Current status and future prospects of mesenchymal stem cell therapy for liver fibrosis
J Zhejiang Univ-Sci B (Biomed & Biotechnol)
Current status and future prospects of mesenchymal stem cell therapy for liver fibrosis*
Yang GUO 0
Bo CHEN 0
Li-jun CHEN 0
Chun-feng ZHANG 0
Charlie XIANG 0
0 (State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University , Hangzhou 310003 , China)
1 Project supported by the National High-Tech R & D Program (863) of China (No. 2015AA020306) ORCID: Yang GUO
Liver fibrosis is the end-stage of many chronic liver diseases and is a significant health threat. The only effective therapy is liver transplantation, which still has many problems, including the lack of donor sources, immunological rejection, and high surgery costs, among others. However, the use of cell therapy is becoming more prevalent, and mesenchymal stem cells (MSCs) seem to be a promising cell type for the treatment of liver fibrosis. MSCs have multiple differentiation abilities, allowing them to migrate directly into injured tissue and differentiate into hepatocyte-like cells. Additionally, MSCs can release various growth factors and cytokines to increase hepatocyte regeneration, regress liver fibrosis, and regulate inflammation and immune responses. In this review, we summarize the current uses of MSC therapies for liver fibrosis and suggest potential future applications.
Liver fibrosis; Cell therapy; Mesenchymal stem cells http; //dx; doi; org/10; 1631/jzus; B1600101 CLC number; R575
Liver fibrosis is a pathologic process that occurs
between liver injury and liver cirrhosis. After liver
injury, several processes occur including cell
apoptosis, inflammation, and scarring, resulting in the
deposition of the extracellular matrix (ECM). In the
early stages, the ECM deposition can be hydrolyzed
by proteolytic enzymes such as matrix
metalloproteinases (MMPs). However, continuous damage will
ultimately lead to excessive matrix deposition and the
alteration of the normal liver structure (Lichtinghagen
et al., 2001).
Liver fibrosis can be triggered by viruses,
alcohol abuse, drug abuse, and auto-immunity, among
other conditions. When liver fibrosis is not well
controlled, it can develop into liver cirrhosis, which is the
end-stage of liver disease. Currently, liver
transplantation is the main effective therapy for liver cirrhosis,
but this treatment method is associated with many
problems, such as immunological rejection, donor
shortages, surgical complications, and high costs
(Eom et al., 2015). Therefore, finding new
therapeutic strategies for liver fibrosis is essential.
In addition to liver transplantation, cell therapy
is a common treatment method for liver disease. For
instance, hepatocyte transplantation can be used to
restore liver function because of the regeneration
abilities of these cells. However, the utility of this
treatment is limited because hepatocytes easily lose
their viability and function when they are cultured in
vitro or when they are preserved cryogenically (Eom
et al., 2015). Thus, other types of cells have been
explored in an effort to find an ideal treatment for
liver diseases. Such research has shown that stem cell
transplantation is an effective therapy for liver fibrosis
(Kakinuma et al., 2009; Kharaziha et al., 2009)
Among the different types of stem cells,
mesenchymal stem cells (MSCs) in particular have obvious
advantages in regenerative repair because of their
high potential for multipotent differentiation, capacity
for self-renewal, and low immunogenicity (Jang et al.,
2014). MSCs are fibroblast-like and plate-adhering
cells, which have the ability to self-renew and to
differentiate into adult cells from different germ layers,
such as neurocytes from ectoderm, osteoblasts and
myocytes from mesoderm, and hepatocyte-like cells
from endoderm (Chan et al., 2014). Recently, MSCs
have been isolated from a variety of tissues including
umbilical cord blood, adipose tissue, liver, lung,
dermis, amniotic membrane, and menstrual blood
(Erices et al., 2000; Campagnoli et al., 2001; Jiang
et al., 2002; de Ugarte et al., 2003; Mou et al., 2013).
Additionally, MSCs can secrete a series of cytokines
and signaling molecules, such as hepatocyte growth
factor (HGF), interleukin 6 (IL-6), tumor necrosis
factor alpha (TNF-α), epidermal growth factor (EGF),
nitric oxide, prostaglandin E2 (PGE2), and
indoleamine 2,3-dioxygenase (Ortiz et al., 2007; Kiss et al.,
2008; Puglisi et al., 2011), which can regulate
inflammatory responses, stimulate hepatocyte
proliferation, and maintain hepatocyte function (Lin et al.,
2011; Sharma et al., 2014) (Fig. 1).
In general, MSC therapy for liver fibrosis is
effective and promising, and many studies have been
performed in this field. Thus, in this review, we
discuss the current research regarding the mechanisms
and uses of MSC therapy for liver fibrosis and the
associated limitations, and we suggest some potential
future applications of this therapy.
2 Mechanisms of fibrogenesis in the liver
The mechanisms of liver fibrosis are complex
and involve a variety of cytokines, growth factors,
and signaling pathways. Although many studies have
been performed, the exact mechanisms of
fibrogenesis remain unknown. It has been established that an
imbalance between ECM production and degradation
is the precipitating cause of liver fibrosis (Ek et al.,
2007). However, how the imbalance happens is unclear.
The universally accepted key mechanism
involved in ECM accumulation is the activation of
transforming growth factor beta (TGF-β)/Smad
(Wrana, 1999; Berardis et al., 2015)
, which is
mediated by transmembrane serine/threonine kinase
receptors including type I and type II. In the injured
liver, the microenvironment promotes the activation
of Kupffer cells, which in turn exert proinflammatory
cytokines such as TNF-α, TGF-α, TGF-β, and
plateletderived growth factor (PDGF), among others. The
increased TGF-β, combined with the type II receptor,
activates the type I receptor and forms a complex.
Then, the complex phosphorylates, for the
downstream signal transduction molecules Smad2/3, are
translocated into the nucleus where they regulate
transcriptional responses such as collagens.
The activated hepatic stellate cells (HSCs),
which can transform into myofibroblast-like cells,
also play a critical role in the production of ECM
(Berardis et al., 2015)
. When stimulated by lipid
peroxides, products from injured hepatocytes, or
biochemical signals from Kupffer cells, HSCs can
become activated and exhibit the following: high
expression of alpha smooth muscle actin (α-SMA),
tissue inhibitors of metalloproteinases (TIMPs)-1/2,
the secretion of collagen-1, and increased
(Iredale et al., 1992; Friedman, 1993;
Benyon et al., 1996)
. The activation can occur
through autocrine and paracrine signaling pathways;
one of the main pathways is the PDGF-β signaling
pathway. The PDGF-β signaling pathway can activate
other pathways, such as the Ras-mitogen-activated
protein kinase, phosphoinositide 3-kinase-AKT/protein
kinase B, and protein kinase C pathways, resulting in
HSC proliferation (Kelly et al., 1991). In addition to
HSCs, other cell types, such as circulating fibrocytes,
portal fibroblasts, and bone marrow-derived cells, are
believed to contribute to ECM deposition (Forbes
et al., 2004; Wells et al., 2004).
Recent studies have shown that the development
of liver fibrosis is accompanied by the expression of
MMPs (Lichtinghagen et al., 2001). MMPs are
critical for the regression of fibrogenesis in which they can
degrade collagens and are involved in the early stages
of tissue remodeling (Milani et al., 1994; Benyon et al.,
1996). Moreover, TIMPs, which can be produced by
activated HSCs, are believed to induce ECM
deposition by slowing the breakdown of collagens (Arthur,
1995; 1997). It is known that the expression of TIMPs
is mainly induced by inflammation responses.
Inflammation factors like IL-1β and TNF-α can
promote TIMP expression. Thus, the expression level
and activity of TIMPs can be used as indicators to
measure the disease process. In general, the balance
between MMPs and TIMPs plays an important role in
liver fibrosis (Bӧker et al., 2000).
Thus, it is clear that the TGF-β/Smad signaling
pathway plays a critical role in ECM accumulation.
When this pathway is activated, the downstream
factors notably increase, which induces the
expression of fibrosis-related genes including the genes for
collagen-1, α-SMA (a surface marker of HSCs), and
TIMPs. As a result, the number of activated HSCs
increases, whereupon the degradation cannot match
the production of ECM, resulting in liver fibrosis
It is known that MSCs have the capacity to dif
ferentiate into various progenitor cells from different
cell lines, including hepatic progenitor cells. Indeed, a
variety of studies
(Banas et al., 2007; Ishii et al., 2008;
Kakinuma et al., 2009; Puglisi et al., 2011; Hang
et al., 2014)
have demonstrated the ability of MSCs to
differentiate into hepatocyte-like cells by examining
the expression of specific hepatocyte markers such as
albumin, α-fetoprotein, and cytokeratin-19, among
The ability of MSCs to differentiate into
hepatocyte-like cells makes them an ideal alternative
method for treating liver fibrosis. Therefore, many
studies have examined the mechanisms underlying
the differentiation ability of MSCs. Several recent
studies (Yoshida et al., 2007; Ishii et al., 2008; Liu
et al., 2015) have demonstrated that Wnt/β-catenin
signaling plays an important role in regulating the
hepatic differentiation of human MSCs. Upon Wnt
signaling activation, β-catenin will translocate into
the nucleus and coactivate downstream transcription
factors to regulate the differentiation of MSCs.
Furthermore, mesenchymal-epithelial transition and the
reverse, epithelial-mesenchymal transition are critical
developmental processes that play fundamental roles
in the differentiation of multiple tissues (Hay, 2005).
Epigenetic modifications, such as DNA methylation
and histone acetylation, have also been shown to
participate in the differentiation of MSCs (Snykers
et al., 2007).
Additionally, some studies have tried to enhance
the efficiency of MSC differentiation, since during
regular differentiation, MSCs have low metabolic
activity and low expression of functional proteins (Ek
et al., 2007). For instance,
Mohsin et al. (2011)
demonstrated that pretreating MSCs with injured liver
tissue enhances their differentiation ability owing to
the growth factors and cytokines that are released by
the injured tissue, such as HGF, insulin-like growth
factor (IGF), EGF, and basic fibroblast growth factor
(bFGF), among others (Liu et al., 2015).
While it is clear that MSCs have
multidifferentiation abilities including the ability to
differentiate into hepatocyte-like cells, it remains
unclear whether MSCs can adopt a mature hepatic fate,
as no reliable and detailed results of mature
hepatocytic gene expression have been reported. Many
researchers believe that MSCs can become hepatocytes
both morphologically and functionally. For instance,
Banas et al. (2007) and Yin et al. (2015)
demonstrated that adipose tissue-derived MSCs could be
induced into transplantable and mature
hepatocytelike cells both in vivo and in vitro. Moreover, these
studies showed that differentiated MSCs express
hepatocyte-specific markers including albumin and
α-fetoprotein and share liver functions such as
lowdensity lipoprotein uptake, glucose storage, and
In contrast, other researchers have opposed the
opinion that MSCs can adopt a mature hepatic fate by
claiming that only early specific markers have been
detected and noting that little credible data on the
detection of mature hepatocyte markers exist.
Campard et al. (2008) conducted a study to detect the
differentiation ability of umbilical cord matrix stem
cells; the results showed that the differentiated
umbilical cord matrix stem cells exhibited
hepatocytelike morphologies, specific liver markers (e.g.,
albumin, α-fetoprotein, cytokeratin-19, connexin-32), and
some hepatic functions including glucose storage,
low-density lipoprotein uptake, and urea production.
However, the cells did not express hepatocyte nuclear
factor 4 or HepPar1, two specific hepatic makers. In
addition, the differentiated MSCs still contained some
MSC-specific makers. Collectively, these findings
suggested that the differentiated MSCs did not
express enough markers of mature hepatocytes,
implying that MSCs cannot fully become hepatocytes. Lian
et al. (2006) demonstrated that bone marrow (BM)
hematopoietic stem cells expressed several hepatic
markers but could not be efficiently converted into
hepatocyte-like cells, as one of the mature hepatic
markers (anti-trypsin) was not detected. Another
study (Hengstler et al., 2005), based on drug
metabolism, showed that it is unlikely that MSCs fully
differentiate into hepatocytes, and it has also noted
that the use of different protocols for hepatic
differentiation and different detection methods are
problematic. Therefore, specific criteria are needed to
define hepatocyte-like cells derived from MSCs. It
has been suggested that the definition should not only
be based on qualitative analyses but also on
quantitative analyses including analyses of enzyme activity.
Overall, MSCs have the potential to differentiate
into immature hepatocyte-like cells that exhibit some
early specific hepatic markers and functions.
Regardless, MSCs are an optimal choice for treating
liver fibrosis because of their paracrine effects and
immunologic regulation in addition to their
4 Paracrine effect of MSCs
MSCs have the ability to migrate into injured
tissues via chemotaxis due to cytokines that are
released from the injured organ or tissues (Golzar et al.,
2015; Lourenco et al., 2015). Under stimulation of the
microenvironment in injured tissue, like some
inflammation factors, MSCs can release various growth
factors and cytokines, which promote the
proliferation of endogenous hepatocytes, reduce hepatocyte
apoptosis, enhance liver function, and repress
inflammatory responses (Zhou et al., 2009; Lin et al.,
Research shows that MSCs secrete cytokines,
such as HGF, EGF, IL-6, and TNF-α, which can
stimulate hepatocyte proliferation and enhance liver
function, as indicated by the high levels of albumin
and urea secretion. For instance, Kim et al. (2014)
overexpressed HGF by transducing MSCs with an
adenovirus vector carrying the HGF gene. Their
results showed a decrease in collagen and lower mRNA
levels of the fibrogenic cytokines PDGF-bb and
TGF-β1, suggesting that MSCs that overexpress HGF
are effective in the treatment of liver fibrosis.
Additionally, MSCs can release other cytokines such as
IGF-1, stromal cell-derived factor-1 (SDF-1), and a
vascular endothelial growth factor (VEGF), which
inhibit cell apoptosis mainly by regulating the
SDF-1/CX chemokine receptor-4 (CXCR-4) axis
(Lin et al., 2011). IGF-1 is an important factor in body
metabolism, and has been demonstrated to be
antiapoptotic to hepatocytes and increase the secretion of
HGF in the cirrhotic liver
(Bonefeld and Møller,
. Fiore et al. (2015) used a recombinant
adenovirus overexpressing IGF-1 in BM-MSCs to
ameliorate liver fibrosis in mice. The application of
BMderived AdIGF-I-MSCs resulted in the reduced
activation of HSCs, increased IGF-I and HGF
expression, reduced fibrogenesis, and increased hepatocyte
Moreover, researches (Siller-López et al., 2004;
Snykers et al., 2007) have demonstrated that MSCs
overexpressing MMPs promote the regression of liver
fibrosis. MSCs have the potential to reverse the
fibrotic process by inhibiting collagen deposition
through high levels of MMPs including MMP-8,
MMP-9, and MMP-13. MMPs have been shown to
degrade the ECM directly in order to balance the
increased TIMPs that are induced by activated HSCs,
thus contributing to the regression of fibrogenesis
(Lin et al., 2011). In addition, the blockades of
MSC-derived IL-10 and TNF-α exhibit minimal
inhibitory effects on HSC proliferation and collagen
synthesis, demonstrating the anti-fibrogenic effects of
IL-10 and TNF-α (Parekkadan et al., 2007).
It is known that cytokines are important factors
that participate in inflammation, as they can mediate
inflammatory responses and prevent inflammatory
effects. TNF-α, IL-1, and IL-6 are familiar
proinflammatory factors that play critical roles in
activating immunocytes and in regulating tissue metabolism
(Liu et al., 2013; Huang et al., 2015). In injured tissue,
TNF-α is one of the first factors to be released, which
then activates neutrophil granulocytes and
lymphocytes and induces the secretion of other inflammation
factors. In a lung injury model, MSCs have been
shown to express an IL-1 receptor antagonist that
blocks the release of TNF-α from activated
macrophages, thus preventing tissue damage (Ortiz et al.,
5 MSC therapy and immunoregulation
It has been established that MSCs possess
remarkable immunosuppressive properties that inhibit
the proliferation and function of immune cells from
both the adaptive and innate immune systems (Shi
et al., 2011). The immunomodulatory effects of MSCs
are mediated through both a cell-cell contact and
secreted factors such as PGE2, nitric oxide, and
TGF-β. MSCs can also inhibit the proliferation of T
lymphocytes through cell contact (Tse et al., 2003;
Sotiropoulou et al., 2006) and through soluble
cytokines such as HGF, IL-1β, TGF-β1, interferon gamma
(IFN-γ), and indoleamine 2,3-dioxygenase (di Nicola
et al., 2002; Meisel et al., 2004; Groh et al., 2005;
Krampera et al., 2006), which is indicated by an
increase in the number of cells in the G0/G1 phase
(Glennie et al., 2005) and by the up-regulated
expression of p27 (Krampera et al., 2003). Further,
MSCs can inhibit CD4+ T cells, CD8+ T cells (Glennie
et al., 2005), T-helper lymphocytes (Th1/Th17)
(Aggarwal and Pittenger, 2005; Zappia et al., 2005)
and cytotoxic T cells (Potian et al., 2003; Rasmusson
et al., 2003). The suppression effect of MSCs on T
cells can indirectly act on B lymphocytes because B
cell activation mainly depends on T cells. Additionally,
MSCs can directly inhibit the proliferation of B
lymphocytes, the production of antibodies, and chemotaxis
when co-stimulating with anti-immunoglobulin
antibodies, anti-CD40L and IL-4 in humans (Corcione
et al., 2006).
MSCs also have suppression effects on cells
belonging to the innate immune system, including
natural killer (NK) cells, dendritic cells (DCs),
monocytes, and macrophages. Studies have shown that
MSCs can only partially suppress the proliferation of
activated NK cells. Some cell factors such as TGF-β1
and PGE2 are believed to participate in the
suppression of NK cell proliferation (Rasmusson et al., 2003;
Krampera et al., 2006). In addition, MSCs can affect
the production of DCs by inhibiting the
differentiation of monocytes, as MSCs can block the maturation
signals and co-stimulatory molecules (Zhang et al.,
2004; Jiang et al., 2005; Nauta et al., 2006). On the
other hand, MSCs reduce the proinflammatory ability
of DCs by decreasing the secretion of TNF-α, IFN-γ,
and IL-12 and increasing IL-10 secretion (Zhang et al.,
2004; Jiang et al., 2005).
In summary, the ability of MSCs to regulate
immune responses is an important advantage for cell
therapy and allogeneic transplantation. It is known that
MSCs have low immunogenicity because they lack
human leukocyte antigen class II and co-stimulatory
molecules such as CD80, CD86, and CD40 in the
cytomembrane (Reinders et al., 2013). In addition,
the sources of MSCs are various and abundant. MSCs
also have direct migration abilities and a high
differentiation capacity. Considering all of these
characteristics, MSCs are the ideal transplant donors in
6 MSC therapy and CRISPR/Cas9
Currently, genome editing is widely used in
studies involving functional genomics, transgenic
animals, and gene therapy. Genome editing is based on
programmable and highly specific nucleases, which
generate site-specific cleavage and subsequently
induce cellular DNA repair (Zhang et al., 2014).
Multiple artificial nuclease systems have been developed for
genome editing, including zinc-finger nucleases,
transcription activator-like effector nucleases, and
clustered regularly interspaced short palindromic repeats
(CRISPR) and the CRISPR-associated (Cas) protein 9.
Zinc-finger nucleases and transcription activator-like
effector nucleases, based on protein-DNA interactions,
are more complex and time-consuming compared with
CRISPR/Cas9, which is easier and more efficient when
using guide RNA (gRNA) and DNA targeting.
CRISPR/Cas9 is widely used in genetic
modification, transcription regulation, and gene therapy
studies. Researches have demonstrated that CRISPR/
Cas9 can be used to conduct genomic editing in many
organisms, including in bacteria (Jiang et al., 2013),
drosophila (Gratz et al., 2013), zebrafish (Hruscha
et al., 2013), mice (Wang H. et al., 2013),
Caenorhabditis elegans (Friedland et al., 2013), and
Bombyx mori (Wang Y. et al., 2013). Furthermore, in
terms of the development of stem cell therapy,
CRISPR/Cas9 has been widely applied in the accurate
and complex genetic manipulation of stem cells to
enhance their reprogramming, differentiation, and
other functions. Mandal et al. (2014) successfully
silenced the expression of the genes B2M and CCR5
in human hematopoietic cells using CRISPR/Cas9
with minimal off-target mutagenesis. Additionally,
Wettstein et al. (2016) transfected two paired CRISPR
single guide RNAs (sgRNAs)-Cas9 plasmids into
mouse embryonic stem cells, which resulted in the
knock-out of the targeted gene.
CRISPR/Cas9 provides us with a more efficient
way to optimize MSC therapy for liver fibrosis. We
can transform MSCs using different aspects to
enhance their vitality and function, including their
proliferation and differentiation ability, chemotaxis for
injured tissue, and anti-inflammatory capacity. To aid
in this, Schmidt et al. (2015) successfully built an
arrayed sgRNA library that can target one critical
exon of almost every protein-coding gene in humans.
Therefore, by using the sgRNA library, we can find
genes related to the various characteristics of MSCs,
and then knockout the specific gene to optimize the
It is also possible to take advantage of
homologous recombination to overexpress targeted genes
through CRISPR/Cas9. As mentioned above,
genetically engineered MSCs that overexpress certain
genes such as the genes for HGF and IGF-1 have
therapeutic effects on liver fibrosis. However, it is
unclear how we can overexpress specific genes stably
without affecting the MSC function or the expression
of other genes. This problem is critical. Currently, the
use of recombinant virus infection is fervent,
including the use of non-integrating viruses like RNA
viruses, modified lentiviruses, and integrating
adenoviruses (Seah et al., 2015). The efficiency of virus
infection and the level of gene expression are both
high; however, there are still some problems with this
method. Non-integrating viruses will not integrate
into the cell genome; therefore, the heterologous
gene will not be stably expressed as cell proliferation.
Thus, integrated adenoviruses are a good vector for
targeted gene overexpression. However, adenoviruses,
lentiviruses, and RNA viruses are all viruses,
meaning that they are associated with pathogenic risks in
clinical treatments. Therefore, finding a new method
CRISPR/Cas9 is a promising tool that may allow
us to transform MSCs in order to overexpress targeted
genes. Currently, our lab is performing some related
experiments. We have constructed a donor vector that
contains the targeted gene, and next we will transfect
it with the CRISPR sgRNAs-Cas9 plasmid into MSCs.
Taking advantage of homology-direct repair, targeted
genes can be combined into the genomic DNA of the
MSCs and stably expressed through proliferation
(Fig. 3). Our goal is to obtain the targeted gene in a
stably expressed cell line, which can then be used to
treat liver fibrosis. However, the transfection
efficiency is not high; hence, additional research is
needed to improve the efficiency.
In general, CRISPR/Cas9 can be used to reform
stem cells. Additionally, stem cell therapy combined
with genomic editing will be a promising method for
many diseases in the future.
7 Current problems and future prospects
The transplantation of MSCs for the treatment of
liver fibrosis is an effective and promising method,
considering the targeted migration ability, release
capacity, and low immunogenicity of MSCs. MSCs
can directly interact with the fibrogenic liver by
differentiating into hepatocyte-like cells or by fusing
with hepatocytes. Additionally, MSCs have the
potential to release different growth factors and
cytokines, which can regulate the microenvironment
and immune system to enhance their therapeutic
effects on liver fibrosis. MSCs can also be combined
with gene engineering to create a new method that can
obviously regress fibrogenesis, promote regeneration,
and restore the liver function. Therefore, MSC
therapy for liver fibrosis is an optimal choice. However,
many issues with these methods still need to be
resolved. For instance, several different types of MSCs
exist, which each have their respective advantages
and disadvantages. The isolation of BM-MSCs is
strenuous and traumatic. In contrast, MSCs derived
from adipose tissue-derived MSCs are abundant and
easily obtained, but the therapeutic effect is inferior to
that of BM-MSCs (Liu et al., 2015). Moreover, we
still do not fully understand the mechanisms
underlying the therapeutic effects of MSCs. Therefore, the
oncogenic potential and the risks of using MSCs
remain unknown. In addition, when combining MSCs
with gene engineering, the transfection problem
exists, which will require finding a better transfection
condition to increase the efficiency. In general, there
is still much for us to explore regarding the use of
MSCs in the treatment of liver fibrosis.
We thank Dr. Qiu-rong DING (Chinese Academy of
Sciences in Shanghai, China) for his guidance on genome editing
techniques. Additionally, we thank Editage (https://www.
editage.com) for assistance with the English language editing.
Compliance with ethics guidelines
Yang GUO, Bo CHEN, Li-jun CHEN, Chun-feng ZHANG,
and Charlie XIANG declare that they have no conflict of
This article does not contain any studies with human or
animal subjects performed by any of the authors.
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