Accepted: 12 September 2017

DOI: 10.1111/exd.13451

LETTERS TO THE EDITOR

Exosomes from human adipose-­derived stem cells promote

proliferation and migration of skin fibroblasts
Abstract 2 | QUESTIONS ADDRESSED
This study was undertaken to evaluate whether exosomes from
human adipose-­derived stem cells (ASC-­exo) can stimulate the re- We asked whether ASC-­derived exosomes can stimulate the skin der-
generation of human dermal fibroblasts (HDFs). Immunoblotting mal fibroblasts’ proliferation and migration, and by what mechanism
and FACS analyses showed that ASC-­exo was positive for exosome these exosomes play such functions.
markers. Fluorescence tracking revealed that the contents of ASC-­
exo were transferred into the HDFs. ASC-­exo treatment also stimu-
lated the proliferation and migration of HDFs in a dose-­dependent 3 | EXPERIMENTAL DESIGN
manner. Similarly, the expression levels of genes involved in skin
cell proliferation were increased by ASC-­
exo. Microarray analy- See supplementary information.
sis showed an enrichment of microRNAs that have regenerative
function. We suggest that the ASC-­
exo can stimulate skin cell
proliferation. 4 | RESULTS

Transmission electron microscopy (TEM) and nanoparticle tracking

1 | BACKGRO UND analysis (NTA) showed that ASC-­exo isolated from the supernatants

It has been well reported that adipose tissue-­d erived stem cell
(ASC) secretes various soluble factors, that is growth factors,
cytokines, exosomes that can rescue damaged cells. [1] Exosome,
a 30-­ to 150-­n m-­s ized extracellular vesicle that is secreted from
most cell types, is formed within endosomal compartments and
released into the extracellular milieu. [2] It has been demon-
strated that exosomes possess similar functional properties of
mesenchymal stem cells (MSCs) from which they are derived, [3]
and due to such functions, exosomes are regarded now as a
communicator between tissues. [4] Functionally, exosomes can
reduce the immune recognition, so that the integrity of cell
membrane can be maintained. [5] A study of stem cell transplan-
tation therapy demonstrated that the role of MSCs in cell-­t o-­
cell communication was exerted through a paracrine mechanism
and that exosomes play a major role in this process. [6] Other
findings also described that the conditioned media of ASC
(ASC-­C M) [7] or exosomes [8] were able to promote the migration
of dermal fibroblasts during the process of wound healing. [7] F I G U R E   1   Proliferation and migration effect of human ASC-­exos
on HDFs. (A) Gene expression profiling of collagen type 1, elastin,
Although the mechanism of the exosomes’ role on proliferation
KGF, CD34 and VEGF in HDFs treated with vehicle and those treated
and migration of fibroblasts was demonstrated, [8] no study has either 5 or 10 μg/mL of ASC-­exo (*P < .05). (B) Assessment of the
been conducted to identify the expressional changes of microR- migratory capacity of HDFs after being incubated with 5 or 10 μg/mL
NAs, which play major role in various cellular responses, includ- of ASC-­exo. Assays were repeated three times. (C) The effect of ASC-­
ing proliferation. exo treatment on HDF cell cycle (*P < .05) (N = 3)

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© 2017 The Authors. Experimental Dermatology Published by John Wiley & Sons Ltd

1170  |  wileyonlinelibrary.com/journal/exd Experimental Dermatology. 2018;27:1170–1175.

LETTERS TO THE EDITOR
F I G U R E   2   (A) Scatter plot showing the microRNAs that
are differentially expressed between ASC and ASC-­exo. The
x-­axis and y-­axis represent the normalized expression level of
ASC ASC-­exo, respectively. Each miRNA, having a fold change
more than two, is represented by a dot. (B) Heatmap analysis
of microarray results showing hierarchical clustering of 292
differentially expressed miRNAs between ASC and ASC-­exo.
Red (high) or green (low) colours indicate upregulated or
downregulated microRNA intensity. Mean signal values were
background-­corrected and 0. Genes showing a minimum of 2-­fold
changes with P value <.05 at the 95% confidence level were
considered as significant. (C) Biological processes abundantly
expressed by microRNAs. The 718 validated target genes
of the top 10 upregulated miRNAs in ASC-­exo are grouped
according to the gene ontology (GOTERM_BP_FAT) with an
enrichment EASE score < 0.05 and more than 10 target genes.
(D) Schematic representation of miRNAs with their validated
targets contributing to reduced cellular senescence and, ageing-­
associated genes, deregulated cell growth, ageing-­related disease.
NPM1, nucleophosmin; PDCD4, programmed cell death 4; CCL5,
C-­C motif chemokine ligand 5; NUP62, nucleoporin 62 kDa
of the ASC culture showed the typical morphology of exosomes that
are within the range of 70-­150 nm (Fig. S1A). Western blot analysis
revealed that ASC-­exo expresses exosome markers HSP70, CD63
and CD9 (Fig. S1B), while these markers were absent in ASC lysate.
Flow cytometry analysis also showed that ASC-­exos were positive for
CD9, CD63 and CD81 (Fig. S1C). We next asked whether the con-
tents of ASC-­exos could be transmitted to HDFs. After co-­incubation,
|
      1171
for 24 hours, of PKH26-­labelled ASC-­exos with HDFs, it was found
that red fluorescence of PKH26 was localized within the cytoplasm
of HDFs (Fig. S1D), showing that ASC-­exo can be internalized into
HDFs. Also, quantitative real-­time PCR showed that the expression
of genes related with skin regeneration, such as CD34, collagen type
1, elastin and keratinocyte growth factor (KGF), was increased after
being treated with ASC-­exos in a dose-­dependent manner (Figure 1A).
We also found that ASC-­exo stimulated the proliferation of HDFs
and that such effect was significantly higher in 10 μg/mL compared
with those treated with 5 μg/mL (Fig. S2). In line with this finding, we
found that the migration of HDFs was increased in a similar manner
(Figure 1B). Similarly, cell cycle analysis by FACS demonstrated that
ASC-­exo treatment significantly increased the population of those
within S-­phase (Figure 1C).
We next conducted a comprehensive analysis of microRNA ex-
pression between ASC and ASC-­exo by microarray. We selected the
microRNAs expressed differentially (>2-­folds, Figure 2A) and found
that 292 among 333 microRNAs were differentially expressed (199
and 93 upregulated and downregulated, respectively, in ASC-­
exo)
(Figure 2B). These differentially expressed microRNAs were grouped
based on function, as referenced with miRTarBase (http://http://mir-
tarbase.mbc.nctu.edu.tw/). The 718 validated gene targets of top 10
upregulated miRNAs were grouped according to the gene ontology
(GOTERM_BP_FAT) with an enrichment EASE score < 0.05 and 10
more than number of target genes (Figure 2C). The role of ASC-­exo
microRNAs with their corresponding validated target genes was sche-
matically represented in Figure 2D. From this results, it can be con-
cluded that exosomes from ASC contain microRNAs that can inhibit
genes including NPM1, PDCD4, CCL5 and NUP62, thereby contribut-
ing to the regeneration of skin fibroblasts by stimulating the prolifera-
tion of dermal fibroblasts.
5 | CONCLUSION
Recent studies have demonstrated that exosomes are involved in the
paracrine activity of stem cells.[9] This study demonstrates that the
exosomes derived from ASCs play a prosurvival role in human dermal
fibroblasts by stimulating its cell cycle and that microRNAs that are
known to have regenerative potentials were enriched in ASC-­exo. Our
finding may be of useful for developing novel therapeutic methods for
the enhancing wound healing.
CO NFL I C T O F I NT ER ES T
The authors have no conflict of interest to declare.
AU T HO R CO NT R I B U T I O NS
EW Choi performed the research, designed the study, analysed the
data and wrote the manuscript. MK Seo, EY Woo, SH Kim and EJ.
Park designed the study and performed the research. S Kim edited
the manuscript.
 |
1172       LETTERS TO THE EDITOR
Keywords
extracellular vesicle, microRNA array, skin regeneration, wound
healing
O RCI D
Sue Kim  http://orcid.org/0000-0002-1818-0551
Eun Wook Choi
Min Koo Seo
Eun Young Woo
Suk Hyung Kim
Eun Joo Park
Sue Kim
Prostemics Research Institute, Seongdong-gu, Seoul, South Korea
Correspondence
Sue Kim, Stem Cell Center, ASAN Institute for Life Sciences, ASAN
Medical Center, Songpa-gu, Seoul, South Korea.
Email: sue2949@gmail.com
REFERENCES
[1] W. S. Kim, B. S. Park, S. H. Park, H. K. Kim, J. H. Sung, J. Dermatol. Sci.
2009, 53, 96.
[2] A. Marote, F. G. Teixeira, B. Mendes-Pinheiro, A. J. Salgado, Front
Pharmacol. 2016, 7, 231.
[3] R. C. Lai, F. Arslan, M. M. Lee, N. S. Sze, A. Choo, T. S. Chen, M. Salto-
Tellez, L. Timmers, C. N. Lee, R. M. El Oakley, G. Pasterkamp, D. P. de
Kleijn, S. K. Lim, Stem Cell Res. 2010, 4, 214.
[4] N. Vyas, J. Dhawan, Cell. Mol. Life Sci. 2017, 74, 1567.
[5] P. Vader, E. A. Mol, G. Pasterkamp, R. M. Schiffelers, Adv. Drug Deliv.
Rev. 2010, 106, 148.
Accepted: 26 September 2017
DOI: 10.1111/exd.13453
RNA-­seq analysis of Lgr6+ stem cells and identification of an
Lgr6 isoform
Abstract
We studied Lgr6+ stem cells in experimental UV carcinogenesis in
hairless mice. For further characterization through RNA-­seq, these
stem cells were isolated by FACS from transgenic hairless mice bear-
ing an EGFP-Ires-CreERT2 reporter cassette inserted into exon 1 of
the Lgr6 gene (purity confirmed by human ERT2 expression). Between
Lgr6/EGFP+ and Lgr6/EGFP− basal cells (Tg/wt), 682 RNAs were dif-
ferentially expressed, indicating stemness and expression of cancer-­
related pathways in Lgr6/EGFP+ cells. We discovered that suspected
“Lgr6 null” mice (Tg/Tg) expressed RNA of an Lgr6 isoform (delta-­Lgr6,
lacking 74 N-­terminal aa) which could be functional and explain the
lack of a phenotype.
[6] S. Raik, A. Kumar, S. Bhattacharyya, Biotechnol. Appl. Biochem. 2017,
https://www.ncbi.nlm.nih.gov/pubmed/28321921. [Epub ahead of
print].
[7] W. S. Kim, B. S. Park, J. H. Sung, J. M. Yang, S. B. Park, S. J. Kwak, J. S.
Park, J. Dermatol. Sci. 2007, 48, 15.
[8] L. Hu, J. Wang, X. Zhou, Z. Xiong, J. Zhao, R. Yu, F. Huang, H. Zhang, L.
Chen, Sci. Rep. 2016, 6, 32993.
[9] G. D. Kusuma, J. Carthew, R. Lim, J. E. Frith, Stem Cells Dev. 2017, 26,
617.
S U P P O RT I NG I NFO R M AT I O N
Additional Supporting Information may be found online in the
­supporting information tab for this article.
FIGURE S1 Characterization of human ASC-­exos. (A) A representative
image of the size-­distribution plot of NTA, and the inlet is an image of
TEM analysis. (B) Detection of HSP70, CD63, and CD9 in ASC-­exos by
Western blot analysis. (C) Flow cytometry analysis showing that ASC-­
exo are positive for CD9, CD63 and CD81. (D) The uptake of internal
contents of ASC-­exo into HDFs. PKH-­26-­labled ASC-­exos were incu-
bated with HDFs for 24 h and the fluorescence was detected
FIGURE S2 Effect of ASCs derived exosomes on the cell growth of
HDFs. HDFs were treated with 5 μg/mL or 10 μg/mL of ASCs derived
exosomes (ASC-­Exo) for 48 h and HDFs proliferation was determined
by MTS assay (n = 3). Data are expressed as the means ± SD. Asterisk
indicates a statistically significant difference between two groups of
cells (P < .05)
TABLE S1 Primers used in the real-­time RT-­PCR analysis
APPENDIX S1 Experimental design
DATA S1 ASC and ASC-exo miRNA array results
1 | INTRODUCTION
We investigated the fate of a novel class of Lgr6+ proliferative stem
cells in UV carcinogenesis in hairless mice, a model closely mimick-
ing the development of cutaneous squamous cell carcinoma in man.[1]
Using transgenic mice with an EGFP-Ires-CreERT2 reporter cassette
inserted into exon 1 of Lgr6, we found—similar to what was found in
haired mice[2]—that Lgr6/EGFP+ stem cells appeared in large clusters in
the interfollicular epidermis (IFE) and in very small clusters in the rem-
nants of hair follicles of hairless mice.[3] Contrary to our expectation,
the Lgr6/EGFP+ cells dwindled in numbers in the hyperplastic IFE under
chronic UV exposure and did not become tumor-­initiating cells (TICs).