Page 90 - EXOSOMES

1426 uMSC Exosomes Suppress Scar Formation

SMAD2 (p-SMAD2) levels have been proposed as a positive prog- dressing. Skin contracture was serially assessed at 10, 14, and 25 days
nostic marker in myofibroblast differentiation [2, 5]. In this regard, by measuring passive extension and skin collection at 25 days.
interfering with the activity of the TGF-b/SMAD2 signaling path- For uMSC-Exo treatment of the model, HydroMatrix (Sigma-
way might suppress myofibroblast differentiation and overaggre- Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) was used as
gation to reduce excessive fibrosis or scar formation. a scaffold, and uMSC-Exo was dissolved into the hydrogel following
Mesenchymal stem cells (MSCs) have been reported to be suit- the manufacturer’s instructions. In brief, 100 mg/ml uMSC-Exo dis-
ablefortreatingtissuedefectsandexcessivefibrosisbecauseoftheir solved in phosphate-buffered saline (PBS) is prepared in one tube
ability to migrate to the site of injury, their potential to differentiate first, and 1% (10 mg/ml) hydrogel is prepared using sterile water
into cells needed for tissue repair, and their relative ease of expan- in another tube. The two components were mixed immediately at
sion in vitro. Nevertheless, with the recognition that only a small a ratio of 1:1 and injected around the wound 48 hours after wound-
number of MSCs are retained in the injury site after MSC treatment, ing. PBS, HEK-293-exosome (100 mg/ml), and uMSC exosome-free
many investigators [6], including our group, have suggested that a supernatant(UEFS;theconcentratedmediumleftafterexosomere-
strongparacrinecapacityofMSCsmightbetheprincipalmechanism moval) served as controls. At the indicated time points, the wounds
responsible for the clinical benefits of stem cell-based therapies [7]. were photographed and quantified using Adobe Photoshop soft-
Recently, exosomes have been identified as a new kind of ma- ware (Adobe Systems, New York, NY, http://www.adobe.com).
jor paracrine factor released by the outward budding of various
types of cells, including MSCs, and important for various cellular Cell Culture
functions. Exosomes are a type of membrane vesicle with diame-
Umbilical cords and skin were obtained from the Changhai Hos-
ters of 40–150 nm that are surrounded by a phospholipid bilayer
pital affiliated to the Second Military Medicine University. Pri-
[8]. They have been reported to be an important mediator of
mary culture of uMSCs and dermal fibroblasts were established
cell-to-cell communication. They protect the bioactive substances
using standard procedures. In brief, umbilical cords and skin were
they carry from high temperatures, a variety of pH environments,
washed with Dulbecco’s modified Eagle’s medium (DMEM;
repeated freezing and thawing, and other adverse conditions. Exo-
HycloneLaboratories;ThermoFisherScientificLifeSciences,Waltham,
somes have been found to play key roles in normal physiology and
MA, http://www.thermofisher.com) to remove excess blood.
in diseases such as myocardial fibrosis, renal fibrosis, and hepatic After another wash with 70% ethanol, the tissues were minced
fibrosis [9–12]. However, it is still unclear precisely whether into small pieces (2–4 mm) and incubated with standard culture
MSC-derived exosomes can mediate myofibroblast functions, es- medium in dishes at 37°C. When the fibroblasts and uMSCs
pecially in the process of wound repair.
reached 80% confluence, they were trypsinized and prepared
PreviousstudieshaveimplicateduMSC-derivedexosomes(uMSC-
for subculture. Thereafter, the medium was changed every 3
Exos), which contain proteins, mRNAs, and microRNAs (miRNAs), to
days. Only uMSCs and fibroblasts in passages 2–5wereused.
have functions in diverse biological processes [13–15]. We identified
HEK293T cells were purchased from American Type Culture Col-
thatuMSC-Exos-derivedmiRNAsmainlyfunctionthroughsuppressing
lection (ATCC, Manassas, VA, http://www.atcc.org) and main-
the differentiation of fibroblasts to myofibroblasts. In the present
tained in DMEM (Thermo Fisher Scientific Life Sciences)
study, through high-throughput sequencing, we identified a group
containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific
of specific miRNAs carried by uMSC-Exos as key components contrib-
Life Sciences) at 37°C with 5% CO 2 . Fibroblast culture medium
uting to the fibroblast/myofibroblast transition by inhibiting excess
consisted of high-glucose DMEM supplemented with 10% FBS
a-smooth muscle actin (a-SMA) and collagen deposition associated
and 100 mg/ml streptomycin and penicillin (Thermo Fisher Scien-
with activity of the TGF-b/SMAD2 signaling pathway. Thus, our find-
tific Life Sciences). uMSC culture medium consisted of CMRL
ings suggest that applying the uMSC-derived exosomes could be a po-
(Connaught Medical Research Laboratories developed medium,
tentialstrategytopreventscarformationoreventissuefibrosisduring
Thermo Fisher Scientific Life Sciences) with 10% FBS, 2%
wound healing in patients.
antibiotic-antimycotic solution, and 1% L-glutamine.
Exosome Isolation
MATERIALS AND METHODS
Before isolation, the FBS used was depleted of host exosomes by ul-
Mouse Model tracentrifugation at 120,000g for 3 hours at 4°C. Cell suspension me-
All procedures using animal subjects were performed under an insti- dium was collected every 2 days. Collected culture suspension was
tutionally approved protocol deemed in accordance with the guide- transferredtoconical tubes for centrifugation at 300g for 10 minutes
lines of the Institute of Laboratory Animal Resources, the Second at 4°C to pellet the cells. The supernatant was again centrifuged at
Military Medical University. Mice were obtained from the Shanghai 16,500g for 20 minutes at 4°C to further remove cell debris. The su-
Laboratory Animal Research Center (SIPPR-BK Laboratory Animal pernatantwasthenfilteredthrougha0.22-mmfilterandtheflowwas
Corp., Shanghai, China, http://www.sippr.org.cn) and then housed transferredtonewtubesandthenultracentrifugedagainat120,000g
in a specific pathogen-free environment with 12-hour photoperiods for 70 minutes at 4°C in a SW32Ti rotor (Beckman Coulter, Inc., Pasa-
and ad libitum access to standard chow and water. Adult male ICR dena,CA,http://www.beckman.com)topellettheexosomes.Thesu-
mice (Swiss-Hauschka mice) and nude mice (BALB/c-n)wereused pernatant was immediately aspirated on completion of the first
for the present study. In brief, the mice were anesthetized using ultracentrifugationandthenultracentrifugedagainasdescribedpre-
10% chloral hydrate (0.3 ml/100 g). After hair was removed from viously. For maximal exosome retrieval, the exosome-enriched pel-
the dorsal surface, 1.5 cm of skin, uniform in diameter, was removed let was resuspended in a small volume (approximately 100 ml) of an
fromthebackofmicetocreatefull-thicknessskindefects.Thewounds appropriate buffer. This buffer depends on the downstream exper-
were dressed using Tegaderm (3M, St. Paul, MN, http://www.3m. iments planned after exosome isolation. The exosomes were
com) for 1 day after surgery and subsequently maintained with open measuredfortheirproteincontentusingtheBCAproteinassaykit
©AlphaMed Press 2016 STEM CELLS TRANSLATIONAL MEDICINE