Accepted Manuscript

Historical Perspectives and Advances in the Mesenchymal Stem Cell Research forthe Treatment of Liver Diseases

Chien-Wei Lee, Yu-Fan Chen, Hao-Hsiang Wu, Oscar K. Lee

PII: S0016-5085(17)36280-7DOI: 10.1053/j.gastro.2017.09.049Reference: YGAST 61495

To appear in: GastroenterologyAccepted Date: 27 September 2017

Please cite this article as: Lee C-W, Chen Y-F, Wu H-H, Lee OK, Historical Perspectives and Advancesin the Mesenchymal Stem Cell Research for the Treatment of Liver Diseases, Gastroenterology (2017),doi: 10.1053/j.gastro.2017.09.049.

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Historical Perspectives and Advances in the Mesenchymal Stem Cell Research for the

Treatment of Liver Diseases

Short Title: Stem Cell Therapy for Liver Diseases

Chien-Wei Lee1,2,#

, Yu-Fan Chen2,3,#

, Hao-Hsiang Wu2,4,#

and Oscar K. Lee2,3,5,6,*

1Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei,

Taiwan;

2Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan;

3Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan;

4Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan;

5Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan;

6Taipei City Hospital, Taipei, Taiwan.

#These authors contributed equally to this manuscript

Grant Support: The authors acknowledge financial support from the Ministry of Science and

Technology, Taiwan (MOST103-2314-B-010-053-MY3, MOST 106-2321-B-010-008, MOST

106-2911-I-010-502 and MOST 106-3114-B-010-002). This study was also supported by

Aiming for the Top University Plan, a grant from Ministry of Education.

Abbreviations: BAL, bioartificial liver system; GVHD, graft-versus-host disease; HESC, human

embryonic stem cell; HGF, hepatocyte growth factor; HLC, hepatocyte-like cell; HPSC, human

pluripotent stem cell; HSC, hepatic stellate cell; IDO, indoleamine 2,3-dioxygenase; iPSCs,

induced pluripotent stem cells; MSC, mesenchymal stem cell; PSC, pluripotent stem cell

*Correspondence: Oscar K. Lee, MD, PhD

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Taipei City Hospital, No. 145, Zhengzhou Road, Datong District, Taipei 10341, Taiwan

Tel.: +886-2-2559-6131; Fax: +886-2-2559-9051; E-mail: DAV47@tpech.gov.tw

Conflicts of Interest: The authors disclose no conflicts.

Author Contributions: Chien-Wei Lee, Yu-Fan Chen, and Hao-Hsiang Wu wrote the

manuscript; Oscar K. Lee provided feedback and edited the manuscript; Oscar K. Lee

supervised the literature search.

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Abstract

Liver transplantation is the only effective therapy for patients with decompensated cirrhosis

and fulminant liver failure. However, due to a shortage of donor livers and complications

associated with immune suppression, there is an urgent need for new therapeutic strategies

for patients with end-stage liver diseases. Given their unique function in self-renewal and

differentiation potential, stem cells might be used to regenerate damaged liver tissue.

Recent studies have shown that stem cell-based therapies can improve liver function in a

mouse model of hepatic failure. Moreover, acellular liver scaffolds, seeded with hepatocytes,

produced functional bioengineered livers for organ transplantation in pre-clinical studies.

The therapeutic potential of stem cells or their differentiated progenies will depend on their

capacity to differentiate into mature and functional cell types after transplantation. It will

also be important to devise methods to overcome their genomic instability, immune

reactivity, and tumorigenic potential. We review directions and advances in the use of

mesenchymal stem cells and their derived hepatocytes for liver regeneration. We also

discuss the potential applications of hepatocytes derived from human pluripotent stem cells

and challenges to using these cells in treating end-stage liver disease.

Keywords: Stem Cell Therapy; Pluripotent Stem Cells; Hepatocytes; Regenerative Medicine

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Despite the success of transplantation of hematopoietic stem cells, few other cell-based

therapies have been successfully translated into clinical application. The dearth of cell-based

therapies is unfortunate as the potential and demand for such therapies are apparent. One

field with exciting developments in cell-based therapies is the field of liver diseases.

Acute liver failure, end-stage liver diseases, and inherited metabolic disorders usually

lead to morbidity and mortality, and are associated with a variety of etiologies (toxic injury,

viral infections, and autoimmune and genetic disorders). In the United States, approximately

30,000 deaths each year are caused by chronic liver disease; the incidence of such deaths

increases by 3 % each year.1 The only curative treatment for end-stage liver disease is liver

transplantation. However, there are more than 15,000 patients on the wait list for a liver in

the United States—approximately 50% of them will not receive a transplant. Liver disease is

the leading cause of premature death in the United Kingdom, resulting in the loss of a

greater number of life-years than many other causes.2 Liver transplantation, the best

treatment option, is limited by high cost and shortage of donor organs. Moreover,

long-term survival is hampered by the graft vs host disease (GVHD) and side effects of

life-long immunosuppression.3

Transplantation of hepatocytes has been considered as an alternative to

transplantation of organs, but faces multiple problems, including the shortage of high

quantity cell sources, challenges to expansion in vitro, concerns of allogeneic rejection and

xenozoonosis, and the fact that hepatocytes spontaneously and quickly lose hepatic

characteristics in vitro.4-6

Stem cells, including induced pluripotent stem cells (iPSCs),

mesenchymal stem cells (MSCs), fetal liver stem cells, fetal biliary tree stem cells,

chemically-induced liver progenitors, and bone marrow CD45+ cells, could provide

alternatives to organ and hepatocyte transplantation, and may offer solutions to the

above-mentioned problems.7, 8

Among them, MSCs have been investigated in detail and

hold great promise for clinical application, as a personalized cell therapy. This is because

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they can be conveniently isolated and expanded in culture, are immune modulatory, and

allogeneic cells to not pose a xenozoonosis risk. MSCs also provide trophic support, have the

potential to differentiate into hepatocyte-like cells (HLCs), and there are no ethical issues

are associated with their use.9-11

We review the role of MSCs in HLC generation and liver

regenerative medicine, and to point to new technologies for clinical application.

In Treatment of Liver Disease

MSCs were first isolated from the bone marrow in 1968, by Friedenstein et al.12

These cells

can be also isolated from other adult somatic tissues, such as the adipose tissue and

umbilical cord blood. Given the self-renewal abilities of MSC, and their therapeutic potential

in tissue engineering and regenerative medicine, MSCs are considered as an ideal cell source

for transplantation. The safety and feasibility of MSC-based therapies have been

demonstrated in clinical trials for many diseases, including GVHD, heart diseases,

osteoarthritis, bone and cartilage injuries, diabetes and its complications, cancer, spinal cord

injury, multiple sclerosis, liver cirrhosis, respiratory disorders, Crohn’s disease, autoimmune

diseases, and others.13

MSCs can localize to damaged liver and eliminate liver fibrosis. Migration of MSCs is

facilitated by release of several molecules from the damaged liver that interact with

different receptors expressed by the MSCs.14-16

MSCs might be used to treat different types

of liver disease because they trans-differentiate in vitro and MSC-derived HLCs can

substitute for injured hepatocytes to restore liver function, have immunosuppressive and

anti-inflammatory effects, promote survival of resident hepatocytes, and direct

differentiation in vivo (Figure 1).

Hepatocyte Differentiation

Transplanted MSC-derived HLCs increase hepatic function at the engraftment site in models

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of acute and chronic liver failure. In 2004, Shu et al17

demonstrated the differentiation of rat

bone marrow MSCs into albumin- and cytokeratin 18-expressing HLCs. In the same year, we

reported a 2-step protocol for the induction of HLCs from human bone marrow MSCs—these

cells have hepatocyte-specific gene expression patterns and hepatocyte-specific functions,

including urea and glycogen production.18

Based on these findings, successful differentiation

of MSCs from other tissue origin into HLCs was also reported. HLCs have been derived from

umbilical cord blood and bone marrow-derived MSCs.19, 20

Furthermore, adipose

tissue-derived MSCs were also successfully induced to differentiate into HLCs.21

In the last decade, different groups established several hepatic differentiation protocols

for MSCs (summarized in Table 1). MSCs are more efficiently induced into HLCs in mice via a

2-step hepatic differentiation protocol18,21

than by the hepatic differentiation protocol

described by Shu et al.17

Transplantation of MSC-derived hepatocytes can restore liver

function to mice with carbon tetrachloride (CCl4)-induced liver failure.22

Co-culture of MSCs

with primary liver cells induces differentiation of MSCs into HLCs 23

. MSC-derived HLCs,

formed by co-culture with primary hepatocytes, integrate into injured liver and reduce

fibrosis 24

. Incubation of MSCs with injured liver tissue was reported to increase expression

of hepatocyte-specific genes in MSCs.25

These observations provide evidence for alternative

methods of inducing differentiation of MSCs into HLCs, via interactions with liver cells, and

the possibility of tissue integration after stem cell transplantation.

Small Inhibitors in Hepatocyte Differentiation

Differentiation of MSCs into hepatocytes involves in the mesenchymal to epithelial transition.

An inhibitor of RAC1 (NSC23766) maintains the epithelial morphology of MSCs, accelerates

upregulation of hepatocyte marker genes, and induces hepatocyte cell functions, by altering

actin polymerization during differentiation.26

Chromatin-remodeling agents, such as valproic

acid, 5-aza-2'-deoxycytidine, and trichostatin A, promote differentiation of MSCs into

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hepatocytes, indicating epigenetic regulation of this transition. hepatocytes.27-29

Manipulating hepatocyte differentiation of MSCs, via intracellular and extracellular factors,

might promote hepatic maturation, accelerate cell differentiation, or increase proliferation.

Mechanical Forces and Microenvironment Architecture

Mechanical forces and the architecture of the microenvironment affect differentiation

of MSCs into hepatocytes. Continuous flow of fluid over microfluidic chips increases the

ability of MSCs to uptake low-density lipoprotein as they differentiate into hepatocytes 30

.

Low shear stress increases differentiation of MSCs into hepatocytes.31

To mimic the

3-dimensional (3D) microenvironment and architecture of the liver, some groups fabricated

artificial 3D scaffolds and investigated whether they induce differentiation of MSCs more

efficiently than 2D culture. MSCs were reported to increase differentiation into hepatocytes

on poly-ε-caprolactone/collagen/polyethersulfone nanofiber scaffolds 32

. Poly-L-lactic acid

nanofiber scaffolds were also reported to increase the expression of hepatocyte-specific

genes by differentiating MSCs.33

Interestingly, during the differentiation of MSCs into

hepatocytes on artificial 3D scaffolds, MSCs form hepatic spheroids that express markers of

hepatocytes and undergo glycogen storage, urea production, albumin secretion, and

cytochrome P450 activity.34

Despite reports indicating the potential of MSCs to differentiate into hepatocytes,

MSC-derived HLCs are similar to fetal hepatocytes and do not reach full maturity in vitro 33

.

Moreover, different types of stem cell sources for the induction of HLCs have been reported;

these were generated using a modified differentiation protocol.35

Therefore, the maturation

process of MSCs during hepatic differentiation requires improvement.

Anti-inflammatory Effects

The liver is an immunologically complex organ. It contains specialized immune cells,

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including Kupffer cells; antigen-presenting liver sinusoidal endothelial cells and dendritic

cells; and liver-resident lymphocytes.36

In addition, the hepatocytes and hepatic stellate cells

(HSCs) affect the immune response in the liver by activating T cells.37-39

The liver

microenvironment and immune response maintain liver homeostasis.

Inflammation is one of the most characteristic features of different liver diseases and is

present at all disease stages.40

MSCs modulate innate and adaptive immune responses.41

MSC-derived prostaglandin E2 and indoleamine 2,3-dioxygenase (IDO) reduces proliferation,

cytolytic activity, and cytokine secretion of natural killer cells.42

MSCs also repress the

activity of Kupffer cells, reducing secretion of the inflammatory cytokine tumor necrosis

factor;43

they also convert M1 macrophages (inflammatory) to M2 macrophages

(anti-inflammatory), by secreting prostaglandin E2.44

MSC-derived human leukocyte antigen

G inactivates dendritic cells;45

MSC-derived interleukin 6 (IL6) and hepatocyte growth factor

(HGF) inhibit the maturation of monocytes into dendritic cells, reducing their inflammatory

potential and changing activated CD4+ T cells from T-helper 1 to T-helper 2 phenotypes.

46

MSCs also inhibit proliferation of CD8+ T cells and increase the rate of conversion of CD4+ T

cells from T-helper 1 to T-helper 2 phenotypes by secreting heme oxygenase 1 and IDO.47-50

Furthermore, MSCs induce the development of regulatory T cells to modulate CD4+ T cell

polarization.51

MSCs directly inhibit B cell proliferation and terminal differentiation through soluble

factors and cell contacts.52

MSCs also act as immunosuppressors following liver

transplantation in mice.53

Only a few studies reported the use of immunosuppression during

liver transplantation but they indicated that liver transplantation with MSC infusion might

prevent acute rejection by regulating T-cell expansion and increasing serum levels of

IL10.54-57

MSC-derived IDO increases survival of patients with refractory chronic GVHD by

stimulating CD5+ regulatory B cells to produce IL10; this is associated with reduced

inflammatory cytokine production by T cells.58

Collectively, MSCs convert the overall

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immune response to less inflammatory (or more tolerant) response by altering the

secretome of the immune cells.59

Most of the immunomodulatory features of MSCs are attributed to their secretome and

proteome. However, the spectrum of secretory factors of MSCs differs from that of

MSCs-derived hepatocyte-like cells and hepatocytes.27, 60

Although immunoregulation by

MSCs participates in liver repair and liver transplantation43, 54, 61

, the immunomodulatory

features of MSCs are typically reported in other organs rather than in the liver. Therefore,

further investigations are indispensable to determine whether immunomodulation by MSCs

is crucial in liver regeneration.

Anti-fibrotic Effects

Clinical chronic liver inflammation caused by viral infection, alcoholic liver disease,

nonalcoholic steatohepatitis, and autoimmune diseases results in liver fibrosis and

cirrhosis.62

Hepatic inflammation activates HSCs to produce extracellular matrix; this

precedes and promotes progression of liver fibrosis. MSCs reduce or reverse liver fibrosis via

their immunomodulatory and anti-inflammatory effects.63, 64

On the other hand, MSCs

directly inactivate HSCs by secreting IL10 and tumor necrosis factor, and induce HSC

apoptosis through HGF, nerve growth factor signaling via p75, and Fas–FasL pathway.65

MSCs

might therefore be used in treatment of hepatic fibrosis.65

MSCs can degrade ECM directly,

by secreting matrix metalloproteinases,66

or indirectly, by stimulating the immune cells to

increase the production of these enzymes.67

MSCs were shown to reduce liver fibrosis in

mice by secreting cytokines and matrix metalloproteinases.68-72

DLK1 contributes to HSC

activation; MSC transplantation reduced activation of HSCs by DLK1 and liver fibrosis.73

Liver Regeneration and Direct MSC Differentiation In Vivo

The immunomodulatory activities and anti-fibrotic effects of MSCs are important for liver

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regeneration. MSCs are also directly involved in maintaining the hepatocyte pool, by

repressing hepatocyte apoptosis and stimulating hepatocyte proliferation, and by secreting

factors such as HGF that maintain liver function.74

Christ et al. reported an in vitro

differentiation protocol for generating hepatocytes from bone marrow and adipose tissue

MSC.75

They showed that MSC-derived hepatocyte-like cells engraft more efficiently than

undifferentiated cells.76

In addition, transplantation of MSCs, MSC-derived brown adipocytes,

or MSC lysates into obese mice reduced fat accumulation and hepatocyte lipotoxicity,

restoring liver functions.77, 78

Although rare, transplanted MSCs can spontaneously

differentiate into hepatocytes in rat79

and this may also, at least partially, contribute to liver

regeneration.

MSC-based Treatments

MSC-derived microvesicles

Microvesicles are extracellular vesicles that contain exosomes and microparticles. Exosomes

are small extracellular vesicles (30–120 nm in diameter) derived from budding membranes

of multi-vesicular bodies; microparticles are large cell-derived vesicles (100 nm to 1 µm)

derived from budding of the cell membrane. Microvesicles can interact with local or distal

cells and transfer molecules such as RNAs, microRNAs (miRNAs), and proteins, for

intercellular communication through a paracrine effect.

Stem cell-derived exosomes and micro-vesicles have been tested in models of liver

diseases.80-84

Injection of MSC-derived exosomes reduced fibrosis in livers of mice following

CCl4 injections, by suppressing expression of collagen and tumor growth factor beta 1.85

MSC-derived exosomes upregulate expression of priming-phase genes during liver

regeneration, leading to increased number of proliferating cell nuclear antigen-positive cells

and levels of cyclin D1 in livers of mice with CCl4-induced fibrosis. MSC-derived exosomes

also inhibit acetaminophen- and H2O2-induced apoptosis of hepatocytes.86

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Exosome miRNAs contribute to development of liver disease in animal models. 87

Exosome miRNA125b, derived from MSCs, reduces activation of the hedgehog pathway in

HSCs, by reducing levels of Smo expression and subsequent CCl4-induced liver fibrosis in

rat.88

MSC-derived exosomes also increase expression of anti-inflammatory cytokines and

the proportion of T-regulatory cells in mice with concanavalin A-induced liver injury,

supporting their immunosuppressive effects.89

MSC-derived exosomes might therefore be

developed for treatment of liver diseases, but additional studies are needed to elucidate

mechanisms of MSC-derived exosomes in liver regeneration. Studies with large animal

models are necessary to translate the findings into clinical practice.

Bioartificial liver (BAL) systems

In 1956, researchers 90

demonstrated that a fresh bovine liver homogenate metabolizes

salicylic acids and ketone bodies, and produces urea from ammonium chloride. In 1967,

some case reports indicated that cross-circulation and exchange transfusion supported livers

of patients in acute comas.91

Accordingly, BAL was developed as a therapy of acute liver

failures.

BAL systems are extracorporeal systems consisting of a bioreactor and biocomponents

(hepatocytes), connected to patient circulation and supporting liver functions. Many studies

indicate that BAL systems improve the survival in animal models.92-94

Matsumura et al

reported clinical application of a BAL system in 1987.95

Eleven BAL device systems are now

available. Among them, the Extracorporeal Liver Assist Device and the HepatAssist are being

tested in clinical trials.96, 97

Since human hepatocyte sources are limited, stem cell-derived HLCs might be used as

substitutes in the BAL system. A BAL system with human fetal fibroblast-derived HLCs was

reported to prolong survival of pigs with acute liver failure in 2016.98

Highly functional HLCs

have been generated from human pluripotent stem cells .99, 100

Human pluripotent stem cell

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(HPSC)-derived HLCs are an ideal cell source for a clinical-grade BAL system. BAL systems

might be developed as alternative strategies for patients with acute liver failure. The use of

BAL systems could also extend the wait time for a suitable liver donor for patients with

end-stage liver disease.

Organ bioengineering

Organ bioengineering involving decellularized organs has recently enabled the development

of a bone fide ECM architecture for stem cell differentiation. Researchers have induced

hepatic differentiation of MSCs on ECM sheets derived from decellularized liver, and

demonstrated efficient transplantation of these cells into mice with CCl4-induced liver

failure101

. Direct infusion of MSCs into de-cellularized liver could be more efficient in

induction of hepatic differentiation.102

Loss of biliary structure or impaired blood supply in

donor livers suppresses liver regeneration after transplantation 103

, so re-cellularization of

liver tissues with a combination of liver sinusoidal endothelial cells, bile duct cells, and

hepatocytes is essential. This strategy might be used in transplantation.

Although the use of MSC-based therapy and recellularized liver tissue is a more

cost-and time-effective option than whole-organ transplantation, the recelluarization

protocol should be optimized and the limitations need to be addressed before future clinical

applications.104, 105

These include, the route of administration or recellularization (e.g., direct

injection or continuous perfusion at a physiological pressure), potential immunogenicity, lack

of hepatic non-parenchymal cells, the possibility of small-for-size syndrome, and differences

in the vascular infrastructure of recellularized and normal liver tissues.106-108

Trials of MSC-Based Therapies

Anti-inflammatory and anti-fibrotic effects of MSCs have been observed in animal models,109,

110so several clinical trials of human MSCs had been conducted in patients with liver

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fibrosis.7, 111-114

To evaluate MSC transplantation in patients, serum creatinine and bilirubin

are used as a therapeutic index in patients with advanced liver fibrosis.115, 116

Evaluation of

the endpoints in these studies revealed the safety and efficacy of human MSC

transplantation. Jang et al reported a reduction of collagen deposition in the histological

analyses of the liver from patients after MSC transplantation in 2014 114

. In 2016, MSC

transplantation was tested in a phase 2 trial of patients with alcoholic cirrhosis.117

MSC

transplantation was reported to reduce collagen deposition and model for end-stage liver

disease scores and increased liver function in these patients. Based on the results of these

studies, we anticipate phase 3 clinical trials in the near future. Although these studies

indicate the therapeutic potential of MSC transplantation for patients with liver fibrosis,

studies are needed to determine the appropriate therapeutic window, MSC dosage, and

mechanisms of fibrosis reduction.

HPSC Administration

Advantages

In 1998, Thomson et al118

reported isolation of human embryonic stem cells (HESCs) from

blastocysts and confirmed that these cells maintained the potential to differentiate into all 3

embryonic germ layers. These cells became powerful tools used in developmental biology,

drug discovery, and regenerative medicine because of their ability to self-renew indefinitely

and their ability to differentiate into any cell lineage. Despite their great medical potential,

there was controversy over use of HESCs, because generation of HESCs involves the

destruction of a human embryo. Another important issue is the immunogenicity of HESCs.

In 2006, Takahashi et al119

reported reprogramming of fibroblasts into iPSCs by

expression of 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). The generation of

human iPSCs was reported by same group in 2007.120

iPSC generation does not involve

destruction of human embryos, and these cells can be generated in an autologous,

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patient-specific manner, and differentiated into various cell types. The unlimited supply of

these cells provides the patients with an alternative autologous, rather than allogeneic,

transplantation option.

Patients with acute liver failure or chronic liver disease are ideal candidates for

cell-based transplantation. Patients with acute liver failure require an immediate restoration

of the liver function by liver or hepatocyte transplantation, but only 10% or fewer of

required liver transplantations are performed.121

As an immediately available and

inexhaustible cell source, PSC-derived hepatocytes might be used to treat these patients.

Transplantation of PSC-derived hepatocytes by a direct splenic injection improves liver

function and prolongs survival of mice with CCl4-induced liver injury.122-124

MSCs are available from the human bone marrow, fat, umbilical cord, dental pulp, and

PSCs. Among these alternatives, human PSCs with extended in vitro culture are the best

sources for MSC production.125

In addition, PSC-derived MSCs have higher telomerase

activity,125

are less sensitive to inflammatory cytokines such as interferon gamma,126

and

have stronger immunomodulation than bone marrow-derived MSCs.127

PSC-derived MSCs

therefore provide an ideal source for clinical application. Researchers have generated

decellularized liver tissues with 3D anatomical structures, including a microvascular network,

that is difficult to manufacture in vitro.128

HPSC-derived hepatic cells could therefore provide

an ideal cell source to develop a BAL system, because of their similarity to primary human

hepatocytes. By studying the liver development, scientists might identify the factors

required for direct differentiation of HPSCs into hepatocytes.129

Challenges

It is important to maintain the genomic stability of PSCs to be used in cell-based therapies.

Cultured PSCs and reprogrammed cells acquire genomic abnormalities, such as large

chromosomal aberrations,130, 131

variations in gene copy numbers,132-134

and somatic

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mutations in coding regions.135, 136

For example, shortly after derivation, 10% of PSCs contain

at least 1 large chromosomal aberration, indicating that large chromosomal aberrations

occur early in the PSC culture process. Interestingly, levels of serum response factor, a

transcription factor, were reported to be reduced in diploid HPSCs. So reduced expression of

serum response factor could contribute to chromosomal instability in these cells.137

A similar

mechanism might operate during the initiation of chromosome instability in diploid cells.

Similarly, DNA analysis studies indicated that iPSC cell lines already contain copy number

variations during early stages of passage, so copy number variations might be induced by the

reprogramming process. Genomic instability is a feature of cancer, so the maintenance of

PSC genomic stability is important.

Some challenges of using HPSCs for therapeutic purposes are linked with the availability

of a homogeneous supply of mature PSC-derived cells. Alarmingly, Zhao et al138

showed that

cells derived from iPSCs induced immune response in syngeneic mice. Providing

HPSC-derived cells for use in clinical therapies requires further, substantial development.

Nonetheless, the utility of PSC-derived cells has been demonstrated in treatment of diabetes,

liver diseases, retinal diseases, muscular dystrophies, and heart diseases.139

Future Directions

An unlimited supply of hepatocytes is needed for cell-based, regenerative treatments for

patients with liver disease. HPSCs are more suitable candidates for generation of functional

hepatocytes than other types of stem cells. Nevertheless, genomic aberrations and the need

for rapid generation of mature and functional hepatocytes challenge their clinical application.

MSCs can suppress activated immune cells, and have anti-inflammatory and anti-fibrotic

effects, so they have been studied for their effects during immune rejection following

allogeneic transplantation and liver regeneration.

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MSCs might also protect the liver from injury, by producing trophic factors (IL10 and

HGF).110

MSC transplantation has been tested, with promising results, in several clinical trials,

and is a possible alternative approach for treatment of patients with liver diseases. MSCs can

reduce collagen deposition in patients with alcoholic liver cirrhosis.114

Although studies have

found MSC transplantation to be safe in preclinical and clinical trials,140, 141

some

chromosomal aberrations have been reported in cultured cells.142-144

Long-term culture

expansion may cause genomic instability and lead to transformation of mouse MSCs,

although this is rarely observed in human MSCs.145-148

Stem cell therapies hold a great

promise for liver regenerative medicine. Mechanistic and clinical studies should foster a

closer collaboration between the academic and industry researchers, to make the most of

the therapeutic potential of MSCs.

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Table 1. Current MSC hepatic differentiation protocols

Cell Source Differentiation Protocol Cell Characteristics Reference

Rat bone marrow

DMEM-LG and MCDB-201

(6:4) supplemented with 0.5%

FBS, 1 µM dexamethasone,

10 ng/mL EGF, and ITS+

(Insulin-Transferrin-Selenium)

premix

Round cells expressing

CK18 and albumin [17]

Human bone marrow

Induction: IMDM

supplemented with 20 ng/mL

HGF, 10 ng/mL FGF2, and

0.61 g/L nicotinamide

Maturation: IMDM

supplemented with 20 ng/mL

oncostatin M, 1 µM

dexamethasone, and 50

mg/mL ITS+ premix

Cuboidal cells with

abundant intracellular

granules, expressing

liver-specific genes;

glycogen storage, urea

production, and LDL uptake

[18]

Human umbilical cord blood

Induction: IMDM

supplemented with 10% FBS,

0.5 µM dexamethasone, 50

mg/mL ITS+ premix, and 50

ng/mL HGF

Maturation: IMDM

supplemented with 10% FBS,

0.5 µM dexamethasone, 50

mg/mL ITS+ premix, and 50

ng/mL oncostatin M

Round cells expressing

liver-specific genes; LDL

uptake

[19]

Human adipose tissue

Hepatic commitment:

HCM-modified William’s E

medium supplemented with

150 ng/mL HGF, 300 ng/mL

FGF1, and 25 ng/mL FGF4

Maturation: HCM-modified

William’s E medium

supplemented with 30 ng/mL

oncostatin M and 0.2 µM

dexamethasone

Round cells expressing

liver-specific genes;

glycogen storage, urea

production, and LDL uptake

[21]

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Rat bone marrow Co-cultured with primary liver

cells and injured liver tissue

Spheroids expressing

liver-specific genes;

glycogen storage

[23,24,25]

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