Current status and future prospects of mesenchymal stem cell therapy for liver fibrosis - PDF Flipbook

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Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841 831

Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
www.zju.edu.cn/jzus; www.springerlink.com
E-mail: [email protected]

Review:

Current status and future prospects of mesenchymal
stem cell therapy for liver fibrosis*

Yang GUO†, Bo CHEN, Li-jun CHEN, Chun-feng ZHANG, Charlie XIANG†‡

(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)

†E-mail: [email protected]; [email protected]
Received Mar. 3, 2016; Revision accepted May 14, 2016; Crosschecked Oct. 10, 2016

Abstract: 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, immu-
nological rejection, and high surgery costs, among others. However, the use of cell therapy is becoming more prev-
alent, 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.

Key words: Liver fibrosis, Cell therapy, Mesenchymal stem cells

http://dx.doi.org/10.1631/jzus.B1600101 CLC number: R575

1 Introduction other conditions. When liver fibrosis is not well con-
trolled, it can develop into liver cirrhosis, which is the
Liver fibrosis is a pathologic process that occurs end-stage of liver disease. Currently, liver transplan-
between liver injury and liver cirrhosis. After liver tation is the main effective therapy for liver cirrhosis,
injury, several processes occur including cell apop- but this treatment method is associated with many
tosis, inflammation, and scarring, resulting in the problems, such as immunological rejection, donor
deposition of the extracellular matrix (ECM). In the shortages, surgical complications, and high costs
early stages, the ECM deposition can be hydrolyzed (Eom et al., 2015). Therefore, finding new therapeu-
by proteolytic enzymes such as matrix metallopro- tic strategies for liver fibrosis is essential.
teinases (MMPs). However, continuous damage will
ultimately lead to excessive matrix deposition and the In addition to liver transplantation, cell therapy
alteration of the normal liver structure (Lichtinghagen is a common treatment method for liver disease. For
et al., 2001). instance, hepatocyte transplantation can be used to
restore liver function because of the regeneration
Liver fibrosis can be triggered by viruses, alco- abilities of these cells. However, the utility of this
hol abuse, drug abuse, and auto-immunity, among treatment is limited because hepatocytes easily lose
their viability and function when they are cultured in
‡ Corresponding author vitro or when they are preserved cryogenically (Eom
* Project supported by the National High-Tech R & D Program (863) et al., 2015). Thus, other types of cells have been
of China (No. 2015AA020306) explored in an effort to find an ideal treatment for
liver diseases. Such research has shown that stem cell
ORCID: Yang GUO, http://orcid.org/0000-0002-2125-0637 transplantation is an effective therapy for liver fibrosis
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2016

832 Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841

(Kakinuma et al., 2009; Kharaziha et al., 2009). In general, MSC therapy for liver fibrosis is ef-
Among the different types of stem cells, mesenchy- fective and promising, and many studies have been
mal stem cells (MSCs) in particular have obvious performed in this field. Thus, in this review, we dis-
advantages in regenerative repair because of their cuss the current research regarding the mechanisms
high potential for multipotent differentiation, capacity and uses of MSC therapy for liver fibrosis and the
for self-renewal, and low immunogenicity (Jang et al., associated limitations, and we suggest some potential
2014). MSCs are fibroblast-like and plate-adhering future applications of this therapy.
cells, which have the ability to self-renew and to dif-
ferentiate into adult cells from different germ layers, 2 Mechanisms of fibrogenesis in the liver
such as neurocytes from ectoderm, osteoblasts and
myocytes from mesoderm, and hepatocyte-like cells The mechanisms of liver fibrosis are complex
from endoderm (Chan et al., 2014). Recently, MSCs and involve a variety of cytokines, growth factors,
have been isolated from a variety of tissues including and signaling pathways. Although many studies have
umbilical cord blood, adipose tissue, liver, lung, been performed, the exact mechanisms of fibrogene-
dermis, amniotic membrane, and menstrual blood sis remain unknown. It has been established that an
(Erices et al., 2000; Campagnoli et al., 2001; Jiang imbalance between ECM production and degradation
et al., 2002; de Ugarte et al., 2003; Mou et al., 2013). is the precipitating cause of liver fibrosis (Ek et al.,
Additionally, MSCs can secrete a series of cytokines 2007). However, how the imbalance happens is unclear.
and signaling molecules, such as hepatocyte growth
factor (HGF), interleukin 6 (IL-6), tumor necrosis The universally accepted key mechanism in-
factor alpha (TNF-α), epidermal growth factor (EGF), volved in ECM accumulation is the activation of
nitric oxide, prostaglandin E2 (PGE2), and indoleam- transforming growth factor beta (TGF-β)/Smad sig-
ine 2,3-dioxygenase (Ortiz et al., 2007; Kiss et al., naling (Wrana, 1999; Berardis et al., 2015), which is
2008; Puglisi et al., 2011), which can regulate in- mediated by transmembrane serine/threonine kinase
flammatory responses, stimulate hepatocyte prolifer- receptors including type I and type II. In the injured
ation, and maintain hepatocyte function (Lin et al., liver, the microenvironment promotes the activation
2011; Sharma et al., 2014) (Fig. 1). of Kupffer cells, which in turn exert proinflammatory

Fig. 1 Sources and function of mesenchymal stem cells (MSCs)
The sources of MSCs are abundant, including umbilical cord blood, adipose tissue, liver, lung, dermis, amniotic membrane, and
menstrual blood, among others. MSCs can differentiate into hepatocytes, secrete various cell factors, and participate in im-
munoregulation. IFN-γ: interferon gamma; IL: interleukin; TGF: transforming growth factor; HGF: hepatocyte growth factor;
IDO: indoleamine 2,3-dioxygenase; PGE2: prostaglandin E2; EGF: epidermal growth factor; IGF: insulin-like growth factor;

TNF: tumor necrosis factor; NK cells: natural killer cells

Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841 833

cytokines such as TNF-α, TGF-α, TGF-β, and platelet- Thus, it is clear that the TGF-β/Smad signaling
derived growth factor (PDGF), among others. The pathway plays a critical role in ECM accumulation.
increased TGF-β, combined with the type II receptor, When this pathway is activated, the downstream
activates the type I receptor and forms a complex. factors notably increase, which induces the expres-
Then, the complex phosphorylates, for the down- sion of fibrosis-related genes including the genes for
stream signal transduction molecules Smad2/3, are collagen-1, α-SMA (a surface marker of HSCs), and
translocated into the nucleus where they regulate TIMPs. As a result, the number of activated HSCs
transcriptional responses such as collagens. increases, whereupon the degradation cannot match
the production of ECM, resulting in liver fibrosis
The activated hepatic stellate cells (HSCs), (Fig. 2).
which can transform into myofibroblast-like cells,
also play a critical role in the production of ECM Fig. 2 Signaling pathway of liver fibrogenisis
(Berardis et al., 2015). When stimulated by lipid The injured liver produces various cell factors, which can
peroxides, products from injured hepatocytes, or bi- activate transforming growth factor beta (TGF-β) and
ochemical signals from Kupffer cells, HSCs can be- platelet-derived growth factor beta (PDGF-β), and bone
come activated and exhibit the following: high ex- morphogenetic protein (BMP). Through the TGF-β signal-
pression of alpha smooth muscle actin (α-SMA), ing pathway, Smad2/3 are phosphorylated and activate
tissue inhibitors of metalloproteinases (TIMPs)-1/2, downstream molecules such as extracellular matrix (ECM)
the secretion of collagen-1, and increased prolifera- genes, resulting in the deposition of collagen. Hepatic stel-
tion ability (Iredale et al., 1992; Friedman, 1993; late cells (HSCs) are greatly increased through the PDGF-β
Benyon et al., 1996). The activation can occur pathway and they induce the secretion of collagens and
through autocrine and paracrine signaling pathways; tissue inhibitors of metalloproteinases (TIMPs). Thus, the
one of the main pathways is the PDGF-β signaling degradation of collagens decreases and the secretion in-
pathway. The PDGF-β signaling pathway can activate creases, leading to the deposition of collagens, which results
other pathways, such as the Ras-mitogen-activated in worsened liver disease. α-SMA: alpha smooth muscle actin
protein kinase, phosphoinositide 3-kinase-AKT/protein
kinase B, and protein kinase C pathways, resulting in 3 Differentiation ability of MSCs
HSC proliferation (Kelly et al., 1991). In addition to
HSCs, other cell types, such as circulating fibrocytes, It is known that MSCs have the capacity to dif-
portal fibroblasts, and bone marrow-derived cells, are ferentiate into various progenitor cells from different
believed to contribute to ECM deposition (Forbes cell lines, including hepatic progenitor cells. Indeed, a
et al., 2004; Wells et al., 2004). variety of studies (Banas et al., 2007; Ishii et al., 2008;
Kakinuma et al., 2009; Puglisi et al., 2011; Hang
Recent studies have shown that the development et al., 2014) have demonstrated the ability of MSCs to
of liver fibrosis is accompanied by the expression of
MMPs (Lichtinghagen et al., 2001). MMPs are criti-
cal 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 deposi-
tion by slowing the breakdown of collagens (Arthur,
1995; 1997). It is known that the expression of TIMPs
is mainly induced by inflammation responses. In-
flammation factors like IL-1β and TNF-α can pro-
mote 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).

834 Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841

differentiate into hepatocyte-like cells by examining studies showed that differentiated MSCs express
the expression of specific hepatocyte markers such as hepatocyte-specific markers including albumin and
albumin, α-fetoprotein, and cytokeratin-19, among α-fetoprotein and share liver functions such as low-
others. density lipoprotein uptake, glucose storage, and
ammonia detoxification.
The ability of MSCs to differentiate into
hepatocyte-like cells makes them an ideal alternative In contrast, other researchers have opposed the
method for treating liver fibrosis. Therefore, many opinion that MSCs can adopt a mature hepatic fate by
studies have examined the mechanisms underlying claiming that only early specific markers have been
the differentiation ability of MSCs. Several recent detected and noting that little credible data on the
studies (Yoshida et al., 2007; Ishii et al., 2008; Liu detection of mature hepatocyte markers exist. Cam-
et al., 2015) have demonstrated that Wnt/β-catenin pard et al. (2008) conducted a study to detect the
signaling plays an important role in regulating the differentiation ability of umbilical cord matrix stem
hepatic differentiation of human MSCs. Upon Wnt cells; the results showed that the differentiated um-
signaling activation, β-catenin will translocate into bilical cord matrix stem cells exhibited hepatocyte-
the nucleus and coactivate downstream transcription like morphologies, specific liver markers (e.g., albu-
factors to regulate the differentiation of MSCs. Fur- min, α-fetoprotein, cytokeratin-19, connexin-32), and
thermore, mesenchymal-epithelial transition and the some hepatic functions including glucose storage,
reverse, epithelial-mesenchymal transition are critical low-density lipoprotein uptake, and urea production.
developmental processes that play fundamental roles However, the cells did not express hepatocyte nuclear
in the differentiation of multiple tissues (Hay, 2005). factor 4 or HepPar1, two specific hepatic makers. In
Epigenetic modifications, such as DNA methylation addition, the differentiated MSCs still contained some
and histone acetylation, have also been shown to MSC-specific makers. Collectively, these findings
participate in the differentiation of MSCs (Snykers suggested that the differentiated MSCs did not ex-
et al., 2007). press enough markers of mature hepatocytes, imply-
ing that MSCs cannot fully become hepatocytes. Lian
Additionally, some studies have tried to enhance et al. (2006) demonstrated that bone marrow (BM)
the efficiency of MSC differentiation, since during hematopoietic stem cells expressed several hepatic
regular differentiation, MSCs have low metabolic markers but could not be efficiently converted into
activity and low expression of functional proteins (Ek hepatocyte-like cells, as one of the mature hepatic
et al., 2007). For instance, Mohsin et al. (2011) markers (anti-trypsin) was not detected. Another
demonstrated that pretreating MSCs with injured liver study (Hengstler et al., 2005), based on drug metab-
tissue enhances their differentiation ability owing to olism, showed that it is unlikely that MSCs fully
the growth factors and cytokines that are released by differentiate into hepatocytes, and it has also noted
the injured tissue, such as HGF, insulin-like growth that the use of different protocols for hepatic differ-
factor (IGF), EGF, and basic fibroblast growth factor entiation and different detection methods are prob-
(bFGF), among others (Liu et al., 2015). lematic. Therefore, specific criteria are needed to
define hepatocyte-like cells derived from MSCs. It
While it is clear that MSCs have multi- has been suggested that the definition should not only
differentiation abilities including the ability to dif- be based on qualitative analyses but also on quantita-
ferentiate into hepatocyte-like cells, it remains un- tive analyses including analyses of enzyme activity.
clear whether MSCs can adopt a mature hepatic fate,
as no reliable and detailed results of mature hepato- Overall, MSCs have the potential to differentiate
cytic gene expression have been reported. Many re- into immature hepatocyte-like cells that exhibit some
searchers believe that MSCs can become hepatocytes early specific hepatic markers and functions. Re-
both morphologically and functionally. For instance, gardless, MSCs are an optimal choice for treating
Banas et al. (2007) and Yin et al. (2015) demon- liver fibrosis because of their paracrine effects and
strated that adipose tissue-derived MSCs could be immunologic regulation in addition to their multi-
induced into transplantable and mature hepatocyte- differentiation potential.
like cells both in vivo and in vitro. Moreover, these

Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841 835

4 Paracrine effect of MSCs increased TIMPs that are induced by activated HSCs,
thus contributing to the regression of fibrogenesis
MSCs have the ability to migrate into injured (Lin et al., 2011). In addition, the blockades of
tissues via chemotaxis due to cytokines that are re- MSC-derived IL-10 and TNF-α exhibit minimal in-
leased from the injured organ or tissues (Golzar et al., hibitory effects on HSC proliferation and collagen
2015; Lourenco et al., 2015). Under stimulation of the synthesis, demonstrating the anti-fibrogenic effects of
microenvironment in injured tissue, like some in- IL-10 and TNF-α (Parekkadan et al., 2007).
flammation factors, MSCs can release various growth
factors and cytokines, which promote the prolifera- It is known that cytokines are important factors
tion of endogenous hepatocytes, reduce hepatocyte that participate in inflammation, as they can mediate
apoptosis, enhance liver function, and repress in- inflammatory responses and prevent inflammatory
flammatory responses (Zhou et al., 2009; Lin et al., effects. TNF-α, IL-1, and IL-6 are familiar proin-
2011). flammatory factors that play critical roles in activat-
ing immunocytes and in regulating tissue metabolism
Research shows that MSCs secrete cytokines, (Liu et al., 2013; Huang et al., 2015). In injured tissue,
such as HGF, EGF, IL-6, and TNF-α, which can TNF-α is one of the first factors to be released, which
stimulate hepatocyte proliferation and enhance liver then activates neutrophil granulocytes and lympho-
function, as indicated by the high levels of albumin cytes and induces the secretion of other inflammation
and urea secretion. For instance, Kim et al. (2014) factors. In a lung injury model, MSCs have been
overexpressed HGF by transducing MSCs with an shown to express an IL-1 receptor antagonist that
adenovirus vector carrying the HGF gene. Their re- blocks the release of TNF-α from activated macro-
sults showed a decrease in collagen and lower mRNA phages, thus preventing tissue damage (Ortiz et al.,
levels of the fibrogenic cytokines PDGF-bb and 2007).
TGF-β1, suggesting that MSCs that overexpress HGF
are effective in the treatment of liver fibrosis. Addi- 5 MSC therapy and immunoregulation
tionally, MSCs can release other cytokines such as
IGF-1, stromal cell-derived factor-1 (SDF-1), and a It has been established that MSCs possess re-
vascular endothelial growth factor (VEGF), which markable immunosuppressive properties that inhibit
inhibit cell apoptosis mainly by regulating the the proliferation and function of immune cells from
SDF-1/CX chemokine receptor-4 (CXCR-4) axis both the adaptive and innate immune systems (Shi
(Lin et al., 2011). IGF-1 is an important factor in body et al., 2011). The immunomodulatory effects of MSCs
metabolism, and has been demonstrated to be anti- are mediated through both a cell-cell contact and
apoptotic to hepatocytes and increase the secretion of secreted factors such as PGE2, nitric oxide, and
HGF in the cirrhotic liver (Bonefeld and Møller, TGF-β. MSCs can also inhibit the proliferation of T
2011). Fiore et al. (2015) used a recombinant adeno- lymphocytes through cell contact (Tse et al., 2003;
virus overexpressing IGF-1 in BM-MSCs to amelio- Sotiropoulou et al., 2006) and through soluble cyto-
rate liver fibrosis in mice. The application of BM- kines such as HGF, IL-1β, TGF-β1, interferon gamma
derived AdIGF-I-MSCs resulted in the reduced ac- (IFN-γ), and indoleamine 2,3-dioxygenase (di Nicola
tivation of HSCs, increased IGF-I and HGF expres- et al., 2002; Meisel et al., 2004; Groh et al., 2005;
sion, reduced fibrogenesis, and increased hepatocyte Krampera et al., 2006), which is indicated by an in-
proliferation. crease in the number of cells in the G0/G1 phase
(Glennie et al., 2005) and by the up-regulated ex-
Moreover, researches (Siller-López et al., 2004; pression of p27 (Krampera et al., 2003). Further,
Snykers et al., 2007) have demonstrated that MSCs MSCs can inhibit CD4+ T cells, CD8+ T cells (Glennie
overexpressing MMPs promote the regression of liver et al., 2005), T-helper lymphocytes (Th1/Th17)
fibrosis. MSCs have the potential to reverse the fi- (Aggarwal and Pittenger, 2005; Zappia et al., 2005),
brotic process by inhibiting collagen deposition and cytotoxic T cells (Potian et al., 2003; Rasmusson
through high levels of MMPs including MMP-8, et al., 2003). The suppression effect of MSCs on T
MMP-9, and MMP-13. MMPs have been shown to
degrade the ECM directly in order to balance the

836 Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841

cells can indirectly act on B lymphocytes because B scription activator-like effector nucleases, and clus-
cell activation mainly depends on T cells. Additionally, tered regularly interspaced short palindromic repeats
MSCs can directly inhibit the proliferation of B lym- (CRISPR) and the CRISPR-associated (Cas) protein 9.
phocytes, the production of antibodies, and chemotaxis Zinc-finger nucleases and transcription activator-like
when co-stimulating with anti-immunoglobulin an- effector nucleases, based on protein-DNA interactions,
tibodies, anti-CD40L and IL-4 in humans (Corcione are more complex and time-consuming compared with
et al., 2006). CRISPR/Cas9, which is easier and more efficient when
using guide RNA (gRNA) and DNA targeting.
MSCs also have suppression effects on cells
belonging to the innate immune system, including CRISPR/Cas9 is widely used in genetic modi-
natural killer (NK) cells, dendritic cells (DCs), mon- fication, transcription regulation, and gene therapy
ocytes, and macrophages. Studies have shown that studies. Researches have demonstrated that CRISPR/
MSCs can only partially suppress the proliferation of Cas9 can be used to conduct genomic editing in many
activated NK cells. Some cell factors such as TGF-β1 organisms, including in bacteria (Jiang et al., 2013),
and PGE2 are believed to participate in the suppres- drosophila (Gratz et al., 2013), zebrafish (Hruscha
sion of NK cell proliferation (Rasmusson et al., 2003; et al., 2013), mice (Wang H. et al., 2013), Caeno-
Krampera et al., 2006). In addition, MSCs can affect rhabditis elegans (Friedland et al., 2013), and
the production of DCs by inhibiting the differentia- Bombyx mori (Wang Y. et al., 2013). Furthermore, in
tion of monocytes, as MSCs can block the maturation terms of the development of stem cell therapy,
signals and co-stimulatory molecules (Zhang et al., CRISPR/Cas9 has been widely applied in the accurate
2004; Jiang et al., 2005; Nauta et al., 2006). On the and complex genetic manipulation of stem cells to
other hand, MSCs reduce the proinflammatory ability enhance their reprogramming, differentiation, and
of DCs by decreasing the secretion of TNF-α, IFN-γ, other functions. Mandal et al. (2014) successfully
and IL-12 and increasing IL-10 secretion (Zhang et al., silenced the expression of the genes B2M and CCR5
2004; Jiang et al., 2005). in human hematopoietic cells using CRISPR/Cas9
with minimal off-target mutagenesis. Additionally,
In summary, the ability of MSCs to regulate Wettstein et al. (2016) transfected two paired CRISPR
immune responses is an important advantage for cell single guide RNAs (sgRNAs)-Cas9 plasmids into
therapy and allogeneic transplantation. It is known that mouse embryonic stem cells, which resulted in the
MSCs have low immunogenicity because they lack knock-out of the targeted gene.
human leukocyte antigen class II and co-stimulatory
molecules such as CD80, CD86, and CD40 in the CRISPR/Cas9 provides us with a more efficient
cytomembrane (Reinders et al., 2013). In addition, way to optimize MSC therapy for liver fibrosis. We
the sources of MSCs are various and abundant. MSCs can transform MSCs using different aspects to en-
also have direct migration abilities and a high dif- hance their vitality and function, including their pro-
ferentiation capacity. Considering all of these char- liferation and differentiation ability, chemotaxis for
acteristics, MSCs are the ideal transplant donors in injured tissue, and anti-inflammatory capacity. To aid
regeneration diseases. in this, Schmidt et al. (2015) successfully built an
arrayed sgRNA library that can target one critical
6 MSC therapy and CRISPR/Cas9 exon of almost every protein-coding gene in humans.
Therefore, by using the sgRNA library, we can find
Currently, genome editing is widely used in genes related to the various characteristics of MSCs,
studies involving functional genomics, transgenic and then knockout the specific gene to optimize the
animals, and gene therapy. Genome editing is based on MSC function.
programmable and highly specific nucleases, which
generate site-specific cleavage and subsequently in- It is also possible to take advantage of homolo-
duce cellular DNA repair (Zhang et al., 2014). Multi- gous recombination to overexpress targeted genes
ple artificial nuclease systems have been developed for through CRISPR/Cas9. As mentioned above, genet-
genome editing, including zinc-finger nucleases, tran- ically engineered MSCs that overexpress certain
genes such as the genes for HGF and IGF-1 have
therapeutic effects on liver fibrosis. However, it is

Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841 837

unclear how we can overexpress specific genes stably Taking advantage of homology-direct repair, targeted
without affecting the MSC function or the expression genes can be combined into the genomic DNA of the
of other genes. This problem is critical. Currently, the MSCs and stably expressed through proliferation
use of recombinant virus infection is fervent, includ- (Fig. 3). Our goal is to obtain the targeted gene in a
ing the use of non-integrating viruses like RNA vi- stably expressed cell line, which can then be used to
ruses, modified lentiviruses, and integrating adeno- treat liver fibrosis. However, the transfection effi-
viruses (Seah et al., 2015). The efficiency of virus ciency is not high; hence, additional research is
infection and the level of gene expression are both needed to improve the efficiency.
high; however, there are still some problems with this
method. Non-integrating viruses will not integrate In general, CRISPR/Cas9 can be used to reform
into the cell genome; therefore, the heterologous stem cells. Additionally, stem cell therapy combined
gene will not be stably expressed as cell proliferation. with genomic editing will be a promising method for
Thus, integrated adenoviruses are a good vector for many diseases in the future.
targeted gene overexpression. However, adenoviruses,
lentiviruses, and RNA viruses are all viruses, mean- 7 Current problems and future prospects
ing that they are associated with pathogenic risks in
clinical treatments. Therefore, finding a new method The transplantation of MSCs for the treatment of
is necessary. liver fibrosis is an effective and promising method,
considering the targeted migration ability, release
CRISPR/Cas9 is a promising tool that may allow capacity, and low immunogenicity of MSCs. MSCs
us to transform MSCs in order to overexpress targeted can directly interact with the fibrogenic liver by dif-
genes. Currently, our lab is performing some related ferentiating into hepatocyte-like cells or by fusing
experiments. We have constructed a donor vector that with hepatocytes. Additionally, MSCs have the
contains the targeted gene, and next we will transfect potential to release different growth factors and
it with the CRISPR sgRNAs-Cas9 plasmid into MSCs.

Fig. 3 Overexpressing gene in targeted site of genome through CRISPR/Cas9
The cleavage induced by CRISPR/Cas9 produces a double strand break, which will trigger cellular DNA repair processes,
including non-homologous end-joining and homology-directed repairs. The AAVS1 locus is a safe harbor for insertion, and
does not interfere with the expression of the inserted gene or other genes. We constructed a plasmid containing homologous
arms of AAVS1 and inserted genes. Taking advantage of homology directed repair, we can insert certain genes into the specific

site and obtain a stably expressed cell line

838 Guo et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(11):831-841

cytokines, which can regulate the microenvironment http://dx.doi.org/10.1002/hep.21704
and immune system to enhance their therapeutic ef- Benyon, R.C., Iredale, J.P., Goddard, S., et al., 1996.
fects on liver fibrosis. MSCs can also be combined
with gene engineering to create a new method that can Expression of tissue inhibitor of metalloproteinases 1 and
obviously regress fibrogenesis, promote regeneration, 2 is increased in fibrotic human liver. Gastroenterology,
and restore the liver function. Therefore, MSC ther- 110(3):821-831.
apy for liver fibrosis is an optimal choice. However, Berardis, S., Dwisthi Sattwika, P., Najimi, M., et al., 2015. Use
many issues with these methods still need to be re- of mesenchymal stem cells to treat liver fibrosis: current
solved. For instance, several different types of MSCs situation and future prospects. World J. Gastroenterol.,
exist, which each have their respective advantages 21(3):742-758.
and disadvantages. The isolation of BM-MSCs is http://dx.doi.org/10.3748/wjg.v21.i3.742
strenuous and traumatic. In contrast, MSCs derived Bӧker, K.H., Pehle, B., Steinmetz, C., et al., 2000. Tissue
from adipose tissue-derived MSCs are abundant and inhibitors of metalloproteinases in liver and serum/plasma
easily obtained, but the therapeutic effect is inferior to in chronic active hepatitis C and HCV-induced cirrhosis.
that of BM-MSCs (Liu et al., 2015). Moreover, we Hepatogastroenterology, 47(33):812-819.
still do not fully understand the mechanisms under- Bonefeld, K., Møller, S., 2011. Insulin-like growth factor-I and
lying the therapeutic effects of MSCs. Therefore, the the liver. Liver Int., 31(7):911-919.
oncogenic potential and the risks of using MSCs re- http://dx.doi.org/10.1111/j.1478-3231.2010.02428.x
main unknown. In addition, when combining MSCs Campagnoli, C., Roberts, I.A., Kumar, S., et al., 2001.
with gene engineering, the transfection problem ex- Identification of mesenchymal stem/progenitor cells in
ists, which will require finding a better transfection human first-trimester fetal blood, liver, and bone marrow.
condition to increase the efficiency. In general, there Blood, 98(8):2396-2402.
is still much for us to explore regarding the use of http://dx.doi.org/10.1182/blood.V98.8.2396
MSCs in the treatment of liver fibrosis. Campard, D., Lysy, P.A., Najimi, M., et al., 2008. Native
umbilical cord matrix stem cells express hepatic markers
Acknowledgements and differentiate into hepatocyte-like cells. Gastroenterology,
134(3):833-848.
We thank Dr. Qiu-rong DING (Chinese Academy of Sci- http://dx.doi.org/10.1053/j.gastro.2007.12.024
ences in Shanghai, China) for his guidance on genome editing Chan, T.M., Harn, H.J., Lin, H.P., et al., 2014. Improved
techniques. Additionally, we thank Editage (https://www. human mesenchymal stem cell isolation. Cell Transplant.,
editage.com) for assistance with the English language editing. 23(4-5):399-406.
http://dx.doi.org/10.3727/096368914X678292
Compliance with ethics guidelines Corcione, A., Benvenuto, F., Ferretti, E., et al., 2006. Human
mesenchymal stem cells modulate B-cell functions. Blood,
Yang GUO, Bo CHEN, Li-jun CHEN, Chun-feng ZHANG, 107(1):367-372.
and Charlie XIANG declare that they have no conflict of http://dx.doi.org/10.1182/blood-2005-07-2657
interest. de Ugarte, D.A., Morizono, K., Elbarbary, A., et al., 2003.
Comparison of multi-lineage cells from human adipose
This article does not contain any studies with human or tissue and bone marrow. Cells Tissues Organs, 174(3):
animal subjects performed by any of the authors. 101-109.
http://dx.doi.org/10.1159/000071150
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http://dx.doi.org/10.1038/cr.2013.146 题目：间充质干细胞治疗肝纤维化的现况和前景
Wells, R.G., Kruglov, E., Dranoff, J.A., 2004. Autocrine
release of TGF-β by portal fibroblasts regulates cell 概要：许多慢性肝病发展到终末阶段形成肝纤维化进而
growth. FEBS Lett., 559(1-3):107-110.
http://dx.doi.org/10.1016/S0014-5793(04)00037-7 转变成肝硬化，严重威胁人们的健康。目前临床
Wettstein, R., Bodak, M., Ciaudo, C., 2016. Generation of a
knockout mouse embryonic stem cell line using a paired 治疗肝纤维化的有效方法是肝移植，但由于供体
CRISPR/Cas9 genome engineering tool. Methods Mol.
Biol., 1341:321-343. 缺乏、免疫排斥和治疗费用昂贵等诸多缺陷，这
http://dx.doi.org/10.1007/7651_2015_213
Wrana, J.L., 1999. Transforming growth factor-β signaling and 种疗法并不是一种理想的治疗途径。近些年细胞
cirrhosis. Hepatology, 29(6):1909-1910.
http://dx.doi.org/10.1002/hep.510290641 治疗研究火热，间充质干细胞作为一种来源广
Yin, L.B., Zhu, Y.H., Yang, J.G., et al., 2015. Adipose
tissue-derived mesenchymal stem cells differentiated into 泛、可定向迁移和多向分化的治疗载体，在肝纤
hepatocyte-like cells in vivo and in vitro. Mol. Med. Rep.,
维化治疗上具有广阔的应用前景。本文对间充质

干细胞治疗肝纤维化的现状进行归纳，并对其可

行性、局限性及应用前景进行分析。

关键词：间充质干细胞；肝纤维化；细胞治疗

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