Mesenchymal stromal cells in hepatic fibrosis/cirrhosis: from pathogenesis to treatment

Abstract

Hepatic fibrosis/cirrhosis is a significant health burden worldwide, resulting in liver failure or hepatocellular carcinoma (HCC) and accounting for many deaths each year. The pathogenesis of hepatic fibrosis/cirrhosis is very complex, which makes treatment challenging. Endogenous mesenchymal stromal cells (MSCs) have been shown to play pivotal roles in the pathogenesis of hepatic fibrosis. Paradoxically, exogenous MSCs have also been used in clinical trials for liver cirrhosis, and their effectiveness has been observed in most completed clinical trials. There are still many issues to be resolved to promote the use of MSCs in the clinic in the future. In this review, we will examine the controversial role of MSCs in the pathogenesis and treatment of hepatic fibrosis/cirrhosis. We also investigated the clinical trials involving MSCs in liver cirrhosis, summarized the parameters that need to be standardized, and discussed how to promote the use of MSCs from a clinical perspective.

Full text

REVIEW ARTICLE OPEN
Mesenchymal stromal cells in hepatic fibrosis/cirrhosis: from
pathogenesis to treatment
Xue Yang
1,2,3,4,6
, Qing Li
5,6
, Wenting Liu
1,2,6
, Chen Zong
1,2
, Lixin Wei
1,2
, Yufang Shi
3,4
✉and Zhipeng Han
1,2
✉
© The Author(s), under exclusive licence to CSI and USTC 2023
Hepatic fibrosis/cirrhosis is a significant health burden worldwide, resulting in liver failure or hepatocellular carcinoma (HCC) and
accounting for many deaths each year. The pathogenesis of hepatic fibrosis/cirrhosis is very complex, which makes treatment
challenging. Endogenous mesenchymal stromal cells (MSCs) have been shown to play pivotal roles in the pathogenesis of hepatic
fibrosis. Paradoxically, exogenous MSCs have also been used in clinical trials for liver cirrhosis, and their effectiveness has been
observed in most completed clinical trials. There are still many issues to be resolved to promote the use of MSCs in the clinic in the
future. In this review, we will examine the controversial role of MSCs in the pathogenesis and treatment of hepatic fibrosis/cirrhosis.
We also investigated the clinical trials involving MSCs in liver cirrhosis, summarized the parameters that need to be standardized,
and discussed how to promote the use of MSCs from a clinical perspective.
Keywords: Mesenchymal stromal cells; Hepatic fibrosis/cirrhosis; Pathogenesis; Treatment; Clinical application
Cellular & Molecular Immunology; https://doi.org/10.1038/s41423-023-00983-5
INTRODUCTION
Liver diseases present a significant threat to human health
globally, resulting in ~2 million deaths each year. Hepatic cirrhosis
accounts for ~50% of liver disease-associated deaths [1]. Liver
cirrhosis is a complication of liver disease, which is a further step
toward hepatic fibrosis and involves the loss of liver cells and
irreversible liver scarring. Chronic damage due to factors such as
viral hepatitis, alcohol, and drugs impairs hepatocytes. This
damage induces inflammatory cell infiltration, leading to the
excessive accumulation of collagen and extracellular matrix (ECM)
and destroying liver structure and function [2,3]. Activated
hepatic stellate cells (HSCs) are the primary source of ECM and
contribute to hepatic fibrosis [4]. Increasing evidence has
demonstrated that MSCs are involved in the pathological
progression of hepatic fibrosis [5,6] through changes in their
functions and microenvironment regulation.
MSCs were first discovered in bone marrow (BM) by Frieden-
stein and colleagues in 1968 and were described as an adherent,
fibroblast-like population in BM in vitro that could differentiate
into bone in vivo [7]. Subsequently, numerous studies have
revealed the plasticity, high proliferation, multidifferentiation, and
immunomodulatory capacity of these cells. MSCs are also present
in other tissues, including adipose (AD) tissue, umbilical cord (UC),
fetal liver, muscle, and lung [8–12]. MSCs have the ability to
differentiate into multiple lineages, such as chondrocytes,
osteocytes, adipocytes, myocytes, and astrocytes. MSCs express
major histocompatibility complex (MHC) class I but do not express
MHC class II, B7-1, B7-2, CD40, or CD40L. MSCs can be expanded
more than 10
4
-fold in culture without the loss of their multilineage
differentiation potential. Therefore, MSCs could be a potential
source of stem cells for cellular and genetic therapy [9,13].
Exogenous MSCs have been shown to exert antifibrotic effects,
and various mechanisms have been examined. MSCs provide an
alternative for patients with liver cirrhosis because there is no
effective traditional treatment other than liver transplantation,
which is expensive and takes a long time to find a suitable organ.
MSCs are considered to be the most promising cells for cell
therapy due to the following properties: (1) they are easy to obtain
from various tissues and easy to expand in vitro without changing
their properties [8,10] (2) they allow for allogeneic transplantation
because of their low immunogenicity and lack of ethical issues
[14,15]; (3) they migrate to injury sites; [16] (4) they have multiple
differentiation capacity and favorable regeneration and injury
repair effects [9,17]; and (5) they have strong immunoregulatory
abilities [18,19]. There have also been bench studies focusing on
new strategies to improve the therapeutic efficacy of MSCs in liver
cirrhosis. Although MSCs have been used to treat liver cirrhosis in
clinical trials and good outcomes have been observed in most
cases, many problems need to be solved to promote the use of
MSCs in the clinic in the future. In this review, we aimed to
Received: 14 November 2022 Accepted: 29 January 2023
1
Department of Tumor Immunology and Gene Therapy Center, Third Affiliated Hospital of Naval Medical University, Shanghai 200438, China.
2
Key Laboratory on Signaling
Regulation and Targeting Therapy of Liver Cancer, Ministry of Education, Eastern Hepatobiliary Surgery Hospital/National Center for Liver Cancer, Naval Medical University,
Shanghai 200438, China.
3
The Third Affiliated Hospital of Soochow University, Institutes for Translational Medicine, State Key Laboratory of Radiation Medicine and Protection,
Key Laboratory of Stem Cells and Medical Biomaterials of Jiangsu Province, Medical College of Soochow University, Soochow University, Suzhou 215000, China.
4
Department of
Experimental Medicine, TOR, University of Rome Tor Vergata, 00133 Rome, Italy.
5
CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and
Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
6
These authors contributed equally: Xue Yang, Qing Li, Wenting Liu.
✉email: shiyufang2@gmail.com; hanzhipeng0311@126.com
www.nature.com/cmi
1234567890();,:

examine the different roles of MSCs in the pathogenesis and
treatment of hepatic fibrosis/cirrhosis, propose parameters that
need to be standardized or optimized and suggest prospects for
the use of MSCs to treat hepatic fibrosis/cirrhosis.
ENDOGENOUS MSCS IN THE PATHOGENESIS OF HEPATIC
FIBROSIS
Contribution of MSCs to the population and regulation of
myofibroblasts
Myofibroblasts: The heart of fibrosis. Myofibroblasts (MFs) were
first described by the seminal works of Gabbiani et al. in 1971,
who showed that a population of “modified fibroblasts”was
present in granulation tissue during wound healing [20]. The
researchers found that these typical fibroblasts (1) possessed
fibrillar systems within the cytoplasm; (2) had a deformed
nucleus; and (3) were attached to an extracellular layer of
basement membrane-like material [20]. Due to their similarities
with smooth muscle cells, these unusual fibroblasts were termed
muscle-fibroblast intermediate cells and are now referred to as
“myofibroblasts”[21]. The presence of MFs in tissue injury led to a
series of investigations of their roles in wound repair. Myofibro-
blast filamentous fibers were found to integrate with myosin and
α-smooth muscle actin (α-SMA) proteins through actin [22]. This
unique cytoskeleton of MFs allowed them to exert contractile
forces and to contract the wound edge, and α-SMA is now
regarded as a universal marker of MFs [23,24]. ECM production is
another hallmark of MFs. ECM components, including different
types of fibrillar collagens, hyaluronan (HA), fibronectin (FN), and
extra domain A fibronectin, are essential for closing wounds [25]. It
is important to note that dysregulated wound healing, which
frequently occurs in chronic injury, leads to tissue fibrosis. Due to
awareness of the essential role of MFs in tissue repair, researchers
quickly examined MFs in the context of organ fibrosis. They
identified MFs as the primary source of excess ECM deposition in
the fibrotic area [26]. Furthermore, multiple pieces of evidence
suggest that the status of MFs could balance tissue repair and
fibrosis [27]. Therefore, understanding the origin, modulation, and
remission of MFs is central to developing treatments for hepatic
fibrosis/cirrhosis.
MSCs are one of the sources of MFs in the liver. The origins of MFs
have long been a central debate in the field. In the late 1970s, MFs
were initially thought to be derived from smooth muscle cells [28].
Later, investigators showed that MFs could develop directly from
normal fibroblasts, MSCs, epithelial cells, pericytes, and circulating
fibrocytes [29]. These contradictory findings complicated the field,
and a lack of consensus on the sources of MFs was a significant
obstacle to understanding organ fibrosis. The groundbreaking
technological advances in lineage tracing and single-cell analysis
have enabled the detailed characterization of MFs under
homeostatic and disease conditions. The latest conceptual
advance on MFs is that they are a heterogeneous population of
cells whose origins and properties differ in various organs. The
following section focuses on the relationship between MSCs and
MFs during hepatic fibrosis.
The notion that MSCs were a source of MFs first came from
in vitro studies [5]. Transforming growth factor β(TGF-β), which is
a major profibrotic cytokine, was shown to induce α-SMA
expression in MSCs. These α-SMA
+
MSCs produce ECM, validating
the transition of MSCs to MFs. Notably, the inflammatory
properties of MSCs suggest that these in vitro observations may
also occur under pathological conditions. Taking advantage of a
dual fluorescent labeling system, Michael Quante et al. found
that BM-derived MSCs were recruited to the site of chronic
inflammation and gave rise to α-SMA
+
MFs during Helicobacter
felis-induced gastric cancer [6]. The spatiotemporal coexistence of
BM-MSCs and TGF-βduring liver injury repair was likely to produce
BM-MSC-derived hepatic MFs. In addition to BM niches, MSCs are
found in almost all tissues. These tissue-resident MSCs are located
in perivascular niches and are capable of trilineage differentiation
in vivo [30]. Recent studies using lineage tracing in mice showed
that Gli1 is a marker for resident MSCs in the liver, kidney, lung,
heart, and muscle [30]. Gli1
+
MSCs constitute 0.02% of total live
cells in the liver. These liver-resident MSCs also express CD29,
CD44, CD105, Sca1, and CD34. During carbon tetrachloride (CCl
4
)-
induced hepatic fibrosis, hepatic Gli1
+
MSCs dramatically expand
and induce the expression of α-SMA in a TGF-β-dependent
manner [30]. Notably, Gli1
+
MSCs contribute to ~39% of interstitial
α-SMA
+
cells in hepatic fibrosis in Gli1-CreERt2-tdTomato tracing
mice. Ablation of Gli1
+
MSCs was reported to alleviate the severity
of tissue fibrosis.
MSCs regulate myofibroblast generation from hepatic stellate
cells. While the contribution of MSCs to the hepatic myofibro-
blast pool was recently validated, it is generally believed that HSCs
are the primary sources of MFs during hepatic fibrosis. HSCs
are pericyte-like cells with abundant intracellular vitamin A and
lipids [31]. Fate mapping studies using acyltransferase-Cre mice
revealed that more than 80% of hepatic MFs were derived from
HSCs [32]. When we combine the data on Gli1
+
MSCs and HSCs,
there is a potential paradox: the sum of MSC-derived MFs and
HSCs exceeds 100%. A possible explanation for this contradiction
is that MSCs and HSCs have overlapping subpopulations. HSCs
expanded in vitro express MSC-like surface markers and show
adipogenic and osteogenic differentiation potential [33]. Future
studies are required to delineate the similarities between hepatic
MSCs and HSCs.
HSCs are nonproliferative in the steady state, and MF
differentiation in these cells is initiated by inflammatory cytokines
released by injured hepatocytes or immune cells. MSCs have been
reported to regulate the activation of HSCs. MSCs have been
reported to express soluble and membrane-bound TGF-β, which
may provide signals for the myofibroblast differentiation of HSCs
[34]. However, MSCs were shown to directly suppress the
proliferation of HSCs by the surface expression of Notch1, which
downregulates the PI3K/AKT pathway in HSCs in an in vitro
coculture system [35]. It has also been reported that direct contact
with MSCs induces the expression of proapoptotic proteins, such
as Bax and cleaved caspase-3, in HSCs [36]. In addition to cell‒cell
contact, MSCs may affect HSCs through paracrine effects.
Hepatocyte growth factor (HGF) and nerve growth factor (NGF),
which are secreted by MSCs, were shown to impair the activation
of HSCs by inhibiting nuclear factor kappa B (NF-κB) signaling [37].
The rationale for these in vitro findings is that MSCs and HSCs are
both located in perivascular niches, allowing for potential cell‒cell
communication or paracrine effects. However, the reciprocal
interactions between MSCs and HSCs during the pathogenesis
of hepatic fibrosis remain to be determined.
Immunomodulation of MSCs in hepatic fibrosis
Investigations of stem cells have primarily focused on elucidating
the mechanisms underlying their self-renewal and differentiation
properties. However, most studies of MSCs have been related to
their roles in immune regulation [38]. The first paper linking MSCs
with immunoregulation comes from the observation that the
presence of MSCs effectively suppressed mixed lymphocyte
reactions [39]. The immunosuppressive capabilities of MSCs were
then firmly validated by the successful clinical application of MSCs
to treat a severe steroid-resistant GvHD patient in 2004 [40].
However, when MSC-mediated immunosuppression was inten-
sively studied, controversial results also emerged. The first FDA-
approved MSC-based clinical trial to treat GvHD failed to achieve
significant clinical improvements [41]. Later, it was found that the
immunosuppressive effects of MSCs were not intrinsic but were
triggered by inflammatory cytokines [42,43]. Depending on the
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context of the cytokine milieu, MSCs can exert proinflammatory or
anti-inflammatory effects by modulating adaptive and innate
immune cells [38]. Based on the concept that MSCs are plastic in
the context of immunoregulation, we will discuss how the
interaction of MSCs with different types of immune cells affects
the progression of hepatic fibrosis.
Initiation of early inflammation in liver injury. Hepatic fibrosis is a
dysregulated wound-healing response to liver injury [44]. If not
appropriately controlled, the acute phase of liver injury is the
starting point of hepatic fibrosis. Damage-associated molecular
patterns (DAMPs) released from apoptotic or necrotic hepatocytes
initiate the inflammatory response by recruiting innate immune
cells to the site of tissue injury by secreting chemokines, including
C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand
5 (CCL5), C-X-C motif ligand 1, and C-X-C motif ligand 15 [45].
HSCs and liver-resident macrophages are believed to be the
sources of chemokines induced by liver injury [45]. MSCs are also
immune sentinels that produce chemokines in response to danger
signals or cytokines in vivo [46,47]. It has been reported that BM-
MSCs can sense low levels of circulating microbial molecules and
mediate monocyte mobilization by producing CCL2 [46]. Notably,
most types of liver insult are due to viral infection, alcohol, and
nonalcoholic steatohepatitis (NASH), which do not yield high
levels of danger signals [44]. In this context, the possibility that
liver-resident MSCs initiate inflammation in response to liver injury
should be considered since the signaling threshold for chemokine
induction is low in MSCs [48].
Neutrophils are the first leukocytes to respond to chemokines
released from the injured liver, and the infiltration of neutrophils is
commonly observed in patients with steatohepatitis [49,50].
Generally, neutrophils have a short half-life [51]. However, the
viability of neutrophils is increased in the presence of MSCs [52].
MSCs constitutively express interleukin 6 (IL-6) and granulocyte-
macrophage colony-stimulating factors to inhibit neutrophil
apoptosis without affecting the phagocytic function of neutrophils
[52,53]. The postponed death of neutrophils provides a window
to eliminate damaged hepatocytes, which is energy efficient from
an evolutionary perspective.
Resolution of liver inflammation. Acute immune responses
corresponding to the accumulation of neutrophils and T cells
lead to the abundant production of inflammatory cytokines in
the liver [54]. Activated tissue-resident natural killer T cells and
newly arrived neutrophils express high levels of interferon-
gamma (IFN-γ), IL-1, and tumor necrosis factor α(TNF-α)[
55–57].
It has been shown that MSCs are highly immunosuppressive in
response to proinflammatory cues [58]. Treatment of MSCs with
a combination of IFN-γand TNF-αor IL-1βinduced massive
production of immunosuppressive molecules, including nitric
oxide (NO) in rodent MSCs and indoleamine 2,3-dioxygenase
(IDO) in humans [43,59,60], as well as prostaglandin E2
(PGE2) [61]andTGF-β[62], leading to the subsequent inhibition
of T-cell proliferation and the induction of anti-inflammatory
innate immune cells. The immunosuppressive molecules and the
apparent chemokine gradient surrounding activated MSCs
synergistically form MSC-centered immunosuppressive niches
in liver tissue. Indeed, intravital imaging showed that MSCs
colocalized with macrophages in hepatic fibrosis [63]. The
inflammation-induced immunosuppressive functions of MSCs
were thought to reshape the immune microenvironment of the
liver during injury. However, due to a lack of specific markers for
MSCs and their activation status, the MSC-centered immuno-
suppressive population during hepatic fibrosis has not been
elucidated in vivo. Recent advances in mass spectrometry and
imaging are promising for investigating the localization-
dependent immunosuppressive capabilities of MSCs in hepatic
fibrosis [64].
To re-establish immune homeostasis in inflammatory tissues,
MSCs have been shown to enhance apoptosis in T cells, suppress
the differentiation of T helper 17 (Th17) cells, and induce the
generation and accumulation of regulatory T (Treg) cells through
high expression of inducible nitric oxide synthase (iNOS), IDO,
tumor necrosis factor-stimulated gene-6 (TSG6) and matrix
metalloproteinases (MMPs) [43,48,65,66]. These observations
were consistent with the significant increase in Treg cells and a
decrease in Th17 cell infiltration in fibrotic liver tissues after the
transfusion of MSCs [67,68]. It was reported that the presence of
IL-17 could enhance the stability of iNOS and programmed
death ligand 1 (PD-L1) mRNA by modulating ARE/poly (U)-
binding/degradation factor 1 (AUF1), thus promoting the
immunosuppressive abilities of MSCs [69]. Therefore, MSCs
activated by acute inflammatory cues can control excessive
immune responses and sustain tissue homeostasis.
In addition to T cells, macrophages play essential roles in
immune homeostasis in the liver. Recent studies have shown
that MSCs can impart anti-inflammatory properties to macro-
phages during the monocyte-to-macrophage transition [70].
Specifically, these effects depend on insulin-like growth factor 2
(IGF2) production by MSCs. IGF2 preprograms maturing macro-
phages toward metabolic commitment to oxidative phosphor-
ylation (OXPHOS) [70].Eveninthepresenceofproinflammatory
cues, IGF2-preprogrammed macrophages could exert vital anti-
inflammatory effects by upregulating PD-L1 expression [70].
Therefore, the anti-inflammatory macrophages trained by MSC-
derived IGF2 could be another layer in the mechanism
that quenches inflammation during liver injury. Notably, IGF2
has a dose-dependent effect on training macrophages. At low
concentrations, IGF2 prefers to bind the IGF2 receptor on
monocytes to confer macrophages with anti-inflammatory
properties [71]. However, when the amount of IGF2 was
sufficient to ligate the IGF1 receptor on monocytes, the resultant
macrophages were proinflammatory. The balance between IGF2
receptor and IGF1 receptor signaling is critical in determining
the functions of macrophages.
Maintenance of chronic inflammation in hepatic fibrosis. During
the chronic inflammation phase, low levels of inflammatory stimuli
induce MSCs to continuously secrete chemokines and recruit
immune cells to the liver microenvironment [43,48,72]. It was
reported that iNOS and IDO act as switches in MSC-mediated
immunomodulation, and the stimulation intensity triggers a
switch between pro- and anti-inflammatory functions [48,59].
When iNOS in murine MSCs or IDO in human MSCs was genetically
ablated, the immunosuppressive effects of IFN-γ- and TNF-α-
treated MSCs were diminished, and a significant immunostimu-
latory effect was observed [48,59]. As we have previously
discussed, potent inflammation in the acute phase initiates the
immunosuppressive functions of MSCs. While the cytokines in liver
tissue are inadequate to induce the expression of iNOS or IDO,
these MSCs can be activated under suboptimal conditions and
secrete chemokines, such as C-X-C motif ligand 9 (CXCL9) and
CCL5, which enables them with immunostimulatory capabilities
[43,48]. The dual functions of MSCs in immunomodulation might
partially account for the chronic inflammation during the
progression of hepatic fibrosis.
TGF-βis a major pathogenic cytokine in hepatic fibrosis that is
responsible for MF induction. However, TGF-βalso plays an essential
role in immunosuppression, inhibiting the activation of immune
cells and inducing the generation of Treg cells [73]. However, why
TGF-βsignaling fails to resolve chronic inflammation in the liver
remains unclear. Interestingly, investigators found that TGF-βmay
promote immune responses through interactions with MSCs [74].
TGF-βcould induce the immunosuppressive effects of MSCs by
inhibiting inflammatory cytokine-induced iNOS expression in a
SMAD3-dependent manner, thus sustaining inflammation.
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THERAPEUTIC MECHANISMS OF EXOGENOUS MSCS IN
HEPATIC FIBROSIS
As we summarized previously, liver injury-related factors, includ-
ing viruses and alcohol, can lead to hepatocyte damage and
chronic inflammation in the liver. During chronic inflammatory
injury of the liver, the synergistic effects of inflammation and
hepatocyte death promote the development of hepatic fibrosis.
The role and mechanism of MSCs in the treatment of hepatic
fibrosis are thoroughly discussed in this section (Fig. 1).
MSCs in the context of hepatocyte injury
Differentiation into hepatocytes. Continuous hepatocyte death is
an essential pathological phenomenon in hepatic fibrosis. Due to
the multidifferentiation potential of MSCs, targeting the hepatic
differentiation potential of MSCs is expected to become an
alternative strategy for treating hepatic fibrosis. Several studies
have shown the hepatic differentiation of MSCs in experimental
animal models and humans [75–78]. For example, after direct
xenografting into allyl alcohol (AA)-treated rat livers, human bone
marrow-derived MSCs could differentiate into hepatocyte-like
cells [79]. After being infused into mice with CCl
4
-induced
liver injury, MSCs or MSC-derived hepatocytes were found in the
liver [80]. The liver tropism of MSCs in response to hepatocyte
injury is regulated by the coordinated effects of growth factors
and damage signals [81]. HGF is the initial driving force that
recruits MSCs to the liver. The migration of MSCs is terminated
by adenosine, a metabolite that indicates tissue damage, thus
retaining MSCs in the area of hepatocyte injury [81]. The
transplantation of MSC-derived hepatocyte-like cells could effec-
tively attenuate hepatic fibrosis and protect liver function [82].
It has been reported that cocktail treatment of cytokines,
including HGF, epidermal growth factor (EGF), fibroblast growth
factor (FGF), leukemia inhibitory factor, dexamethasone (DEX),
and nicotinamide could effectively induce the differentiation of
MSCs toward hepatocyte-like cells [83–86]. Coculture with liver
cells or pellet culture can also lead to the differentiation of MSCs
into hepatocytes [87,88]. In the absence or presence of growth
factors, a dynamic cultured scaffold stimulated MSCs to express
endodermal and hepatocyte-specific genes and proteins asso-
ciated with improved functions, and the cells exhibited the
ultrastructural characteristics of mature hepatocytes [77]. In
addition, Maryam et al. demonstrated that Wharton’s jelly-
derived MSCs could differentiate into hepatocyte-like cells by
permeabilization in the presence of HepG2 cell extracts [89].
However, compared with their differentiation into hepato-
cytes, the differentiation efficiency of MSCs into MFs is higher,
which limits their application value in clinical therapy. However,
the transdifferentiation of MSCs into hepatocytes has rarely
been observed in animal models [90]. Therefore, it is necessary
to examine a more efficient method to induce the differentiation
of MSCs into hepatocytes. On the one hand, the biomimetic
microenvironment constructed by human E-cadherin fusion
protein (hE-cad-Fc)-coated poly (lactic-co-glycolic acid) (PLGA)
microparticles (hE-cad-PLGAs) in engineered multicellular aggre-
gates was able to promote endoderm differentiation and the
subsequent hepatic differentiation of human MSCs [91]. On the
other hand, Wang et al. found that the viability and hepatogenic
differentiation of BM-MSCs cultured on plates coated with
Matrigel and ECM were significantly enhanced compared with
those of cells cultured on noncoated plates [92]. Posttranscrip-
tional regulation also plays an important role in regulating the
differentiation of MSCs into hepatocytes. Zhou et al. screened
the best miRNA candidates for hepatocyte differentiation and
found that five miRNAs, including miR-122, miR-148a, miR-424,
miR-542-5p, and miR-1246, were essential for hepatocyte
differentiation because omitting anyone from the five miRNAs
prevented hepatic transdifferentiation [93].
Protection of injured hepatocytes. Increasing evidence has shown
that the production of trophic factors, including cytokines,
chemokines, and growth factors, is essential for MSCs to
contribute to damage repair. Most injured hepatocytes undergo
cell death. However, the distinct forms of cell death and cell
death response pathways used by damaged hepatocytes have
long-term consequences on liver repair [94]. Another pathway
by which MSCs participate in liver damage repair involves the
production of paracrine trophic factors by MSCs through the
paracrine pathway [95,96]. It has been reported that MSC-
derived conditioned medium (CM) effectively inhibits hepato-
cyte death and promotes liver regeneration [97]. The adminis-
tration of MSC-CM to rats with acute liver injury dramatically
prevents apoptotic hepatocellular death [97]. HGF, αFGF, EGF,
and TGF-αcontribute to hepatocyte proliferation, and vascular
endothelial growth factor (VEGF) is the most vital pro-vascular
growth factor that plays a crucial role in angiogenesis during
liver regeneration [98–101]. Gokhan Adas et al. showed that
after the injection of MSCs and VEGF-transfected MSCs into the
portal vein following liver resection, the cells engrafted in the
Fig. 1 The role of MSCs in hepatic fibrosis therapy
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liver, increased bile duct and hepatocyte proliferation and
secreted many growth factors, including HGF, TGF-β,VEGF,
platelet-derived growth factor, EGF, and FGF, via paracrine
effects [102]. In addition, hepatic progenitor cells (HPCs), which
are located in the canals of Hering, are also involved in liver
damage repair by differentiating toward hepatocytes or biliary
lineage cells. EGF and HGF have been reported to play important
roles in HPC differentiation [103]. In addition, MSC-derived
exosomes were shown to suppress ferroptosis in CCl
4
-induced
acute liver injury. Mechanistically, MSC-derived exosomes
stabilized the protein level of SLC7A11 in primary hepatocytes
to downregulate prostaglandin-endoperoxide synthase 2 and
lipoxygenases [104]. Interestingly, MSCs were also shown to
protect hepatocytes by promoting hepatocyte autophagy
through paracrine effects. Let-7a-5p, which is enriched in MSC-
derived exosomes, can activate autophagy by targeting
mitogen-activated protein kinase kinase kinase kinase 3
(MAP4K3) in hepatocytes [105]. MSCs can also exert hepatopro-
tective effects against liver damage by upregulating PTEN-
induced kinase 1-dependent mitophagy [106]. Therefore, MSCs
could shift hepatocyte death toward an ordered mode to limit
tissue damage and inflammation, thus reducing the incidence of
hepatic fibrosis.
Inhibition of HSC activation
Studies on fibrosis have always used the strategy of inhibiting the
activation of HSCs to achieve an antifibrotic effect. Therefore, the
impact of MSCs on HSC activation is particularly important for the
effective treatment of hepatic fibrosis. It has been reported that in
a CCl
4
-induced rat hepatic fibrosis model, MSC transplantation
effectively ameliorated hepatic fibrosis by inhibiting HSC pro-
liferation and promoting HSC apoptosis [107]. Several studies
confirmed the suppressive effect of MSCs on HSCs. TGF-β3 and
HGF derived from MSCs lead to arrest in the G0/G1 phase in HSCs
by decreasing extracellular signal-regulated kinase 1/2 phosphor-
ylation and thereby ameliorating hepatic fibrosis [108]. MSCs
can also decrease HSC proliferation by activating the Notch
signaling pathway and downregulating the PI3K/AKT signaling
pathway [35]. It has also been reported that hUC‑MSCs inhibit the
proliferation of HSCs, possibly by inhibiting TGF-β1 and Smad3
expression and increasing Smad7 protein expression [109]. In
addition, MSCs could induce apoptosis in HSCs. Transfer of
circDIDO1 mediated by MSC-derived exosomes suppresses pro-
liferation, reduces profibrotic markers, and induces apoptosis and
cell cycle arrest in HSCs through the miR-141-3p/PTEN/AKT
pathway [110]. Lin et al. demonstrated that NGF produced by
MSCs increased HSC apoptosis via the NF-κB- and BCL-xl-
associated signaling pathways [111]. In addition, HSC activation
is triggered by the inflammatory microenvironment. MSCs are well
known for their immunosuppressive properties, and MSCs can also
alleviate the expression of inflammatory factors that induce HSC
activation and fibrosis [112].
On the other hand, MSCs play key roles in ECM degradation.
MFs can internalize MSC-derived extracellular vesicles (EVs) to
reduce the mRNA levels of type I collagen, thus decreasing
ECM production by MFs [113]. Analysis of the components of
EVs suggested that these effects were likely mediated by miR-21
and miR-29c, which target the signaling hubs for ECM produc-
tion [113]. In addition, MSCs also produce ECM-remodeling
enzymes, including MMP2, MMP9, and their inhibitors tissue
inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2), to
reduce ECM deposition in fibrotic sites [114]. However, there are
controversial results showing that the activities of MMP-2, MMP-9,
and MMP-13 were reduced in cardiac fibrosis after MSC treatment
[115]. This effect could be due to the context-specific regulation of
MMPs by MSCs. Janina Burk et al. cultured MSCs on different ECM
substrates and found that fibrotic-like ECM reduced MMP
production by MSCs [116].
Inflammatory microenvironment regulation by MSCs
Accumulating evidence has shown that the immunomodulatory
potential of MSCs plays an important role in alleviating hepatic
fibrosis. MSCs demonstrated immunosuppressive potential and
effects on innate and adaptive immune responses through cell
contact or the secretion of immunoregulatory factors, including
heme oxygenase-1 (HO-1), NO, PGE2, IDO, IL-6, and HLA-G5
[117–122]. Soluble factors are constitutively produced by MSCs or
released after MSC crosstalk with target cells. On the one hand,
these immunoregulatory factors can reduce liver injury caused by
inflammation. On the other hand, they also provide a suitable
microenvironment for liver damage repair. MSCs can regulate
several kinds of immune cells, such as T and B lymphocytes,
natural killer (NK) cells, macrophages, Treg cells, neutrophils, and
myeloid-derived suppressor cells (MDSCs). Kazuya Sato et al.
demonstrated that NO produced by MSCs is one of the major
mediators of T-cell suppression by MSCs [120]. iNOS deficiency in
MSCs abolishes the therapeutic effects of MSCs in advanced
hepatic fibrosis models [123]. In addition, it has been reported that
MSC-derived PGE2 and IDO mediate the immunosuppressive
potential of MSCs. On the one hand, PGE2 effectively mediates the
inhibition of NK cells by MSCs [124]. On the other hand, PGE2 and
IDO enhance Treg and Th17 cell differentiation and inhibit Th1 cell
differentiation [125,126]. It has also been reported that MSCs
induce the differentiation of Th17 cells toward Treg cells by
producing HGF, which generates an immunosuppressive environ-
ment [68].
Macrophages are critical inflammation-related cells in the
hepatic inflammatory microenvironment that play important roles
in regulating the hepatic inflammatory microenvironment. Anoop
Babu Vasandan et al. found that MSC-derived PGE2 manipulates
metabolic programs in differentially polarized macrophages to re-
educate these cells [127]. MSCs can induce the differentiation of
macrophages to immunosuppressive phenotypes through direct
interactions [128]. MSC-derived IL-6 plays a crucial role in
repressing M1 polarization during inflammation and promoting
M2b polarization under anti-inflammatory conditions [121].
Augello et al. found that direct contact between MSCs and
target cells in a cognate fashion inhibited cell proliferation via the
engagement of the inhibitory molecule programmed death 1 with
its ligands PD-L1 and PD-L2, leading the target cells to modulate
the expression of different cytokine receptors and transduction
molecules associated with cytokine signaling [129]. MSCs suppress
the activation of CD4
+
T cells, downregulate IL-2 secretion, and
induce irreversible hyporesponsiveness and cell death via the
expression and secretion of PD-L1 and PD-L2 [130].
BENCH RESEARCH ON IMPROVING THE THERAPEUTIC EFFECT
OF MSCS ON HEPATIC FIBROSIS
Pretreatment of MSCs
The therapeutic effects of MSCs can be improved by changing the
cell culture conditions to further modulate the homing capacity,
survival, hepatogenic differentiation, and paracrine effects of
MSCs. Different attempts have been made to improve the efficacy
of MSCs by modifying the culture conditions, including changing
the culture media with additives, such as growth factors, chemical
agents, lipids, vitamins, and inflammatory factors, and other
conditions, such as hypoxia. Pretreatment of MSCs with HGF and
FGF4 before transplantation improved MSC homing, enhanced
the transdifferentiation of MSCs into hepatocyte-like cells in the
recipient mouse liver and increased the therapeutic effects on
CCl
4
-induced fibrosis [131]. Melatonin‐pretreated MSCs showed a
high homing capacity to injured liver sites. These cells significantly
improved the percentage of glycogen storage and decreased
collagen and lipid accumulation in fibrotic liver tissue [132]. Fathy
et al. showed that eugenol significantly improved the self-renewal,
migration, and proliferation characteristics of AD-MSCs in vitro and
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greatly enhanced the therapeutic effects against hepatic fibrosis
induced by CCl
4
in rats [133]. Another recent study reported that
vitamin E pretreatment improved the homing of transplanted
Wharton’s jelly-derived allogenic MSCs, prevented oxidative stress,
and alleviated hepatic fibrosis [134]. L-theanine pretreatment
promoted anti-inflammatory and antiapoptotic and HGF secretion
by AD-MSCs, which decreased the fibrosis level in the N-
nitrosodiethylamine-induced liver injury model [135]. TLR4- and
IFN-γ-activated MSCs reduced fibrosis-related actin alpha 2
(ACTA2), collagen type I alpha 1 chain (COL1A1), TGF-β, and TNF
levels in the liver. TLR4- and IFN-γ-activated MSCs could enhance
the Th1-polarized response, which alleviated liver granulomatous
and fibrosis in mice infected with Schistosoma japonicum [136].
Gene modification of MSCs
Gene modification of MSCs through viral or nonviral vectors has
been demonstrated to improve stemness, differentiation, immu-
noregulation, homing capacity, and other repair-related abilities
in vitro and in vivo. A series of genes have been overexpressed in
MSCs; among them, HGF was broadly used to modify MSCs and
improve the therapeutic effects on hepatic fibrosis, since HGF has
been shown to play a critical role in liver regeneration [137–140].
Other genes, such as IGF1, hepatic nuclear factor (HNF) 4α, FGF4,
FGF21, IL-10, SERPINA1, PTP4A1, SMAD7, and ECM1, have been
used to modify MSCs and showed improved therapeutic effects in
the treatment of hepatic fibrosis (Table 1). HNF4α-overexpressing
MSCs inhibited inflammation by enhancing iNOS expression
through the NF-κB signaling pathway in a CCl
4
-induced mouse
liver injury model, ultimately reducing inflammation in the liver
and alleviating hepatic fibrosis [141]. Forkhead box A2 (FOXA2)-
overexpressing MSCs promoted the recovery of fibrotic liver tissue
by enhancing hepatogenic differentiation in MSCs and upregulat-
ing the expression of liver-specific genes, including FOXA2, alpha
fetoprotein, keratin 18 (CK-18), HNF1α, and HGF [142]. ECM1-
overexpressing hair follicle-derived MSCs could had significantly
improved the homing and differentiation into hepatocytes, which
increased liver function and decreased pathological liver injury in
liver cirrhosis. In addition, HSC activation was significantly
inhibited in the MSC treatment groups in vivo and in vitro [143].
In addition to the overexpression of target genes, modifications of
microRNAs could also improve the effects of MSCs. MiR-122
modification enhanced the therapeutic efficacy of AD-MSCs in the
treatment of hepatic fibrosis by suppressing the activation of HSCs
and alleviating collagen deposition [144].
Furthermore, genetically modified MSCs can enhance the
immunomodulation of MSCs, thus increasing the therapeutic
effect of MSCs. IL-10 gene-edited amniotic MSCs inhibited the
activation of HSCs and TNF-αexpression in T cells/macrophages
derived from thioacetamide (TAA)-induced fibrotic livers [145].
Another study indicated that IL-35-overexpressing MSCs had
increased immunosuppressive capabilities, which caused CD4
+
T cells to produce IL-10 [146]. Therefore, modifying specific genes
in MSCs is a potential new strategy for improving their ability to
treat hepatic fibrosis.
MSC spheroids
There are significant differences in cell phenotypes and biological
activities in three-dimensional (3D) and two-dimensional (2D) cell
cultures. MSC spheroid culture has been reported to increase the
therapeutic potential of MSCs [147]. Studies have demonstrated
that MSC spheroids have robustly enhanced therapeutic potential
by improvements in MSC stemness, multipotent differentiation,
anti-inflammatory effects, angiogenesis, and tissue regeneration
through the secretion of high levels of cytokines. AD-MSC
spheroid formation promotes stemness, including self-renewal
and multidifferentiation capacities, and spheroid-derived AD-
MSCs show improved potential to attenuate liver failure [148]. In
an experimental model, UC-MSC-3D treatment exhibited
enhanced hepatogenic differentiation and improved the func-
tional recovery of the liver with marked decreases in ALT and AST
and a mild increase in albumin in a murine model of CCl
4
-induced
liver cirrhosis [149]. Another study demonstrated that AD-MSC
spheroids had enhanced stemness and anti-inflammatory and
immunomodulatory functions, as revealed by the increased
expression of the stem cell markers OCT4, SOX2, and NANOG,
the anti-inflammatory factors IL-10, TSG6, and IDO, and the
immunomodulatory molecules HGF, VEGF, and C-X-C motif
chemokine receptor 4 (CXCR4) [150]. It was also suggested that
3D-cultured MSC spheroids significantly promoted wound healing
through well-organized collagen fibrils and high expression of the
angiogenesis biomarker CD31 [151]. In addition, stem cells from
human exfoliated deciduous teeth (SHED) are an ideal source of
MSCs. SHED-converted hepatocyte-like cell-based spheroids
improved liver dysfunction in association with antifibrotic effects
in CCl
4
-treated mice [152]. Taken together, these findings suggest
that MSC spheroids may replace MSCs as a novel therapeutic
strategy with excellent application prospects to treat hepatic
fibrosis.
MSC sheets
Okano’s team developed a cell sheet engineering technology
using thermoresponsive culture dishes coated with poly(N-
isopropyl acrylamide) [153]. IC-2, which is a derivative of the
Wnt/β-catenin signaling inhibitor ICG-001, efficiently induced
hepatic differentiation in human MSCs. A new therapy combining
IC-2 treatment and MSC sheet technology was developed to
establish a novel therapeutic strategy for liver diseases. Research
has reported that the transplantation of IC-2-engineered BM-MSC
sheets exerted potent regenerative effects and effectively
improved acute liver injury in mice [154]. IC-2 also promoted
MMP-14 secretion by BM-MSCs from elderly patients. Secreted
MMP-14 was a valuable predictive factor in reducing hepatic
fibrosis. In addition, MMP-13 activity and thioredoxin levels in IC-2
sheets were inversely correlated with hepatic hydroxyproline
levels [155]. Similar effects were also evident in a study by Itaba
et al., which indicated that orthotopic transplantation of IC-2-
engineered MSC sheets markedly reduced hepatic fibrosis
induced by chronic CCl
4
administration. Mechanistically, IC-2-
engineered MSC sheets produced high levels of MMP-1 and MMP-
14, as well as thioredoxin, which suppressed HSC activation [156].
These studies indicated that IC-2 sheets could be promising
therapeutic options for treating established hepatic fibrosis.
Cell-free therapy based on MSCs
MSCs secrete many molecules into the culture medium (CM), such
as cytokines/chemokines, free nucleic acids, EVs, and lipids,
effectively repairing tissue injury [157,158]. These MSC-derived
secretomes and EVs avoid the potential tumorigenicity, rejection
of cells, emboli formation, undesired differentiation, and infection
transmission of MSC-based therapies. MSC-CM inhibits HSC
activation by reducing the expression of profibrotic genes such
as α-SMA, COL1A1, and MMP2 in vitro, reducing collagen
accumulation and inflammation, and increasing hepatocyte
survival in a mouse hepatic fibrosis model [159,160]. In addition,
proteomic analysis of MSC-CM revealed a broad spectrum of
molecules involved in immunomodulation and liver regeneration
in Gal-N-induced liver-injured rats [161].
Notably, EVs are important paracrine factors secreted by MSCs.
EVs are membrane-bound vesicles that include apoptotic bodies
(50–4000 nm), microvesicles (100–1000 nm), and EXs (40–100 nm)
[162]. Accumulating evidence shows that EVs derived from MSCs
have therapeutic effects on hepatic fibrosis by reducing inflam-
mation and hepatocyte apoptosis. Human BM-MSC-derived EVs
effectively targeted inflammation and reduced fibrosis in a mouse
model, which was accompanied by decreased serum levels of ALP,
bile acid (BA), and ALT. EVs reduced liver accumulation of
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Table 1. The application of genetically modified MSCs in animal models of liver fibrosis/cirrhosis
Genetic
modification
MSCs sources Animal models Dosages Outcomes Mechanisms References
HGF Bone marrow,
umbilical cord
CCl
4
-induced liver fibrosis in
Rats, DMN-induced liver
fibrosis in Rats
2×10
6
~
1×10
7
Promote liver function
and attenuate liver
fibrosis
Upregulate HGF expression; promote
hepatocyte proliferation and inhibit
hepatocyte apoptosis; decrease collagen;
reduce MMP-9, MMP-13, MMP-14 and TIMP-
1 expression; decrease fibrogenic cytokines
PDGF-bb and TGF-β1
[247–250]
IGF1 Bone marrow TAA-induced liver fibrosis
in mice
5×10
5
Reduce liver fibrosis Reduce HSC activation; enhance hepatocyte
proliferation; suppress oxidative stress;
decrease the expression of pro-
inflammatory and pro-fibrogenic genes;
activate hepatic macrophages; induce the
expression of growth factors and MMP2 in
hepatic macrophages
[251], [252]
HNF4αBone marrow CCl
4
-induced liver fibrosis
in mice
1×10
6
Improve liver function
and inhibit liver
cirrhosis
Promote the expression of NF-κB signaling;
enhance anti-inflammatory and immune
regulatory effects of MSCs
[253]
FOXA2 Bone marrow CCl
4
-induced liver fibrosis
in rats
/ Improve liver function
and inhibit liver
fibrosis
Promote hepatic differentiation of MSCs [254]
FGF4 Bone marrow CCl
4
-induced liver fibrosis
in rats
/ Improve liver function Promote the location of MSCs; enhance
proliferation of hepatocytes by increasing
the expression of PCNA, EpCAM, and
Jagged1
[255]
FGF21 Adipose tissue TAA-induced liver fibrosis
in mice
1.5 × 10
6
Improve liver function
and inhibit liver
fibrosis
Reduce serum HA, α-SMA, collagen, and
TIMP1; suppress p-JNK, NF-κB, and p-
SMAD2/3 signaling pathways
[256]
IL10 Amniotic tissue TAA-induced liver fibrosis
in mice
1×10
6
Improve liver function
and ameliorate liver
fibrosis
Promote secretion IL10; suppress activation
of HSCs and the inflammation in
fibrotic liver
[257]
SERPINA1 Adipose tissue CCl
4
-induced liver fibrosis
in mice
2.5 × 10
5
Improve the survival
time of animals with
liver cirrhosis
Promote hepatic differentiation of MSCs [258]
PTP4A1 Placenta BDL-induced liver cirrhosis
in rats
2×10
6
Alleviate liver fibrosis Increase ATP production and mitochondrial
biogenesis; enhance the metabolism of
mitochondria via increased mtDNA and ATP
production
[259]
SMAD7 Bone marrow CCl
4
-induced liver fibrosis
in rats
3–5×10
6
Improve liver function
and ameliorate liver
fibrosis
Reduce collagen I and III, TGFβ1, TGFBR1, α-
SMA, TIMP1, laminin, and hyaluronic acid,
increase MMP1.
[260], [261]
ECM1 Hair follicle CCl
4
-induced liver fibrosis
in mice
1×10
6
Improve liver function Promote hepatic differentiation of MSCs;
inhibit HSC activation; inhibit TGF-β/SMAD
signaling pathway
[262]
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granulocytes and T cells and inhibited VCAM-1 expression [163].
Another study showed that EVs from amnion-derived MSCs
ameliorated inflammation and fibrogenesis in a rat model of
NASH and hepatic fibrosis, potentially by attenuating HSC and KC
activation [164]. Similarly, human embryonic stem (ES) cell-derived
MSC-EVs exerted immunomodulatory and antiapoptotic effects on
TAA-induced chronic rat liver injury by upregulating anti-
inflammatory cytokines (TGF-βand IL-10) and antiapoptotic genes
(BCL-2) and downregulating major contributors to fibrosis,
including COL1A1, α-SMA, and TIMP1 [165].
EXs from MSC-CM have advantages over corresponding MSCs;
they are smaller and less complex than cells, so they are easier to
produce and store and can potentially avoid some of the
regulatory issues associated with MSCs. Several studies have
focused on the beneficial therapeutic effects of MSC-derived
exosomes in animal models of hepatic fibrosis. An in vivo study
showed that hBM-MSC-EXs effectively alleviated hepatic fibrosis,
which was characterized by a reduction in collagen accumulation,
enhanced liver functionality, inhibition of inflammation, and
increased hepatocyte regeneration. hBM-MSC-EX treatment could
ameliorate CCl
4
-induced hepatic fibrosis by inhibiting HSC
activation through the Wnt/β-catenin pathway [166]. Tan et al.
found that MSC-derived exosomes contained beclin 1 (BECN1),
which promoted HSC ferroptosis by suppressing glutathione
peroxidase 4 (GPX4) expression. Knockdown of BECN1 in MSCs
inhibited ferroptosis and the antifibrotic effects of MSC-derived
EXs in HSCs and fibrotic livers [167]. Several studies have indicated
that MSC-EXs contain miRNAs such as miR-125b, miR-150-5p, and
miR-486-5p, which suppress the activation of HSCs and reduce
fibrosis [168–170]. In addition, EXs produced by MSCs could be
modified to improve their therapeutic effects on liver failure. MiR-
122-modified AD-MSCs expressed high levels of miR-122, which
could transfer miR-122 to HSCs and further affect the expression
levels of miR-122 target genes, such as insulin-like growth factor
receptor 1, cyclin G1, and prolyl-4-hydroxylase α1, which are
involved in HSC proliferation and collagen maturation [144]. Tang
et al. reported that EXs derived from clinical-grade fibroblast-like
MSCs were engineered to carry antisense oligonucleotide target-
ing STAT3 and could suppress STAT3 levels and ECM deposition in
mice with established hepatic fibrosis and significantly improve
liver function [171]. These animal model-based studies suggested
that EVs may represent effective cell-free therapeutic agents for
treating liver diseases. MSC-derived EVs exert beneficial therapeu-
tic effects. However, there are currently no clinical trials on MSC-
derived EVs in liver diseases. Many major obstacles, such as the
application strategies and therapeutic efficacies, as well as stable
and consistent isolation of EVs, remain to be resolved.
CLINICAL APPLICATIONS OF MSCS IN LIVER CIRRHOSIS
TREATMENT
Overview of MSCs in the treatment of liver cirrhosis in clinical
trials
Based on the well-known properties of MSCs, including ease of
isolation and expansion in vitro, self-renewal and multiple
differentiation capacity, injury tropism, immunomodulation, and
low immunogenicity, these cells are considered the most
promising candidates for cell therapy. MSCs have been used in
clinical trials to treat many diseases[172] related to inflammation
and injury. Liver diseases are associated with immune responses
and tissue injury. Therefore, liver diseases are ideal targets for MSC
therapy. There are 68 clinical trials for diseases involving MSC
intervention registered at https://clinicaltrials.gov/ as of October
2022, and most these were focused on liver cirrhosis (n=47)
(Fig. 2A). Ten trials had been completed, and only six of them
posted results. Thus, limited data are available to evaluate the
therapeutic effects of MSCs. We also collected 14 published
clinical trial articles on MSC treatment of liver cirrhosis, which
overlapped with the 6 completed clinical trials that had results.
Therefore, we referred to these 14 published articles to evaluate
the outcomes of MSC treatment of liver cirrhosis.
Among the 14 articles, 12 revealed exciting effects of MSCs on
liver cirrhosis by evaluating liver function, liver volume, model for
end-stage liver disease (MELD) scores, and cytokine expression.
Most of these studies followed up with patients for more than
6 months and verified the safety of MSC transplantation. We
believe that a Phase II study about HBV-related decompensated
liver cirrhosis published in 2021 is the most convincing [173]
because it had the longest follow-up and largest sample size. In
this study, 219 patients were recruited and divided into a control
group (n=111) and a UC-MSC-treated group (n=108). UC-MSCs
were transplanted into patients monthly three times in total, and
the patients were followed up for 75 months. No obvious side
effects were observed, and there was a significantly increased
overall survival rate and improved liver function in the UC-MSC-
treated group. However, not every study showed positive results.
Two clinical trials did not observe any therapeutic effects of MSCs
on liver function [174,175]. These studies recruited 27 patients
and 12 patients, respectively. This outcome is probably because
the sample size was not very large, and individual differences can
affect the results.
Among the clinical trials, two used hepatocytes derived from
MSCs to treat liver cirrhosis, which provided another possible MSC
application in liver cirrhosis. MSCs were induced to differentiate
into hepatocytes by using HGF, DEX, and oncostatin M (OSM) for
~28 days before being injected [176]. After a follow-up of
24 weeks, liver function was improved, as verified by decreases
in MELD score, prothrombin complex levels, and serum creatinine
and bilirubin and an increase in serum albumin without any
adverse effects. However, during the treatment, which was
~2 months, ten patients died because of the severity of their
illness. In another phase II trial, the researchers compared the
effects of undifferentiated MSCs and hepatocyte-differentiated
MSCs [177]. Follow-up of patients at 3 and 6 months post-infusion
revealed partial improvements in liver function tests with
increases in prothrombin concentrations and serum albumin
levels and decreases in the elevated bilirubin levels and MELD
scores in both groups. However, statistical comparisons between
the undifferentiated and differentiated MSC groups did not
indicate any significant differences in clinical and laboratory
findings. Animal experiments showed some contradictory conclu-
sions. In a TAA-induced liver cirrhosis model, MSC therapy could
Fig. 2 Clinical trials of MSCs in liver diseases
X. Yang et al.
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regenerate cirrhotic liver tissue and improve liver function and
hepatic encephalopathy. Moreover, hepatogenic partially differ-
entiated MSCs were more efficient than undifferentiated MSCs.
The superiority of partially differentiated MSCs was most likely
due to the increased homing and differentiation abilities of these
cells [178]. Hanan El Baz et al. compared the therapeutic effect of
undifferentiated human cord blood MSCs, in vitro hepatogenically
differentiated MSCs, or freshly isolated rat hepatocytes in a CCl
4
-
induced liver cirrhosis model [179]. The undifferentiated MSC-
treated group had better outcomes than the in vitro prediffer-
entiated hepatocyte-like cell-treated group. Considering the
outcomes of clinical trials and the animal experiments mentioned
above, it is difficult to conclude whether predifferentiation is
better. However, we should keep in mind that undifferentiated
MSCs have more functions, including immunoregulation and
suppressing extracellular matrix accumulation through various
mechanisms, and these cells can also secrete HGF, which favors
hepatocyte differentiation. To determine a superior strategy for
the use of MSCs, more bench research and clinical trials are
needed. Furthermore, considering that time is precious to
patients, highly efficient MSC differentiation procedures should
be optimized if differentiated MSCs are used.
Parameters that need to be standardized for clinical
applications
In these clinical trials of liver cirrhosis, most (57.45%) are in Phase
II, and there is only one in Phase IV (Fig. 2B). Only ten clinical trials
have been completed (21.28%). Most are recruiting or not yet
recruiting (Fig. 2C). The slow progress in the clinical use of MSCs
may be due to some imprecise treatment standards. As shown in
Fig. 3, there are many clinical parameters used in clinics regarding
both cells and patients, including the sources, cell preparation
procedure, dosages, engraftment routes of MSCs, and condition of
patients, that must be standardized to promote the clinical
application of MSCs.
Sources. Although MSCs can be isolated from various tissues,
such as UC [180], BM [181], AD [182], UC blood [183], placenta [11],
amniotic fluid [184], amniotic membrane [185], dental pulp [186],
synovium [187], peripheral blood, liver, lung [188], skeletal muscle
[189,190], and hair follicles [191], UC-MSCs and BM-MSCs have
been primarily used in clinical trials (Fig. 4A). This is probably
because these cells have been most thoroughly studied and are
relatively easy to obtain. Few studies have focused on the
therapeutic effects of MSCs derived from different sources in the
same study. BM-MSCs are mainly suitable for autologous
transplantation. However, there are some limitations to BM-MSC
transplantation. Invasive isolation causes injury and inflammation
in donors, and the efficiency is low compared with MSCs from
other sources [192]. The amount and therapeutic effect of BM-
MSCs are also affected by the condition of the donors. For
instance, the number and differentiation potential of BM-MSCs
decrease with age [193]. Compared with BM-MSCs, UC-MSCs have
many advantages. They are easy to obtain and easy to expand to
the required amount. UC-MSCs are at an early phase of organic
development and show increased self-renewal and differentiation
capacity [194]. UC-MSCs show higher hepatic differentiation
potential than BM-MSCs, suggesting that UC-MSCs may be
advantageous over BM-MSCs for treating end-stage liver disease
[195]. In addition, UC-MSCs have lower immunogenicity than bone
marrow MSCs [196,197]. AD-MSCs are also easy to obtain, but
these cells have poor proliferation and anti-inflammatory abilities
[198]. Therefore, UC-MSCs seem to be the most suitable option for
transplantation.
Autologous or allogeneic transplantation is another problem
challenging doctors. From the clinical trial data, we can see that
Fig. 3 The parameters need to be standardized precisely in clinical application of MSCs in liver cirrhosis
Fig. 4 Clinical trials of liver cirrhosis based on MSCs application
X. Yang et al.
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there is more autologous transplantation than allogeneic trans-
plantation (Fig. 4B). However, due to the advantages of UC-MSCs,
allogeneic transplantation is probably better than autologous
transplantation. Donor variations (age, sex, genetics, and health
status) are important factors that contribute to the clinical
outcomes of MSCs [58]. MSCs isolated from neonatal tissues
exhibit a longer active life and better proliferation and differentia-
tion potential than those derived from adult tissues. Furthermore,
MSCs derived from unhealthy donors may result in negative
clinical outcomes [199].
Cell preparation. Several studies have shown that several
parameters during cell product preparation affect the clinical
outcomes of MSC treatment, including the isolation methods,
culture conditions, cryopreservation and thawing, and product
heterogeneity [200,201]. From the beginning, various isolation
methods have led to robust differences in the potency of MSCs.
For example, techniques used to isolate MSCs from human UC and
placenta vary widely, and there are differences in whether the
specific sections of perivascular tissues are removed [201]. It was
also reported that in UC, MSCs from different parts of the cord
were different [202]. Regardless of the isolation method,
functional and molecular differences exist among MSCs produced
by different centers even when the starting material is the same
and MSCs are isolated from aliquots [203]. Therefore, developing a
worldwide standard to propagate MSCs is very important.
In general, MSCs should be amplified in vitro for at least several
weeks to obtain enough cells for cell therapy; therefore, the
culture conditions, including media, serum, growth factors,
scaffolds and culture systems, are important to maintain the
potency of MSCs [200,204]. Fetal bovine serum (FBS) is commonly
used in research labs to provide nutrients and favor cell
adherence. However, there are several severe limitations in the
use of FBS to culture clinical-grade MSCs. The risk of contamina-
tion and transmission of infectious agents [205], aberrant immune
reactions caused by residual animal proteins [206], antibodies
against FBS produced by MSCs [207], ethical concerns [208] and
batch-to-batch variability [209] make FBS inadequate for culturing
MSCs for clinical applications. Several culture media supplements
have been developed to replace FBS, including human AB serum,
human platelet lysate and chemically defined media (CDM).
However, these alternatives have their own limitations [201].
Serum-free, xenobiotic-free CDM is the preferred alternative to
FBS and has been shown to improve the differentiation and
expansion rates of MSCs [210–212]. The oxygen content of the
culture conditions is another important parameter that should be
considered. Typically, 20% O
2
is used in normal culture conditions
in vitro. However, the O
2
concentration in the tissue environment
of native MSCs ranges between 1% and 7%. High oxygen levels
may compromise the therapeutic benefits of MSCs [213]. Culturing
MSCs in hypoxic conditions enhances their proliferation, homing,
antiapoptotic, and proangiogenic capacities and minimizes
spontaneous differentiation [214–216].
Cryogenic banking is needed to store MSCs before transplanta-
tion, and alterations in MSCs induced by cryopreservation and
subsequent thawing are another challenge regarding the clinical
use of MSCs. Cryopreservation has been demonstrated to stunt
immunosuppressive capabilities [217], postinfusion biodistribu-
tion, engraftment, and clearance kinetics [78,218]. Furthermore,
freeze–thaw steps and transient warming also affect MSC potency
[217]. Thawed MSCs exhibit diminished structural integrity upon
rewarming, resulting in the cells being recognized and deleted by
activated T cells and significantly diminishing the lifetime of MSCs
in patients following infusion [78]. However, freshly thawed MSCs
can be fully recovered after 1–3 days, at least 24 h of postthaw
culture [219,220].
MSC products have robust heterogeneity not only because of
their natural subpopulation diversity but also because of the
donor, cell source, and variable parameters during preparation
[221–223]. Heterogeneous cells showed different proliferation,
differentiation, and immunoregulatory capacities [224,225]. To
overcome the heterogeneity of MSCs and control the therapeutic
potency of MSC products, highly homogeneous MSCs were
obtained by using induced pluripotent stem cells (iPSCs) [226].
iPSCs have been shown to be safe and effective without any sign
of tumorigenesis in a clinical trial of GvHD (NCT02923375). On the
other hand, iPSC-MSCs can be used at a low passage number. It
has been demonstrated that long-term expansion of human UC-
MSCs in vitro (passage 11) reduces their functions [227]. Another
study showed that human AD-MSCs at passage 9 exhibited a
senescent phenotype and impaired therapeutic effects in a
murine hindlimb ischemia model [228]. Expanding hMSCs on soft
poly(ethylene glycol) hydrogel matrices [229] and three-
dimensional (3D) [230] culture systems is also beneficial for
maintaining the early-passage MSC phenotype during expansion.
A bioactive hydrogel scaffold has also been proven to improve the
therapeutic effect of MSCs [231]. Fibroblast contamination is
probably another source of heterogeneity because these cells can
express most MSC markers and adhere to plastic [232]. However,
the increased expression of CD166 and decreased expression of
CD9 in MSCs could be used to purify these cells from fibroblasts
by flow-activated cell sorting (FACS) [201]. Taken together,
developing best practices for MSC preparation by considering all
of the crucial parameters is essential for the successful clinical
translation of MSCs.
Routes of administration. In clinical trials, various engraftment
routes have been used for MSC transplantation, primarily the
hepatic artery, followed by intravenous administration and the
portal vein (Fig. 4C). There have also been clinical trials
transplanting MSCs via the intrasplenic route [233]. It is hard to
say which route is the best. A study on acute liver failure
concluded that intraportal injection was better for repairing liver
injury than hepatic intra-arterial injection, peripheral intravenous
infusion, and in situ intrahepatic injection [234]. Unfortunately, no
similar study has been performed on liver cirrhosis. We cannot
draw a conclusion by comparing different studies.
Considering the convenience and side effects, intravenous
injection would be the most convenient and safest route.
However, ~60% of cells accumulate in the lung and are cleared
by the immune system, as indicated by animal experiments [235].
Thus, more cells should be prepared if the cells will be
transplanted intravenously. Intraportal, intrahepatic, and intras-
plenic injection can deliver MSCs faster and avoid off-target
effects. However, we should bear in mind the complications of
each injection route. These injection routes all require surgery and
cause trauma, and some patients will develop a fever [176]. The
condition of the patient should also be considered. Therefore, it is
difficult to determine which method of administration is optimal.
How to choose the injection route should depend on each
patient’s comprehensive condition.
Doses. Dose selection for MSC transplantation in current clinical
trials is highly irregular. MSC transplantation is performed according
to patient body weight in most clinical trials (0.5–4×10
6
/kg), while
other clinical trials administer MSCs according to the quantity of
cells (1 × 10
7
–5×10
8
cells). Whether MSC therapy should follow
other medicine dosing strategies deserves consideration. Several
clinical trials have focused on the effect of different doses, but they
have not posted the data yet (NCT02705742, NCT03626090,
NCT05080465, NCT05227846, NCT05155657). However, based on
the published data, we can see that as few as 1 × 10
7
cells
administered by intrasplenic injection have apparent therapeutic
effects on liver cirrhosis for six months [233].
Most clinical trials transplant MSCs in one dose, while others
transplant MSCs in several doses, ranging from two to four doses
X. Yang et al.
10
Cellular & Molecular Immunology

with different intervals. One Phase II study compared the effect
of single and two-time transplantation with a monthly interval.
The results showed no significant difference between single and
double transplantation [236]. Another study showed that one
dose of MSCs could improve liver function for up to two years of
follow-up. No significant therapeutic effect was observed after 2
years [237]. It was reported that multiple infusions of MSCs may
enhance the effect of transplantation in animal experiments
[238,239]. Based on these studies, several injections with long
intervals are probably beneficial for improving the therapeutic
effect of MSCs.
Patient condition. We noticed no trials grouping patients
according to their condition in the clinical trials and published
clinical articles. The properties of MSCs could be affected by their
microenvironment to a great extent, including migration, differ-
entiation, and immunoregulation. MSCs and the microenviron-
ment, especially the inflammatory microenvironment, have
substantial crosstalk and interactions [58]. Therefore, the micro-
environment of patients is a crucial parameter that needs to be
considered in clinical trials. The cause of liver cirrhosis, the
inflammation level, liver function and the treatment strategies
affect the microenvironment of the patient [240,241]. It should be
mentioned that patient age is another important parameter that
has been demonstrated to affect the therapeutic effect of
MSCs [242]. Based on animal experimental data, we can also see
that the age of the host adversely affects hMDSC-mediated bone
regeneration [243]. In most clinical trials, the enrolled patients are
~18–30 years old to 65–80 years old (clinicaltrials.gov). This age
span is too broad, resulting in significant differences in the
microenvironment within a single group. The aging microenvir-
onment, including the senescence-associated secretory pheno-
type (SASP), has been proven to impair the function of
transplanted MSCs [244,245]. We can obtain much more accurate
results if patients are grouped by narrow age ranges. In one
previous study, we found that chronic stress inhibited the
therapeutic effects of MSCs in a mouse model [246]. Another
study showed that DEX abrogated the therapeutic effects of MSCs
on fibrin deposition, serum levels of bilirubin, albumin, and
aminotransferases, and T-lymphocyte infiltration [123]. Combining
the comprehensive conditions of patients and the therapeutic
effects, we can evaluate whether a patient is suitable for MSC
treatment before MSC transplantation. We can also do something
to keep the patients in the best condition to receive MSC
transplantation.
CONCLUSION
In this review, we examined the paradoxical roles of MSCs in the
pathogenesis and treatment of hepatic fibrosis/cirrhosis. On the
one hand, MSCs are involved in the pathogenesis of hepatic
fibrosis through various mechanisms, such as differentiation into
MFs and the promotion of HSC activation. MSCs can also affect
hepatic fibrosis by regulating the inflammatory microenviron-
ment. However, in animal experiments and clinical trials, MSCs
are used to treat hepatic fibrosis/cirrhosis. Increasing evidence
has shown that MSC transplantation can alleviate hepatic
fibrosis and improve liver function through several mechanisms,
including hepatocyte differentiation and protection, inhibiting
HSC activation, antifibrotic factors, and immunoregulation. The
contradictory effects of MSCs on hepatic fibrosis mainly result
from whether these cells are endogenous or exogenous. Most of
the data suggesting the involvement of MSCs in the pathogen-
esis of hepatic fibrosis were obtained from endogenous MSCs.
However, the antifibrotic effects of MSCs were mainly observed
after the transplantation of exogenous MSCs. Thus, the long-
term fibrotic microenvironment alters the properties and
functions of MSCs.
Based on studies on MSCs in hepatic fibrosis regression, several
new methods to enhance the therapeutic effects of MSCs have
been developed, including pretreatment, gene modification, cell-
free therapy, cell sheets, and spheroids. Further study is warranted
to determine whether these are new strategies for use in the
clinic. However, it is necessary to perform more bench research to
verify the validity and safety of these new strategies.
MSCs are the most promising candidates for cell therapy due to
their many advantages. Although some satisfactory outcomes in
some patients with cirrhosis have been observed in clinical trials,
various parameters need to be optimized from cells to recipients,
including the sources and preparation of MSCs and the routes and
doses for MSC transplantation. Patient condition, which is much
easier to ignore, is another important factor that affects the
therapeutic efficacy of MSCs. However, more future work needs to
be done to advance the development of MSC therapy.
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ACKNOWLEDGEMENTS
This project was supported by the National Key R&D Program of China (Grant No.
2018YFA0107500), the National Natural Science Foundation of China (Grant Nos.
82173276, 81972599, 81930085, 82073032, 82073037, and 81872243) and Grant No.
2022YFA0807300.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Correspondence and requests for materials should be addressed to Yufang Shi or
Zhipeng Han.
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