment (12 weeks). Photographs taken by a digital
camera (Nikon D90; Nikon) were obtained at each visit.
For self-assessment, participants answered questionnaires
regarding efficacy and adverse events 12 weeks after study
initiation. The questionnaires included grading of overall
treatment satisfaction from 0 (dissatisfied) to 5 (most
satisfied). In addition, participants were asked to report
any side effects during the study. Objective clinical
assessments consisted of 2 dermatologists blinded to the
study design and treatment comparing pre- and post-
treatment photographs separately on each side of the face.
The evaluations were graded by quartile as follows: grade
1, 0%∼25%, minimal to no improvement; grade 2, 26%∼
50%, moderate improvement; grade 3, 51%∼75%, marked
improvement; and grade 4, 75%∼100%, near total im-
provement.
Non-invasive objective skin color measurements
Prior to all measurements, participants were acclimatized
to a temperature- (20
o
C) and humidity-controlled (40%)
room, and the instruments were calibrated according to
the manufacturer’s instructions. A narrow-band simple
reflectance meter, Mexameter (MX18; Courage＋Khazaka
Electronic GmbH, Köln, Germany), was used to quanti-
tatively evaluate color changes after treatments; this
instrument uses arrays of light emitting diodes (LEDs) that
emit light at 3 defined wavelengths: 568 (green), 660
(red), and 880 nm (infrared). The melanin index (MI) and
erythema index (EI) were measured in triplicate on the
same malar area on each side of the face, and mean
values were used for analysis.
Non-invasive objective wrinkle measurements
To evaluate the effects of treatments on collagen regener-
ation, each participant’s periorbital wrinkles were objectively
measured by using a skin replica and microrelief
instrument (Visiometer SV600; Courage＋Khazaka Electronic
GmbH) at baseline and 12 weeks. The Visiometer SV600
can measure skin roughness and the depth of furrows by
measuring the light transmission through a very thin skin
replica. The roughness parameters investigated in this
study were R2 (maximum roughness) and R3 (average
roughness).
Statistical analysis
Paired t-tests were performed to analyze temporal changes
in all parameters at each visit. Data were analyzed using
SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). The level of
significance was set at p＜0.05.
RESULTS
Cutaneous absorption experiment
The proteins in hESC-EPC CM labeled with the Alexa
Fluor 488 Protein Labeling Kit were visualized in both the
epidermis and dermis (Fig. 1).
Clinical assessments
All 25 participants completed the 12-week study protocol.
No serious adverse events were encountered. Mild pain
and temporary erythema during and after treatments were
tolerable in all participants. The only minimal adverse
event reported in one participant was mild desquamation,
which resolved spontaneously within 1 week. Participants’
overall satisfaction scores for microneedling alone and
microneedling plus hESC-EPC CM were 2.72±1.45 and
3.25±1.26, respectively (p＜0.05; Table 2). The mean
grades of objective clinical improvement of pigmentation
based on photographs for microneedling alone and
microneedling plus hESC-EPC CM were 1.32±0.62 and
1.54±0.57, respectively (p＜0.05; Table 2). The mean
grade of objective clinical improvement of wrinkles based
on photographs for microneedling alone and microneedling
plus hESC-EPC CM were 1.49±0.48 and 1.92±0.42,
respectively (p＜0.05; Table 2). Representative photographs
showed greater improvements in wrinkles and dilated
Microneedling Plus hESC-EPC CM for Rejuvenation
Vol. 26, No. 5, 2014
587
Fig. 1. Transdermal penetration of human embryonic stem cell-derived endothelial precursor cell (hESC-EPC) conditioned medium
(CM). (A∼C) microneedling alone (control); (D∼F) microneedling plus hESC-EPC CM. Proteins in hESC-EPC CM labeled with Alexa
Fluor 488 (green color) are visible in both the epidermis and dermis. Scale bar＝200 μm.
Table 2. Clinical assessments
Microneedling alone Microneedling plus hESC-EPC
Participant’s overall satisfaction scores 2.72±1.45 3.25±1.26
Physician’s global assessments for pigmentation 1.32±0.62 1.54±0.57
Physician’s global assessments for wrinkle 1.49±0.48 1.92±0.42
Participant’s overall satisfaction scores: from 0, not satisfied; to 5, most satisfied. Physician’s global assessments: grade 1, 0%∼25%
＝
minimal to no improvement; grade 2, 26%∼50%＝moderate improvement; grade 3, 51%∼75%＝marked improvement; and grade
4, 75%∼100%＝near total improvement. hESC-EPC: human embryonic stem cell-derived endothelial precursor cell.
pores following microneedling with hESC-EPC CM than
microneedling alone (Fig. 2, 3).
Pigmentation improvement
At baseline, the MI determined by Mexameter revealed no
statistically significant difference in pigmentation between
microneedling alone and microneedling plus hESC-EPC
CM (p＝0.15). However, treatment with microneedling
plus hESC-EPC CM resulted in a significantly greater
decrease in the MI than microneedling alone (p＜0.05,
Fig. 4A). The mean MI of the sides treated with micro-
needling alone decreased from 143±11.1 at baseline to
136±12.8 two weeks after the final session (p＝0.052).
Meanwhile, the mean MI of the microneedling plus
hESC-EPC CM sides decreased significantly from 138±
14.2 at baseline to 113±12.1 two weeks after the final
session (p＜0.05), demonstrating the efficacy of hESC-EPC
CM for skin lightening.
Erythema improvement
Treatment with microneedling plus hESC-EPC CM signifi-
cantly decreased the EI from 271±24.2 at baseline to
HJ Lee, et al
588 Ann Dermatol
Fig. 2. Photographs at baseline and after 5 treatments. Clinical photos showed greater improvements of wrinkles and dilated pores
following microneedling with human embryonic stem cell-derived endothelial precursor cell conditioned medium (A, baseline; B,
after 5 treatments) than microneedling alone (C, baseline; D, after 5 treatments).
243±21.1 two weeks after the final session (p＜0.05, Fig.
4B). The mean EI of the sides treated with microneedling
alone decreased from 268±25.1 at baseline to 255±29.8
two weeks after the final session (p＞0.05).
Wrinkle improvement
Treatment with microneedling plus hESC-EPC CM resulted
in a significantly greater decrease in the R2 and R3 values
measured by Visiometer than microneedling alone (p＜
0.05; Fig. 4C, D). The mean R2 value of the sides treated
with microneedling alone decreased from 0.52±0.07 at
baseline to 0.50±0.1 two weeks after the final session,
but the changes were not significant (p＞0.05). Of note,
the R2 values of the sides treated with microneedling plus
hESC-EPC CM decreased significantly from 0.58±0.1 at
baseline to 0.46±0.09 two weeks after the final session (p＜
0.05). Similarly, the mean R3 value of the sides treated
with microneedling alone decreased from 0.38±0.06 at
baseline to 0.34±0.1 two weeks after the final session (p＝
0.51). Meanwhile, the mean R3 values of the sides treated
with microneedling plus hESC-EPC CM decreased signi-
ficantly from 0.4±0.1 at baseline to 0.31±0.06 two weeks
after the final session (p＜0.05).
DISCUSSION
Skin aging is mediated by the effects of both the natural
aging process (i.e. intrinsic aging) and environmental
factors (i.e. extrinsic aging) on cellular and extracellular
components. Cell-based therapies using the body’s own
stem cells and growth factors have recently been used as
an alternative therapeutic strategy to repair damaged
tissue, including skin rejuvenation. Stem cells may exert
their beneficial effects on tissue regeneration through
complex paracrine mechanisms in addition to their pro-
posed direct cellular effect
11
. Furthermore, stem cells
Microneedling Plus hESC-EPC CM for Rejuvenation
Vol. 26, No. 5, 2014
589
Fig. 3. Photographs focusing on the periorbital areas at baseline and after 5 treatments. Periorbital wrinkles exhibited greater
improvements following microneedling with human embryonic stem cell-derived endothelial precursor cell conditioned medium (A,
baseline; B, after 5 treatments) than microneedling alone (C, baseline; D, after 5 treatments).
synthesize and secrete a variety of extracellular matrix
proteins, cytokines, growth factors, and other bioactive
proteins that contribute to the healing process; the local
environment created by these secreted factors may govern
the fate and function of individual stem cells
11-13
.
A previous study revealed that hESC-EPC CM accelerates
wound healing and increases the tensile strength of
wounds after topical treatment and subcutaneous injec-
tion
1
. In vitro, hESC-EPC CM significantly improved the
proliferation and migration of dermal fibroblasts and
epidermal keratinocytes, and also increased collagen
synthesis by fibroblasts
2
. Analysis of hESC-EPC CM by
using a multiplex cytokine array system revealed that
hESC-EPCs secrete cytokines and chemokines such as
EGF, bFGF, fractalkine, GM-CSF, IL-6, IL-8, PDGF-AA, and
VEGF, which are important in normal angiogenesis and
wound healing
2
. In addition, conditioned media from
adipose-derived stem cells (ADSC CM) have shown to
inhibit melanogenesis by downregulating tyrosinase and
tyrosinase-related protein-1 expression in B16 melanoma
cells, demonstrating the whitening effects of ADSCs
14
.
Similarly, hESC-EPC CM inhibited melanogenesis in B16
melanoma cells (unpublished data). Therefore, we hypo-
thesized that hESC-EPC CM improves the signs of skin
aging such as wrinkles and pigmentation. The present
study demonstrated that 5 sessions of hESC-EPC CM
application significantly improved skin pigmentation and
wrinkles.
Transdermal penetration and epidermal-dermal commun-
ication are important regarding the cutaneous application
of growth factors for skin rejuvenation. Despite the 500-
Da rule for the skin penetration of chemical compounds,
there are several potential routes by which small quan-
tities of large molecules can penetrate the stratum cor-
neum, such as the follicular route
15,16
. Vaccines larger
than 100 kDa were recently found to be able to exert an
immunologic response when applied topically, probably
because of the penetration of a very small amount of
HJ Lee, et al
590 Ann Dermatol
Fig. 4. Objective non-invasive skin measurements. (A) Pigmentation; (B) erythema; (C, D) wrinkles. R2: maximum roughness (C); R3:
average roughness (D). *p＜0.05, post-treatment comparison between microneedling alone and microneedling plus human embryonic
stem cell-derived endothelial precursor cell (hESC-EPC) conditioned medium (CM); **p＜0.05, pre- and post-treatment in microneedling
plus hESC-EPC CM.
protein through intact skin
17
. Penetration into the upper-
most layer of viable epidermal keratinocytes may produce
a signaling cascade of growth factors that affects cells
deeper in the dermis such as fibroblasts.
In the present study, microneedling was performed to
enhance the skin penetration of hESC-EPC CM. Micro-
needling, which is a collagen-induction therapy, is a
method that creates pinhole wounds using many micro-
needles. It stimulates wound healing and improves scars
and wrinkles
18
. Moreover, microneedles have been used
in transdermal and dermal drug delivery for more than a
decade
19
. We confirmed that proteins in hESC-EPC CM
can directly penetrate the epidermis when combined with
0.25-mm microneedling. Therefore, the presence of
hESC-EPC CM in the dermis is expected to exert direct
effects on the dermal extracellular matrix.
The main limitations of this study are the small number of
participants and lack of the long-term follow-up after final
treatment. However, the randomized split-face study
design enhances the reliability of the data as it enabled us
to obtain significant results with a relatively small group of
participants.
Although there are reports of the roles of soluble factors of
stem cells in photoaging, this is the first randomized
controlled study, which demonstrates the efficacy of the
soluble factors of stem cells on skin rejuvenation in vivo.
ACKNOWLEDGMENT
This study was supported by a grant of the Korean Health
Technology R&D Project, Ministry of Health & Welfare,
Republic of Korea (#A110159).
Microneedling Plus hESC-EPC CM for Rejuvenation
Vol. 26, No. 5, 2014
591
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