Decoding therapeutic roles of adipose tissue-derived stromal cells and their extracellular
vesicles in liver disease
Afsharzadeh, Danial
DOI:
10.33612/diss.121499227
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Publication date:
2020
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Afsharzadeh, D. (2020). Decoding therapeutic roles of adipose tissue-derived stromal cells and their
extracellular vesicles in liver disease. University of Groningen. https://doi.org/10.33612/diss.121499227
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CHAPTER
3
Adipose tissue-derived
stromal cells suppress
liver fibrosis in an
extracellular
vesicle-mediated fashion
Danial Afsharzadeh
1Svenja Sydor
2Emilia Gore
3Julian Friedrich
4Guido Krenning
4Patrick Van Rijn
5Ali Canbay
2Peter Olinga
3Lars P. Bechmann
2Martin C. Harmsen
4Klaas Nico Faber
1,6Departments of 1Hepatology and Gastroenterology,
4Pathology and Medical Biology, 3Pharmaceutical
Technology and Biopharmacy, Biomedical Engineering5,
and 6Laboratory Medicine, Center for Liver, Digestive and
Metabolic Disease, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
2Department of Gastroenterology, Hepatology and
Infectious Diseases, Otto von Guericke University Magdeburg, Magdeburg, Germany.
ABSTRACT
Background & Aim: Liver fibrosis develops in response to chronic liver injury
and is mainly driven by activated hepatic stellate cells and portal myofibroblasts
that produce excessive extracellular matrix proteins. Human adipose
tissue-derived stromal cells (hASC) are able to suppress fibrogenesis, but the underlying
mechanisms are largely unknown. Here, we investigated the anti-fibrotic potential
of hASC-derived extracellular vesicles (EVs) in in vitro, ex vivo and in vivo models
of hepatic fibrosis.
Methods: Primary rat hepatic stellate cells (rHSC) and portal myofibroblasts
(rPMF) were transwell-cocultured with isolated primary hASC, cultured in
hASC-conditioned medium (hASC-CM) or treated with hASC-derived EVs.
CCl
4-treated (4 weeks) mice were treated (i.v.) with hASC-derived EVs. Human
precision-cut liver slices (hPCLS) were exposed to hASC-derived EVs. Cultured
cells, mouse livers and hPCLS were analysed for markers of fibrosis. hASC-derived
EVs were analysed for their miRNA content.
Results: hASC, hASC-CM and hASC-derived EVs suppressed proliferation and
Collagen I and alpha-smooth muscle actin (αSMA) expression of activated rHSC
and rPMF, while the EV-free fraction of hASC-CM did not. hASC-derived EVs
contained more than 1,000 types of miRNAs, the most abundant of which are
known to mediate fibroblast function and fibrosis.
Administration of
hASC-derived EVs to CCl
4-treated mice reduced serum markers of liver injury (ALT and
AST), as well as hepatic expression of Col1a1, Acta2, Tgf-β1, Timp1 and Timp2.
Accordingly, EV-treatment reduced hepatic αSMA protein levels and collagen
deposition. Ex vivo, hASC-derived EVs suppressed the expression of COL1A1,
ACTA2 and PAI1 in hPCLS.
Conclusion: hASC-derived EVs show potent anti-fibrotic potential in vitro, ex
vivo and in vivo and are therefore promising entities for the treatment of liver
3
3.1. INTRODUCTION
Hepatic fibrogenesis is a wound-healing response, which represents a ubiquitous
reaction of the liver to one of many causes of chronic injury, such as alcoholic,
non-alcoholic, viral and autoimmune hepatitis, as well as exposure to toxins or drugs
1-3. Chronic liver injury leads to the activation of hepatic fibroblasts, in particular
hepatic stellate cells (HSC) and portal myofibroblasts (PMF), which produce
excessive amounts of extracellular matrix (ECM) proteins, such as collagens and
fibronectins
4-6.In response to liver injury, HSC undergo a phenotypic switch
from a quiescent, vitamin A-storing cell type residing in the healthy liver, into a
migratory and proliferative, α-smooth muscle actin-positive and ECM-producing,
myofibroblast-like cell
7. Liver fibrosis may progress to liver cirrhosis, in which
liver vasculature and architecture are largely destroyed, which predisposes to liver
cancer and end-stage liver disease
6,8. Emerging data from animal and human
studies support the notion that liver fibrogenesis is a dynamic and bidirectional
process and reversal of fibrosis is therefore an important therapeutic target
9-12.
Nevertheless, drugs to reverse fibrosis in patients are not available yet and liver
transplantation remains the only cure for patients with advanced cirrhosis
13.
Encouragingly, therapeutic application of mesenchymal stem cells (MSC) have
shown promising results in treatment of fibrosis in murine models
14-17, as well
as in patients with advanced fibrosis
18,19. MSC are adult stem cells that reside
in virtually all organs of the body, except for neural tissue and cartilage. MSC
from bone marrow, umbilical cord blood, trabecular bone, synovial membrane
and adipose tissue have already been used in MSC-based therapies
20-22. Human
adipose tissue-derived stem cells (hASC) are being considered for MSC-based
therapies to treat chronic liver diseases, as they lack human leukocyte
antigen-DR expression, suppress proliferation of activated allogenic lymphocytes and
inhibit inflammatory cytokine production
23. Moreover, the high abundance of
hASC in adipose tissue and the relative ease of obtaining significant quantities
compared to other tissue-specific MSC make hASC an attractive and practical
source for cell therapy
24. hASC have already been shown to reverse toxin-
25,26and
diet-
16induced liver fibrosis in rodent models. MSC-related therapeutic effects
are considered to be based on paracrine signaling
27-32, but the identification and
characterization of the relevant factors remain to be determined. Various trophic
molecules have been considered as the main anti-inflammatory, anti-apoptotic
and anti-fibrotic factors in the secretome of cultured MSC
33-36, while the influence
of MSC-derived EVs has not been thoroughly investigated yet. EVs are carriers
of a variety of RNAs and proteins and are able to deliver this cargo to target cells.
Cargo of EVs may modulate key regulatory processes, such as transcription, post
transcriptional modulation and signal transduction in the recipient cells
37-43.
Particular
microRNAs (miRNAs) have been shown to be enriched in EVs and
may strongly affect the expression of target genes in recipient cells
44,45. Here,
we
investigated the anti-fibrotic potential of hASC-derived EVs in in vitro, ex vivo
and in vivo models of liver fibrosis.
3.2. MATERIAL AND METHODS
3.2.1. Human liver
This study was approved by the Medical Ethical Committee of University Medical
Centre Groningen, according to the Dutch legislation and the Code of Conduct
for dealing responsibly with human material in the context of health research
(https://www.federa.org/codes-conduct), refraining the need of written consent
for the ‘further use’ of coded-anonymous human tissue. Clinically healthy
liver tissue was obtained from surgical excess material acquired from donors
undergoing partial hepatectomy or organ donation. The liver tissue was stored in
ice-cold tissue preservation solution (University of Wisconsin) between 3 to 5 h.
3.2.2. Preparation of human precision-cut liver slices (hPCLS)
hPCLS (5 mm diameter, 250-300 μm thickness and 4-5 mg wet weight) were
prepared as previously described 46. Slices were obtained with a Krumdieck slicer
(Alabama Research and Development, Munford, AL, USA) in 4°C Krebs-Henseleit
buffer supplemented with 25 mM D-glucose (Merck, Darmstadt, Germany), 25
mM NaHCO3 (Merck), 10 mM HEPES (MP Biomedicals, Aurora, OH, USA)
saturated with carbogen (95% O2/5% CO2) at pH 7.42. Slices were incubated
in Williams’ medium E (with L-glutamine, Fisher Scientific, Landsmeer, The
Netherlands) supplemented with 25 mM D-glucose and, 50 μg/ml gentamycin
(Invitrogen). hPCLS were incubated for 48 h at 37°C in an 80% O2/ and 5% CO2
atmosphere, while horizontally shaken at 90 rpm. hPCLS were treated with a final
concentration of 1x106 hASC-derived EVs per ml of culture medium.
3.2.3. Human Adipose tissue–derived Stromal Cell (hASC) isolation and
culture
Human subcutaneous adipose tissue was obtained under informed consent from
healthy donors with BMI below 30 undergoing liposuction (Bergman Clinics,
The Netherlands). Adipose tissue was stored at 4°C and processed within 24 h
post-surgery. hASC were isolated as described 47 and seeded in culture flasks
at 4x104 /cm2, expanded by passing three times and used for experiments. All
3
experiments were performed using a pool of hASC from three donors. The use of
adipose tissue as the source of hASC was approved by the local Ethics Committee
of University Medical Center Groningen, given the fact that it was considered the
use of anonymized waste material.
3.2.4. Preparation of hASC-CM
hASC in passage 3-7 were cultured without serum for 24 h. hASC-CM was
collected and centrifuged at 500 xg for 10 min to remove cell debris and stored at
-80°C until use.
3.2.5. Isolation and identification of hASC-derived extracellular vesicles
(EVs)
hASC-derived extracellular vesicles (EVs) were isolated by the gold standard
differential centrifugation method, essentially as previously described 48. Briefly,
serum-free hASC-CM was centrifuged at 10,000 xg for 20 min to remove apoptotic
bodies. The supernatant was collected and subjected to 100,000 xg for 60 min
(optimal-XPN; Beckman Coulter). The EV-enriched pellet was washed in PBS and
subjected to an additional round of ultracentrifugation at 100,000 xg for 90 min.
EVs were resuspended in PBS and, together with the corresponding EV-depleted
hASC-CM, stored at -80°C. hASC-derived EVs were analyzed by transmission
electron microscopy (FEI Tecnai 12; Philips) and with a nanoparticle tracking
analyzer (NanoSight NS500, Malvern, Worcestershire, UK).
3.2.6. CCl4-induced mouse model of liver fibrosis and injection of
hASC-derived EVs
Inbred C57Bl/6 mice were housed in the animal facility of the University
Hospital Essen (ZTL), University of Duisburg-Essen, Germany according to the
recommendations of the Federation of European Laboratory Animal Science
Association (FELASA). All procedures were approved by the Landesamt für
Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV NRW). To
induce liver fibrosis, mice (four-to-six week-old) were injected intraperitoneally
with 0.6 ml CCl4 per kg (Sigma-Aldrich) in corn oil, twice per week for four weeks
as described earlier 49. 2x108 ASC-derived EVs were resuspended in 100 µl PBS
and injected intravenously once per week (3 hours post-CCl4 injection) during
the last two weeks of the CCl4 treatment (CCl4+EV group) (n =8). The control
groups were administered with CCl4 and PBS (CCl4+PBS group) (n=8) or corn
oil and PBS/EVs (oil+PBS and oil+EV groups) (n= 6). The two-weeks control
mice were subjected to either CCl4 or corn oil for the duration of two weeks
(n=6). At the end of the experiment, blood samples were taken, clotted at room
temperature, and the isolated serum was stored at -80°C for further analysis. Liver
samples were snap-frozen in liquid nitrogen and stored at -80°C until isolation
of RNA or fixed in 4.5% formalin, paraffin-embedded and sectioned following
standard procedures 50.
3.2.7. Isolation, culture and treatment of rat hepatic stellate cells (rHSC) and
portal myofibroblasts (rPMF)
Specified pathogen-free male Wistar rats (350 – 400 g; Charles River Laboratories
Inc., Wilmington, MA, USA) were kept under standard laboratory conditions
with free access to standard laboratory chow and water. All experiments were
performed according to Dutch law on welfare of laboratory animals and
guidelines of the ethics committee of University of Groningen for the care and
use of laboratory animals. Primary rHSC were obtained as described before 51
by perfusion of the liver with pronase (Merck, Amsterdam, the Netherlands)
and collagenase-P (Roche, Almere, the Netherlands) and further purified by
Nycodenz (Axis-ShieldPOC, Oslo, Norway) gradient centrifugation. rPMF were
obtained by outgrowth from intrahepatic bile duct segments 52. rHSC and rPMF
were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with Glutamax
(Invitrogen, Brenda, the Netherlands) supplemented with 20% heat-inactivated
fetal calf serum (Invitrogen), 1 mmol/l sodium pyruvate (Invitrogen), 1x
nonessential amino acids (Invitrogen), 50 µg/ml gentamicin (Invitrogen), 100 U/
ml penicillin (Lonza, Vervier, Belgium), 10 µg/ml streptomycin (Lonza) and 250
ng/ml fungizone (Lonza) in a humidified incubator at 37°C with 5% CO2. rHSC
and rPMF were cultured to a density of ~1.5x104 cells per cm2 and transwell
co-cultured with an equal number of hASC or co-cultured in hASC-CM (see “Real-time
monitoring of cell proliferation” below). rHSC and rPMF were treated with a final
concentration of 1x106 hASC-derived EVs per ml of culture medium or cultured
in the EV-depleted hASC-CM, which was supplemented with serum.
3.2.8. RNA isolation and qPCR
RNA was isolated using TRI reagent (Sigma-Aldrich) according to the
manufacturer’s instructions. Reverse transcription was performed on 2.5 µg
total RNA using random nanomers (Sigma-Aldrich) in a final volume of 50 µl.
Real-time semi-quantitative PCR (qPCR) was performed on the 7900HT Fast
Real-TimePCR system (Applied Biosystems Europe, The Netherlands) using the
TaqMan or SYBR Green protocol 53. mRNA levels were normalized to ACTB/
Actb and further normalized to the mean expression level of the control group.
qPCR primers and probes are shown in Supplementary Tables S1 and S2.
3
3.2.9. miRNA isolation, library preparation and sequencing
miRNAs were isolated from hASC and hASC-derived EVs according to the
mirVana PARIS kit manual (Ambion, Carlsbad, CA, USA). Next generation
sequencing libraries were constructed using the TruSeq Small RNA Sample Prep
Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.
Libraries were subsequently sequenced on a Nextseq550 sequencer (Illumina, San
Diego, CA, USA).
3.2.10. Real-time monitoring of cell proliferation
Proliferation of rHSC and rPMF was monitored using the xCELLigence system
(RTCA DP; ACEA Biosciences, Inc., San Diego, CA, USA). rHSC and rPMF
were seeded in E-plates with interdigitated gold microelectrodes to constantly
record the cell proliferation (Δ Cell index/ h-1), according to manufacturer’s
instructions54. rHSC and rPMF were cultured and treated with isolated
hASC-derived EVs as it was described (see “Isolation, culture and treatment of rHSC and
rPMF” above). Results were recorded and analyzed by the xCELLigence software.
3.2.11. Immunofluorescence microscopy
rHSC and rPMF were cultured on coverslips, fixed with 4% paraformaldehyde in
PBS (Merck Millipore) and permeabilized in 1% Triton-X100. Coverslips were
incubated with primary antibodies against rat collagen type 1 (1:400; Southern
Biotech, Birmingham, AL, USA) and rat αSMA (1:400; Sigma-Aldrich, St. Louis,
MO, USA) according to the manufacturer’s instructions. Further, coverslips were
labeled using the GFP-tagged secondary antibody for collagen type 1 (1: 2,000
polyclonal Rabbit Anti-Goat Immunoglobulins, Dako, Denmark) and the
GFP-tagged secondary antibody for αSMA (1: 2,000 polyclonal Rabbit Anti-mouse
Immunoglobulins, Dako, Denmark). Later, all coverslips were mounted in the
fluorescence mounting medium S3023 (DAKO, Heverlee, Belgium). Images were
captured using a Leica DMI6000 and analyzed by ImageJ image analysis software
(ImageJ; National Institutes of Health (NIH), Bethesda, MD, USA) and Adobe
Photoshop CS6.
3.2.12. Histopathological staining
Liver tissues were processed for paraffin embedding and 4-µm-thick sections were
prepared. The sections were stained with picrosirius red and counter-stained with
hematoxylin and eosin according to standard protocols. Picrosirius red staining
was quantified using ImageJ image analysis software (ImageJ; NIH).
3.2.13. Immunohistochemistry
αSMA immunostaining on 4-µm liver sections was carried out using the MOM
kit (Vector labs, USA), according to the manufacturer’s protocol. Briefly, tissue
sections were antigen retrieved by the overnight incubation of liver sections
with the Tris buffer (0.1 M, pH 9) at 80°C. Further, sections were blocked with
MOM mouse Ig blocking reagent. The primary antibody was detected using the
biotin-conjugated anti-mouse IgG antibody, and then incubated with Vectastain
and Novared. The complex was visualized with 3,3-diaminobenzidine reagent for
microscopical examination. Absence of the primary antibody from the procedure
served as negative control. The quantification of αSMA positive areas was
performed using Image-J image analysis software (ImageJ; NIH).
3.2.14. Serum liver damage marker assays
Serum levels of lactate dehydrogenase (LDH), aspartate transaminase (AST) and
alanine aminotransferase (ALT) levels were quantified using the LDH, AST and
ALT Activity Assay Kits (all Abcam), respectively.
3.2.15. Pathway enrichment analysis
To identify pathways enriched with targets of abundant miRNAs in EVs, miRNAs
with a proportion of at least 0.5 % in hASC-derived EVs were selected. For each
miRNA, predicted targets were obtained using miRNAtap and KEGG pathway
enrichment analysis was performed using clusterProfiler55, with a
Bonferroni-Holm adjusted cut-off set to P < 0.1. KEGG pathways with significantly enriched
targets of at least 6 miRNAs were displayed using ggplot2 56. All analysis was
conducted in R (R Foundation for Statistical Computing, Vienna, Austria). The
bigger the size of the circle, the more counts of predicted targets of a certain
miRNA in the pathway. The darker a circle, the lower the adjusted p-value of the
enrichment analysis of a miRNA.
3.2.16. Statistical analysis
Data are expressed as average with standard deviation. Statistical significance was
determined using Student’s t-test or by One-way ANOVA (with Tukey’s
post-hoc test for individual experimental conditions). All tests were performed with
GraphPad Prism (v. 5.0; GraphPad Software, La Jolla, CA, USA). Differences were
considered significant at P < 0.05.
3
3.3. RESULTS
3.3.1. ASC suppress proliferation and activation of HSC and PMF in a
paracrine manner.
Culture-activated primary rat HSC (Figure 1A) or PMF (Figure 1B) were
co-cultured with human ASC (hASC) for 48 h in a transwell system allowing only
paracrine signaling between the two cell types and cell proliferation of HSC/PMF
was monitored using a real-time cell analyzer (xCELLigence). Monocultures of
HSC or PMF served as controls. Proliferation of rHSC and rPMF was significantly
suppressed in the presence of hASC as monitored by a delayed increase in cell
index during the full 48 h (grey lines representing a single experiment and grey
bars for n=4). Especially the proliferation of rPMF appeared highly sensitive to
co-culturing with hASC. The hASC-mediated inhibition of HSC/PMF proliferation
was accompanied by a significant reduction in Col1a1 and Acta2 expression in
rHSC and rPMF, as compared to the rHSC and rPMF monocultures (Figure
1A,B). Next, rHSC and rPMF were cultured for 48 h in 24 h-conditioned medium
produced by hASC (hASC-CM; Figure 1 C,D). Similar as observed for the
co-culture experiments, hASC-CM strongly suppressed proliferation of rHSC and
rPMF, as well as Col1a1 and Acta2 expression, compared to these cells grown
in normal (DMEM) media. In line with the effect on the gene expression, both
co-culture with hASC and exposure to hASC-CM strongly reduced protein
expression of collagen type-1 and αSMA in rHSC and rPMF as analyzed by
immunofluorescence microscopy (Figure 1E). These results show that hASC
secrete factors that suppress activation and proliferation of hepatic fibroblasts.
3.3.2. hASC-derived EVs suppress the proliferation and activation of rHSC
and rPMF.
Next, we analyzed the nature of the hASC-derived factors carrying the
anti-fibrotic properties. The hASC-CM was cleared from (apoptotic) cell remnants
followed by ultracentrifugation (90’@100,000xg) to obtain a pellet fraction with
particle-bound/encapsulated factors, in particular extracellular vesicles (EV), and
a supernatant fraction with soluble factors. The EV-fraction showed high potential
to suppress rHSC and rPMF proliferation (Figure 2A), and reduced Col1a1 and
Acta2 mRNA (Figure 2C) and corresponding collagen type-1 and αSMA protein
levels (Figure 2E). In sharp contrast, soluble factors in hASC-CM did not affect
rHSC and rPMF proliferation (Figure 2B), nor the mRNA (Figure 2D) and
protein levels (Figure 2E) of collagen type-1 and αSMA. These data indicate that
anti-fibrotic factors secreted by hASC are contained in the pelletable entities.
A
B
C
D
Fig
ur
e 1. H
uma
n A
di
pos
e t
iss
ue-d
eriv
ed s
tr
oma
l c
el
ls (hA
SC) s
up
pr
ess p
ro
lif
er
at
io
n a
nd ac
tiva
tio
n o
f r
at HSC a
nd PMF
.
C
on
tin
ue
d o
n n
ext p
ag
e. L
eg
en
d o
n n
ext p
ag
e.
3
3.3.3. Identification and miRNA content of hASC-derived EVs.
Morphological characterization of the pelletable fraction of hASC-CM by
TEM, revealed sphere-shaped vesicles of rather homogeneous size (Figure 3A).
Nanoparticle analysis showed that approximately 85% of vesicles were sized
between 55-220 nm in diameter, with distinguishable peaks at 55, 115 and 165
nm (Figure 3B). This size distribution suggests that most of the purified
derived EVs were exosomes. Isolation and sequencing of miRNA from the
hASC-derived EVs revealed that they contain over 1,000 unique miRNAs (Supplementary
Table S3), but that they were mostly enriched for 27 dominant miRNAs that each
contribute for at least 0.5% to the total exosomal miRNA content (Supplementary
Table S3). Pathway enrichment analysis showed that these miRNAs are predicted
to control biological pathways involved in fibroblast proliferation and activation,
such as MAPK-, FoxO-, PI3K-Akt- and p53-signaling, as well as cellular senescence
and plasticity pathways (Figure 3C). Interestingly, most of these miRNAs were
enriched in the ASC-derived EVs, when compared to the cellular miRNA content
of hASC (Supplementary Table S3). These results suggest that miRNAs in
hASC-derived EVs may contribute to the anti-fibrotic actions on in vitro-cultured rHSC
and rPMF.
Figure 1. Human Adipose tissue-derived stromal cells (hASC) suppress proliferation
and activation of rat HSC and PMF. Cell proliferation and Col1a1 and Acta2 gene
expression in culture-activated rHSC (A) and rPMF (B) after transwell co-culturing with
hASC for 48 h (A,B) and after culturing in hASC-conditioned medium (hASC-CM) (C,D);
(E) Immunofluorescence microscopy images of collagen type I (green, top panels,
counterstained with DAPI blue) and αSMA (green, bottom panels) in culture-activated
rHSC (a-c, g-i) and rPMF (d-f, j-l) after transwell co-culturing with hASC (b,h and e,k) or
culturing in hASC-CM (c,I and f,l) for 72 h compared to control-grown rHSC (a,g) and
rPMF (d,j) (scale bar, 40 µm). Data are presented as mean value ± SD of 4 experiments.
*p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test.
A
B
C
D
Fig
ur
e 2. hA
SC-d
eriv
ed e
xt
rac
el
lu
la
r v
es
icl
es (EVs) s
up
pr
ess p
ro
lif
er
at
io
n a
nd ac
tiva
tio
n o
f c
ul
tur
e-ac
tiva
te
d rHSC a
nd rPMF
.
C
on
tin
ue
d o
n n
ext p
ag
e. L
eg
en
d o
n n
ext p
ag
e.
3
3.3.4. Administration of hASC-derived EVs alleviates liver fibrosis in
CCl4-induced mice.
To test the anti-fibrotic activity of hASC-derived EVs in vivo, mice were exposed
to CCl4 for 4 weeks with 2 therapeutic tail vein injections of hASC-EVs in the
final 2 weeks before sacrifice. As expected, serum levels of liver injury markers
(ALT, AST and LDH) were increased in mice after four weeks of CCl4-exposure
(Figure 4A-C). Administration of hASC-derived EVs did not change serum AST
or LDH levels, but did significantly suppress serum ALT levels (416.5±105.9 U/L
compared to 1256.1± 194.6 U/L in CCl4-treated mice), the latter being considered
the more specific marker for liver injury. Serum liver damage markers were not
affected in mice receiving hASC-derived EVs only (Figure 4). PicroSirius Red
staining revealed that CCl4 exposure induced collagen deposition in mouse
livers when compared to controls (Figure 5Aa1-b1), which was strongly
suppressed by hASC-EV treatment (Figure 5A-c1, quantification in right panel).
No hepatic collagen deposition was observed in EV-only-treated mice (Figure
5A-d1). Similar results were obtained when analyzing αSMA protein levels by
immunohistochemistry; e.g. CCl4 treatment induced αSMA levels, particularly
in the periportal areas, which was strongly suppressed by hASC-EV treatment
(Figure 5Aa2-d2, including quantification in the right panel). Hepatic levels of
Col1a1, Acta2, Timp1 and Timp2 mRNA, all markers of fibrosis, were increased
Figure 2. hASC-derived extracellular vesicles (EVs) suppress proliferation and activation
of culture-activated rHSC and rPMF. Cell proliferation of activated rHSC and rPMF during
exposure to hASC-derived EVs (A) or EV-depleted hASC-CM (B) monitored for 48 h; Col1a1
and Acta2 gene expression in rHSC and rPMF after culturing with hASC-derived EVs (C)
or EV-depleted hASC-CM (D) for 48 h; (E) Immunofluorescence microscopy images of
collagen type I (green, top panels, counterstained with DAPI blue) and αSMA (green,
bottom panels) in culture-activated rHSC (a-c, g-i) and rPMF (d-f, j-l) after 72 h exposure
to hASC-derived EVs (b,h and e,k) or EV-depleted hASC-CM (c,I and f,l) compared to
control rHSC (a,g) and rPMF (d,j) (scale bar, 40 µm). Data are presented as mean value ±
SD of 4 experiments. *p ≤0.05, **p ≤0.01, ***p ≤0.001, Student’s t test.
in CCl4-treated mice, when compared to sham-treated controls (Figure 5B),
which were all suppressed when the CCl4-exposed mice were treated with
hASC-derived EVs. In mice receiving only EVs, hepatic expression of Col1a1, Acta2,
Figure 3. Morphology and miRNA content of hASC-derived EVs. (A) Transmission electron
micrograph of hASC-derived EVs, scale bar = 200 µm; (B) Size distribution histogram
of hASC-derived EVs measured by a nanoparticle tracking analyzer (NTA). (C) Pathway
enrichment analysis of the 27 most dominant miRNA (each with a relative abundance of
at least at least 0.5%) in hASC-derived EVs. KEGG pathways with significantly enriched
targets of at least 6 miRNAs are displayed. The bigger the size of the circle, the more
counts of predicted targets of a certain miRNA in the pathway. The darker a circle, the
lower the adjusted p-value of the enrichment analysis of a miRNA.
C
3
Tgfb1, Timp1 and Timp2 were indistinguishable from sham-treated controls
(Figure 5B).
hASC-derived EVs suppress early onset of fibrosis in human precision-cut
liver slices.
To determine the relevance of our findings for the development of fibrosis in
human liver, we next performed experiments with human precision-cut liver
slices (hPCLS) that were shown earlier to be a relevant model to study the early
onset of liver fibrosis ex vivo 57,58. hPCLS generated from viable non-fibrotic
human liver were therefore cultivated in the absence and presence of hASC for 48
h. As reported before, expression of COL1A1, ACTA2 and PAI1 increased during
the 48 h ex vivo culture period, which were all significantly suppressed when the
hPCLS were co-treated with hASC-derived EVs (Figure 6).
3.4. DISCUSSION
In this study, we show that hASC-derived EVs suppress fibrotic features of hepatic
stellate cells and portal myofibroblasts in vitro, both of which are pivotal in the
development of liver fibrosis. Moreover, hASC-derived EVs suppressed fibrosis
in the in vivo murine CCl4 model of liver fibrosis and suppress the early onset of
fibrosis ex vivo in human precision-cut liver slices. Remarkably, the anti-fibrotic
properties in the hASC-secretome appeared (almost) solely attributable to the EV
fraction, while such features were not detected in the EV-free fraction containing
Figure. 4. hASC-derived EVs ameliorate CCl
4-induced liver injury in mice. Serum levels
of (A) alanine aminotransferase (ALT), (B) Aspartate aminotransferase (AST), and (C)
lactate dehydrogenase (LDH) of mice (n=8 per group) that were exposed to 4-week CCl
4treatment with or without co-treatment with hASC-derived EVs in the last 2 weeks. Data
shows mean value ± SD. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ANOVA (with Tukey’s
post-hoc test for individual experimental conditions).
trophic factors. The hASC-derived EVs carry a great variety of miRNA, the most
dominant of which have been shown earlier to act on fibrosis-related pathways.
Mesenchymal stem cells (MSC) have shown potent therapeutic effects in a vast
variety of diseases, including liver fibrosis. Still, it remains largely unknown what
Figur
e 5.
hASC-d
eriv
ed
EV
s ame
lior
at
e
CCl
4-induced
liv
er
fibr
osis
in
mice
. (A)
Repr
esen
ta
tiv
e
sirius
red
st
aining
(a1-d1;
sc
ale
bar=1
mm)
and
αSMA
immunohis
tochemis
tr
y
(a2-d2;
sc
ale
bar
=
500
µm)
of
liv
er
sections
from
vehicle-tr
ea
ted
(a),
CCl
4-tr
ea
ted
(b),
CCl
4+EV
s
cotr
ea
ted
(c)
and
EV
-only
tr
ea
ted
(d)
mice.
Quan
tific
ation
of
the
st
aining
in
tensity
for
all
animals
(n=8
per
gr
oup)
is
pr
esen
ted
in
the
panels
to
the
righ
t.
(B)
Corr
esponding
hepa
tic
gene
expr
ession
analy
sis
of
Col1a1,
Act
a2,
Tgfb1,
Timp1
and
Timp2
. Da
ta
sho
w
s mean
value
±
SD
. *p
≤0.05,
**p
≤0.01,
***p
≤0.001,
ANO
VA
(with
Tuk
ey
’s pos
t-hoc t
es
t f
or individual e
xperimen
tal c
onditions).
A
B
Upper (g reen) panel Lo w er (br own) panel3
the exact mechanisms are that underlie the therapeutic effect in liver fibrosis,
which may involve 1) stem cell-to-liver cell transdifferentiation, 2) juxtacrine, 3)
paracrine or 4) endocrine signaling. We demonstrate that hASC produce EVs that
suppress the proliferation and activation, e.g. ECM production and cytoskeletal
changes, of culture-activated rHSC and rPMF, thus acting through paracrine/
endocrine signaling. This finding is in line with the current believe that the
secretome of MSC is primary responsible for therapeutic effects of these stem
cells31,59,60. An earlier study reported that conditioned medium produced by
human amniotic epithelial cells suppresses collagen production and proliferation
in human hepatic stellate cells and additionally induced HSC apoptosis61. In line,
others have shown that conditioned medium of MSC suppresses CCl4-induced
liver fibrosis and inflammation in mice62. In part, the anti-fibrotic effect of human
umbilical cord MSC has been attributed through the cytokines that they secrete
rather than by differentiation to hepatocytes63. Remarkably, our results show that
the anti-fibrotic effect of the hASC secretome on in vitro-cultured HSC and PMF
is predominantly, if not solely, dependent on the EVs and is absent in the EV-free
conditioned medium after the 100,000xg centrifugation step. EVs are released by
virtually all cell types, including MSC 64. The 100,000xg pellet fraction of
hASC-CM revealed vesicle sizes ranging from ~55-220 nm, which indicates that most of
them have the size typical for exosomes (30-150 nm). However, a distinct peak of
165 nm vesicles was also detected, which suggest that also other types of EVs are
produced by the hASC. EVs carry a great variety of different proteins and various
types of RNA, such as miRNAs, which may induce functional and phenotypic
Figure 6. hASC-derived EVs suppress early onset of fibrosis in human precision-cut
liver slices (hPCLS). hPLCS were exposed to hASC-derived EVs for 48 h, followed by
gene expression analysis for COL1A1, ACTA2 and PAI1. Spontaneous induction of fibrosis
markers is observed during 48 h of hPLCS culturing (compare T=0 and T=48 h), which
is suppressed by co-treatment with hASC-derived EVs. Data shows mean value ± SD.
*p ≤0.05, **p ≤0.01, ***p ≤0.001, ANOVA (with Tukey’s post-hoc test for individual
experimental conditions).
changes in recipient cells65-68,69,70. Our initial analysis of the hASC-derived
EVs was focused on the miRNA content and revealed that they contain over 1,000
unique types of miRNAs (see Supplementary Table S3). However, most of the
miRNA only appear at very low levels in ASC-derived EVs, where only the top 27
most dominant ones exceeded a relative contribution of 0.5% of the total miRNA
content. Interestingly, ~50% of these abundant miRNAs were enriched in EVs
when compared to the miRNA content of the corresponding hASC, including
21, 181a, 142, 150, 155, 22, 26a, 26b,
27a, 342, 92a, 146b, 16, let-7g, let-7f, let-7a, 30e,
miR-186 and miR-29a. Pathway enrichment analysis indicated various associations
between these miRNAs and pathways regulating fibroblast function and fibrosis,
such as MAP Kinase and FoxO signaling pathways. Mitogen-activated protein
kinases (MAPKs) play central roles in HSC activation and liver fibrosis 71-73 and
MAPK inhibitors alleviate fibrosis in the murine models of liver fibrosis 74-76. In
particular, the various miR-29s have been shown to control MAPK signaling77,78.
miR-29a is the most abundant miR-29 in hASC-derived EVs and suppresses
(cardiac) fibroblast proliferation via modulation of the MAPK pathway79.
Moreover, several EV-associated miRNAs modulate the FoxO signaling pathway,
including let-7a, let-7f, let-7g and miR-29a. As liver inflammation and fibrosis are
augmented by FoxO180,81, it suggests that aforementioned miRNA might have
regulatory roles in anti-fibrotic characteristics of hASC-derived EVs as well. It is
thus likely that the hASC-derived EVs do not act through one unique pathway to
suppress liver fibrosis. The miRNA content would allow modulation of various
pathways controlling hepatic fibroblast proliferation and activation, while other
protein and/or lipid factors contained in the EVs may also contribute to their
overall anti-fibrotic properties.
In the CCl4-treated mice, EVs were administered in the peripheral circulation
while the beneficial effects were apparent in the liver. The biodistribution in vivo
of EVs is similar to that of synthetic membranous nanoparticles, i.e. liposomes,
as they are similar in size and structure of the lipophilic outer layer82-85.
Liposomes (50-200 nm) are able to pass the hepatic endothelium cells because the
sinusoidal endothelial cells are extensively fenestrated with pore sizes of ~50-200
nm86,87.The fact that most of i.v. injected liposomes are captured by the liver and
spleen rather than by other organs88,89, might predict that hASC-derived EVs
accumulate in the mouse liver, even when it is chronically injured by repeated
CCl4 administration. Indeed, Wiklander et al. showed that EVs distribute
predominantly to organs of the mononuclear phagocyte system with highest
accumulation in the liver, followed by spleen, GI-tract and lungs90. Our data show
that hASC-derived EVs ameliorate the early stages of liver fibrosis in mice and
3
reverse it to a nearly normal condition. As the treatment with EVs also suppressed
CCl4-induced serum ALT levels, the effect on fibrosis could come –in part- also
as a secondary effect due to a protective effect on hepatocytes. However, in pilot
experiments preceding the 4-week CCl4 treatment protocol we first analyzed the
effect of hASC-derived EVs on acute –single dosing- CCl4 exposure. This showed
that EVs suppressed serum ALT levels only when given 1 h before CCl4 treatment
and did not do so when given 3 h after the CCl4 injection (Supplementary Figure
S4). In the 4-week CCl4 protocol, we therefore applied the EV treatment 3 h after
the CCl4-injection in the 3rd and 4th week to determine the anti-fibrotic action of
EVs independent of potential effects on hepatocyte viability. Still, we cannot fully
rule out the possibility that the EV treatment may have an additional hepatocyte
protective effect on CCl4 exposure applied 3-4 days later as performed in the
4-week protocol. Importantly, the anti-fibrotic potential of ASC-derived EVs
was also confirmed in the human ex-vivo approach using hPLCS, where all cell
types of the liver are present in their original context. Hepatocytes, Kupffer cells,
endothelial cells, and hepatic stellate cells remain viable and functional in cultured
hPCLS for at least 48 h57,58. Interestingly, early onset of fibrosis occurs during
PCLS culture as demonstrated by increased expression of COL1A1, ACTA2 and
PAI158. As a prelude to future clinical studies on liver fibrosis, we applied EVs
on hPCLS and found that also in this experimental model, the hASC-derived
EVs suppress transcriptional activation of markers of fibrosis, e.g. COL1A1 and
ACTA2.
Our findings are promising for the future use of hASC-derived EVs as treatment
for liver fibrosis. In conclusion, hASC-derived EVs are promising therapeutic
entities for the treatment of liver fibrosis. Clinical application of hASC-derived
EVs is expected to provide a novel alternative therapy for patients with liver
fibrosis.
Acknowledgements
We are thankful to Sebo Withoff, Rutger Modderman and Ineke Tan from the
department of Genetics, University Medical Center Groningen for their technical
support in miRNA analysis. We also thank Johannes Haybaeck and Kirsten
Herrmanns from the department of Pathology, Otto-von-Guericke-university
hospital Magdeburg, for their time and laboratory technical support.
Funding
This study was funded by EASL Entry-Level fellowship, Short-term training
fellowship Andrew K. Burroughs, De Stichting De Cock-Hadders and
Wilhelm-Laupitz foundation.
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3
SUPPLEMENTARY TABLES
Supplementary Table S1. Primers and probes used for real-time quantitative PCR analysis
(Taqman protocol).
Supplementary Table S2. Mouse primers used for real-time quantitative PCR analysis
Supplementary table S3. miRNA content of hASC and hASC-derived EVs
MiRNAs hASC (absolute
values) hASC (relative values) EVs (absolute values) EVs (relative values)
hsa-miR-21-5p 1957493 15.417559 1618929 20.70720178 hsa-miR-181a-5p 1132708 8.921407343 1086132 13.89236618 hsa-miR-142-5p 723206 5.696097599 835995 10.69294401 hsa-miR-22-3p 1257196 9.901896717 653737 8.361740366 hsa-miR-150-5p 364101 2.867723487 367610 4.701981647 hsa-miR-146b-5p 393219 3.097061974 313786 4.01353612 hsa-let-7f-5p 449131 3.537434715 285667 3.653875007 hsa-miR-26a-5p 378414 2.980455191 234806 3.003328263 hsa-miR-92a-3p 321236 2.530111211 179935 2.301490894 hsa-miR-16-5p 181177 1.426981904 175435 2.243932837 hsa-let-7g-5p 131485 1.035598976 133175 1.703398727 hsa-miR-21-3p 199794 1.573612669 132266 1.691771999 hsa-miR-30e-5p 124888 0.983639844 124599 1.59370586 hsa-let-7a-5p 382358 3.011518828 124648 1.594332603 hsa-miR-191-5p 162206 1.277562973 112377 1.437378177 hsa-miR-155-5p 143530 1.130467513 91447 1.169669257 hsa-miR-186-5p 90827 0.71536942 87732 1.122151883 hsa-miR-142-3p 55031 0.433433831 71319 0.912218463 hsa-let-7i-5p 188050 1.48111486 72879 0.932171923 hsa-miR-342-3p 54181 0.426739081 64070 0.819498828 hsa-miR-27a-3p 81227 0.639758132 63558 0.81295 hsa-miR-146a-5p 79218 0.623934895 57334 0.733340812 hsa-miR-28-3p 78058 0.614798531 52145 0.666969976 hsa-miR-29a-3p 60633 0.477556168 49823 0.637270019 hsa-miR-103a-3p 54663 0.430535398 46114 0.589829389 hsa-miR-26b-5p 39686 0.312573913 42524 0.54391085 hsa-miR-181b-5p 52766 0.415594293 40152 0.513571358 hsa-miR-10a-5p 453695 3.573381582 37633 0.481351637 hsa-miR-27b-3p 174210 1.372108587 36171 0.462651664 hsa-miR-25-3p 38499 0.303224892 35089 0.448812149 hsa-miR-148a-3p 64736 0.509872117 31416 0.401831983 hsa-miR-30c-5p 28691 0.22597536 26739 0.342009976 hsa-miR-221-3p 53521 0.421540805 26405 0.337737889 hsa-miR-107 28437 0.223974811 25828 0.330357667 hsa-miR-101-3p 28462 0.224171716 25533 0.326584417 hsa-miR-192-5p 21288 0.167668031 21655 0.276982162 hsa-miR-423-3p 46364 0.365171015 20595 0.263424042 hsa-miR-140-3p 20002 0.157539268 17595 0.225052004 hsa-miR-30d-5p 37024 0.291607533 16368 0.20935784 hsa-miR-146b-3p 17487 0.137730686 15578 0.199253203 hsa-miR-15a-5p 15285 0.120387347 14893 0.190491588 hsa-miR-98-5p 22695 0.17874981 14300 0.182906715 hsa-miR-222-3p 214607 1.690282461 12068 0.154357919 hsa-miR-423-5p 37389 0.294482337 10930 0.139802126 hsa-miR-92b-3p 79682 0.62758944 9389 0.120091689 hsa-miR-93-5p 10797 0.08503907 9423 0.120526572 hsa-miR-361-3p 9065 0.071397534 9398 0.120206805 hsa-miR-23a-3p 17727 0.139620968 8921 0.114105651 hsa-let-7d-5p 13519 0.106478021 8698 0.111253329 hsa-miR-34a-5p 6799 0.053550119 8312 0.106316127 hsa-miR-181a-3p 8398 0.066144124 7799 0.099754508 hsa-miR-29c-3p 6473 0.050982486 7426 0.094983585 hsa-miR-484 8933 0.070357878 7320 0.093627773 hsa-miR-1307-5p 6396 0.05037602 7259 0.092847542
3
hsa-miR-16-2-3p 8225 0.064781546 7419 0.09489405 hsa-miR-106b-3p 7959 0.062686483 7303 0.093410332 hsa-miR-941 7685 0.060528411 6591 0.084303368 hsa-miR-181c-5p 7283 0.057362188 6647 0.085019646 hsa-miR-378a-3p 7406 0.058330958 6393 0.081770813 hsa-miR-320a-3p 11803 0.092962503 6162 0.078816166 hsa-miR-28-5p 7914 0.062332055 5770 0.07380222 hsa-miR-128-3p 7567 0.059599022 5734 0.073341756 hsa-miR-425-5p 5486 0.0432087 5242 0.067048741 hsa-miR-19b-3p 5112 0.040263011 4853 0.062073167 hsa-miR-30e-3p 4835 0.03808131 4850 0.062034795 hsa-miR-148b-3p 9547 0.07519385 4504 0.05760922 hsa-miR-486-5p 10178 0.080163717 4247 0.054322015 hsa-miR-454-3p 3277 0.025810228 4019 0.05140574 hsa-miR-3615 4474 0.035238011 3903 0.049922022 hsa-miR-363-3p 3472 0.027346082 3929 0.050254579 hsa-miR-769-5p 6328 0.04984044 3647 0.046647608 hsa-miR-23b-3p 7679 0.060481154 3412 0.043641798 hsa-miR-182-5p 4311 0.033954194 3576 0.045739469 hsa-miR-20a-5p 3292 0.025928371 3067 0.039229014 hsa-miR-30b-5p 4076 0.032103293 2584 0.033051116 hsa-miR-15b-5p 3550 0.027960424 2746 0.035123206 hsa-miR-130b-3p 4436 0.034938716 2746 0.035123206 hsa-miR-1307-3p 3660 0.028826803 2760 0.035302275 hsa-miR-29b-3p 2995 0.023589147 2666 0.034099951 hsa-miR-223-3p 2206 0.017374844 2567 0.032833674 hsa-miR-421 3052 0.024038089 2493 0.031887164 hsa-miR-374a-3p 3112 0.024510659 2508 0.032079024 hsa-miR-339-3p 2689 0.021179037 2385 0.03050577 hsa-miR-24-3p 4973 0.039168222 2535 0.032424372 hsa-miR-532-5p 3201 0.025211639 2386 0.030518561 hsa-miR-424-3p 4159 0.032757015 2026 0.025913916 hsa-miR-301a-3p 2234 0.017595377 2014 0.025760428 hsa-miR-32-5p 1454 0.01145196 1814 0.023202292 hsa-miR-210-3p 2381 0.018753175 2116 0.027065078 hsa-miR-652-3p 1776 0.013988088 1857 0.023752292 hsa-miR-342-5p 2064 0.016256427 1918 0.024532523 hsa-miR-124-3p 1562 0.012302587 1840 0.02353485 hsa-miR-106b-5p 2210 0.017406349 1803 0.023061595 hsa-miR-17-5p 2238 0.017626881 1754 0.022434852 hsa-miR-19a-3p 2160 0.01701254 1798 0.022997642 hsa-miR-141-3p 1293 0.010183895 1539 0.019684856 hsa-miR-1285-3p 1632 0.012853919 1567 0.020042995 hsa-miR-197-3p 2380 0.018745298 1649 0.02109183 hsa-miR-378c 1217 0.009585306 1515 0.019377879 hsa-miR-99b-5p 40813 0.321450363 1488 0.019032531 hsa-miR-132-3p 5580 0.043949061 1397 0.017868579 hsa-miR-150-3p 1271 0.010010619 1414 0.018086021 hsa-miR-9985 1055 0.008309365 1194 0.015272071 hsa-miR-10399-3p 1157 0.009112735 1266 0.016193 hsa-miR-130a-3p 4989 0.039294241 1199 0.015336025 hsa-miR-501-3p 1397 0.011003018 1166 0.014913932 hsa-miR-125a-5p 40532 0.319237158 1089 0.01392905 hsa-miR-10399-5p 797 0.006277312 1097 0.014031375 hsa-miR-1246 1385 0.010908503 1087 0.013903468 hsa-miR-500a-3p 2764 0.02176975 1030 0.0131744 hsa-miR-589-5p 2432 0.01915486 965 0.012343006 hsa-miR-660-5p 1662 0.013090204 991 0.012675563hsa-let-7b-5p 37884 0.298381044 894 0.011434867 hsa-miR-221-5p 7758 0.061103372 926 0.011844169 hsa-miR-17-3p 1632 0.012853919 823 0.010526729 hsa-miR-374a-5p 847 0.006671121 910 0.011639518 hsa-miR-671-3p 2630 0.020714342 895 0.011447658 hsa-miR-7706 1111 0.008750431 836 0.010693008 hsa-miR-3614-5p 1475 0.011617359 918 0.011741844 hsa-miR-151a-3p 24812 0.195423674 841 0.010756961 hsa-miR-330-5p 818 0.006442712 777 0.009938358 hsa-miR-361-5p 1430 0.011262931 865 0.011063938 hsa-miR-625-3p 1283 0.010105134 824 0.01053952 hsa-miR-223-5p 339 0.002670024 708 0.009055801 hsa-miR-331-3p 923 0.00726971 757 0.009682544 hsa-miR-194-5p 509 0.004008973 718 0.009183708 hsa-miR-26b-3p 1245 0.009805839 725 0.009273243 hsa-miR-148b-5p 1205 0.009490792 751 0.0096058 hsa-miR-27a-5p 3219 0.02535341 700 0.008953476 hsa-miR-766-3p 456 0.003591536 629 0.008045337 hsa-miR-151a-5p 18400 0.144921635 616 0.007879058 hsa-let-7d-3p 1973 0.015539695 607 0.007763942 hsa-let-7i-3p 643 0.005064381 649 0.008301151 hsa-miR-22-5p 753 0.00593076 619 0.007917431 hsa-miR-148a-5p 2222 0.017500863 558 0.007137199 hsa-miR-320b 1168 0.009199373 601 0.007687198 hsa-miR-140-5p 622 0.004898981 597 0.007636036 hsa-let-7c-5p 3830 0.030165753 503 0.006433712 hsa-miR-424-5p 1905 0.015004115 594 0.007597664 hsa-miR-143-3p 426115 3.356156653 573 0.007329059 hsa-miR-374b-5p 701 0.005521199 576 0.007367431 hsa-miR-328-3p 1164 0.009167869 536 0.006855804 hsa-miR-24-2-5p 1573 0.012389225 621 0.007943012 hsa-miR-1273h-3p 463 0.003646669 472 0.006037201 hsa-miR-12136 1182 0.00930964 486 0.00621627 hsa-miR-10b-5p 426981 3.362977421 437 0.005589527 hsa-miR-1843 639 0.005032876 498 0.006369758 hsa-miR-7-1-3p 452 0.003560031 471 0.00602441 hsa-miR-598-3p 442 0.00348127 498 0.006369758 hsa-miR-126-5p 640 0.005040753 499 0.006382549 hsa-miR-744-5p 3466 0.027298825 432 0.005525573 hsa-miR-450b-5p 1475 0.011617359 482 0.006165107 hsa-miR-345-5p 1349 0.010624961 415 0.005308132 hsa-miR-130b-5p 1473 0.011601607 442 0.00565348 hsa-miR-450a-5p 718 0.005655094 447 0.005717434 hsa-miR-576-5p 602 0.004741458 386 0.004937202 hsa-miR-378d 346 0.002725157 432 0.005525573 hsa-miR-330-3p 482 0.003796317 333 0.004259296 hsa-miR-4485-3p 413 0.003252861 402 0.005141853 hsa-miR-18a-5p 471 0.003709679 437 0.005589527 hsa-miR-5701 609 0.004796591 358 0.004579063 hsa-miR-128-1-5p 311 0.002449491 367 0.004694179 hsa-miR-454-5p 418 0.003292241 323 0.004131389 hsa-miR-548bc 281 0.002213205 309 0.00395232 hsa-miR-625-5p 601 0.004733582 408 0.005218597 hsa-miR-7705 288 0.002268339 301 0.003849994 hsa-miR-590-3p 424 0.003339499 310 0.003965111 hsa-miR-212-3p 661 0.005206152 295 0.00377325 hsa-miR-181c-3p 649 0.005111638 320 0.004093017 hsa-miR-3176 329 0.002591262 299 0.003824413
