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Current Stem Cell Research & Therapy, 2014, 9, 00-00 1

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Liver Stem Cells: From Preface to Advancements

Kanwal Rehman1, Muhammad Javed Iqbal

2, Nureen Zahra

3 and Muhammad Sajid Hamid Akash

1,4,*

1Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang

University Hangzhou, China; 2Department of Biochemistry and Molecular Biology, University of Gujrat, Gujrat, Paki-

stan; 3Instititute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan;

4College of

Pharmacy, Government College University Faisalabad, Faisalabad, Pakistan

Abstract: Liver is a major metabolic organ of the body and is known to comprise of two epithelial cell lineages, namely,

hepatocytes and cholangiocytes which are known to originate from hepatoblasts during fetal developing stages. Upon

acute injury, the hepatocytes and cholangiocytes undergo cellular division to compensate the loss, however, chronic dam-

age may suppress this proliferative ability and as a consequence hepatic and extra-hepatic stem cells may contribute for

liver regeneration. Facultative liver stem cells (oval cells) may emerge, proliferate and contribute in replacing damaged

hepatic cells. Similarly, bone marrow and mesenchymal stem cells are also known for contributing in liver regeneration

having their ability of self renewal and differentiation. However, a closer look is still required to bridge the existing

knowledge gaps between functionality and limitations. Thereby, we have discussed the detailed mechanistic insights of

both hepatic and extra-hepatic stem cells including, stem/progenitor cells, adult/fetal hepatocytes, oval cells, bone marrow

and mesenchymal stem cells. We have also focused on few in vitro and in vivo studies elucidating therapeutic applications

and challenges related to the liver stem cells. We believe that such conversations may provide invaluable contribution for

realistic advancement in the state of therapeutic stem-cell transplantation.

Keywords: Bioartificial liver, hepatocellular carcinoma, hepatocytes, oval cells, stem/progenitor cells.

INTRODUCTION

Stem cell research is one among the most fascinating patches of contemporary biology. More than 30 years ago, in

1981 scientists discovered different ways to isolate stem cell

from early mouse embryo [1, 2]. The elaborated molecular analysis of the mouse stem cells led to the discovery of a

technique to isolate and grow stem cells from human embryo

in a laboratory, in 1998 [3]. In 2006, researchers achieved another milestone by enabling stem cell to be “repro-

grammed” genetically. It included genetically reprogram-

ming of adult cells into embryonic stem cells (ESCs) which hold miraculous power to express genes and other factors

that are mandatory for normal ESCs. This new type was

named as induced pluripotent stem cells (iPSCs) [4]. Such types of investigational studies provided a hope for further

discovering and understanding of functional stem cells and

looking them as a therapy for organ disorders. Many years ago, it was believed that some organs such as liver or brain

had no or only a few stem cells that have restricted differen-

tiation abilities [5]. However, during the last decade, re-searchers have revealed the fact that adult stem cells have

potential to differentiate into tissues from which they have

been derived. Undeniable evidences strongly indicated that stem cells derived from adult tissues have ability to repair

*Address correspondence to this author at the Institute of Pharmacology,

Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical

Sciences, Zhejiang University Hangzhou, China and College of Pharmacy,

Government College University Faisalabad, Faisalabad, Pakistan;

Tel: +86-15558171291; Fax: +86-571-88208416;

Email: [email protected]

and replace selective cell types that are already present in their neighborhood. These adult stem cells are generally con-sidered as “multipotent” [6]. The most evolutionary area of this ascertainment is the widely studied example of the abil-ity of hematopoietic stem cells (HSCs) to regenerate organs such as liver. A progenitor/stem cell compartment was rec-ognized in adult livers for its probable ability of being a tar-get for liver regeneration [7]. The therapeutic potential of such cells can prove to be a remarkable way of providing opportunity for the transplantation of cells/tissue, gene ther-apy and drug development.

STEM CELLS/PROGENITOR CELLS

The words, progenitor cell and stem cell are often used interchangeably. “Stem cells” have potential to replenish their own population in addition to differentiate into commit-ted daughter cells. However, the term “progenitor cell” may genetically define as “cell that can divide and has the ability to differentiate”. As a result of these progenitor cells, a prog-eny is produced which can differentiate and possess self re-newal abilities [8].

As ESCs are derived from pluripotent blastocyst, these cells progenerate somatic stem cells that possess potential to differentiate into multipotent tissue specific stem cells. Some stem cells have been shown to possess exceptional capability to differentiate into any cell type within the body. Stem cells have also been further categorized as totipotent and pluripo-tent cells and have been distinguished from the other cells [9] owing to their distinct characteristics as, i) they have mi-raculous renewal or self-maintenance potential, occasionally

2 Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 1 Rehman et al.

after long period of immobility, ii) they generate into organ or tissue specific cells with special functions under specific growth conditions, iii) they acquire serial transplantable dex-terity (plasticity/trans-differentiation). On the other hand, progenitor cells are known to have capability to divide rap-idly but have short term tissue reconstruction [9, 10]. Though, specifically derived adult stem cells are more accu-rately referred to as progenitor until they completely execute according to the characteristics of a stem cells but it is still difficult to distinguish between stem and progenitor cell be-cause of self-renewal and proliferative properties. Hence, here we will refer stem cells as stem/progenitor cells.

STEM/PROGENITOR CELLS IN LIVER REGEN-

ERATION

As far as the liver is concerned, liver transplantation has been thought to be the only convincing therapy to deal with the end stage liver disease, although it has certain disabilities including risk of rejection, which is one of the worldwide health problems [11]. This factor accounts the molecular biologists to identify and isolate exemplary source of stem cells that have attractive innumerable potential to generate large amount of hepatocytes used to repair damaged liver or to make biological artificial liver.

Mounting evidences are available that liver stem/progenitor cell (LSCs) can play regenerative role in the liver which is stimulated by specific pathological or physio-logical stimuli. Most classical demonstration of liver regen-eration was presented in 1997 by a serial transplantation of

-galactosidase marked liver cells in mouse model. This showed that the growth potential of adult mouse hepatocytes was similar to that of HSCs [12]. Recently, the cells derived from bone marrow (BM) in diseased livers of human and rodents have demonstrated that these stem cells may play critical role in the liver repair. BM cells have miraculous regenerative potential to generate several types of hepatic epithelial cells such as hepatocytes, oval cells and duct epi-thelium. It is hypothetically believed that LSCs possess po-tential deriving force to replace liver epithelial cells [13].

However, the characterization and recognition of en-dogenous LSCs and the signals that dictate their differentia-tion and proliferation to adult hepatocytes might direct this fascinating pathway of biology to the development of clini-cally feasible channels to induce liver regeneration from these endogenous cells or to allow maturation of stem/progenitor cells to hepatocytes in vitro and in vivo.

Here we have attempted to discuss in detail the mechanistic insight of liver stem/progenitor cell including their niches, stimulating factors and their derivation from resident and/or extra-hepatic cells along with therapeutic applications and limitations for utilizing LSCs in liver disease.

LIVER STEM/PROGENITOR CELLS

Liver, the essential body organ comprises of complicated structure and various cell types. The liver parenchymal cells are called hepatocytes which are about 80% of the organ and perform important liver functions. However others are non-parenchymal cells of liver like cholangiocytes (which form bile duct), kupffer and endothelial cells [14]. Many reports have defined cells as LSCs which possess the ability to pro-liferate and differentiate into two epithelial cell lineages, namely, hepatocytes and cholangiocytes which are known to originate from similar origin, hepatoblasts during fetal de-veloping stages [15]. As a matter of fact, from the fetal en-doderm foregut, hepatoblasts emerge yielding hepatocytes and cholangiocytes thereby may be known as embryonic liver stem/progenitor cells [16] (Fig. 1). Signaling pathways, TGFb/Activin and Notch have been known to initiate the differentiation of hepatoblasts into cholangiocytes [17]. Whereas at latter stages, parenchymal hepatoblasts expand with the help of mesothelial cells present on their surface [18]. Further, these mesothelial cells bring about the forma-tion of mesenchymal cells in the liver [19]. Several studies during the last decade have identified cells both outside and within the liver exhibiting the properties of hepatic stem cells that can differentiate into hepatocyte and bile duct epithelial cells in culture in addition to their transplantation [20]. Ini-tially, it was believed that adult hepatocytes have limited potential to proliferate but in the last decade ,researches in this area have demonstrated that under specialized condi-tions, hepatocytes have boundless proliferation potential [21]. Upon acute injury, the hepatocytes and cholangiocytes undergo cellular division to compensate the loss of injured cells/tissue; however, chronic damage may suppress this proliferative ability of liver cells. As a consequence, hepatic and/or extra-hepatic stem/progenitor cells may contribute for liver regeneration [14].

Though, the ESCs are totipotent but they are well-known to be tumorigenic as well. On contrary, progenitor cells might be less persuading being committed cells and can be useful for transplantation [22]. Stem cells play a dominant role in tissue homeostasis and tissue repairing after acute injury. However, in mammalian liver, turnover of normal

Fig. (1). Developmental stages of liver cells. From the fetal endoderm foregut, hepatoblasts emerge which proliferate and form two distinct

committed linage which further differentiate yielding hepatocytes and cholangiocytes.

Liver Stem Cells: From Preface to Advancements Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 1 3

epithelial cells is very slow but hepatocytes display rapid cell division and replacement of lost cells within few days as in case of acute liver injury [23].

Several convincing experiments conducted by diverse group of researchers have highlighted the hepatocyte poten-tial for proliferation. In FAH (fumary-lacetoacetate hydro-lase) knockout mice with inclusive and continuous liver in-jury, nearly complete liver repopulation can be achieved with reconstruction of normal function and structure by trans-planted wild type hepatocytes [12]. In another study, trans-plantation of wild type hepatocytes into transgenic mice with extensive and continuous necrosis of host hepatocyte showed 12 or more cell divisions replacing most of the host liver [24].

LIVER STEM/PROGENITOR NICHE

A stem/progenitor cell niche includes supporters in term of self-renewal property of stem/progenitor cells and may comprise of cellular and extra-cellular microenvironment [25]. The inspiring thought about stem cell niche arose from the concept that stem cells may inhabit in the “microenvi-ronment” that stimulate their differentiation. Such niches have been analyzed in BM, skin, brain and intestinal musosa [26-29], where specific signals and factors affecting their behavior of proliferation and differentiation [27]. However, the most favorable candidate for the liver stem cell niche is the “Canal of Hering”. For hepatocytes, their niche is thought to be composed of various cells including hepato-cytes and cholangiocytes themselves, inflammatory cells, kupffer cells, endothelial cells and stellate cells. For instance, a close cross-talk has been suggested to exist between LSCs and hepatic stellate cells [30]. Interestingly, LSCs have most probably thought to be supported by myofibroblasts/ hepatic stellate cells matrix [31]. This matrix is recognized to help LSCs travel for differentiation to compensate the loss of he-patocytes. Similarly, various studies have suggested the regulation of cholangiocytes proliferation to be influenced by different factors including growth factors, steroid hormones and neurotransmitters [32, 33]. In addition, cytokines and chemokines released from inflammatory cells are also known to influence the growth and differentiation of LSCs via dif-ferent signals [34]. In this regard, Jakubowski et al. have suggested that TNF-like weak apoptotic inducer expressed by T-cells may encourage proliferation of LSCs [35]. Simi-larly, according to a study on mouse cholangiocytes, it was found that their proliferation was increased in response to histamine receptor stimulation [36]. Further, the importance of hepatic cell niche have been recognized by carrying out studies involving various cellular and extra-cellular factors like non-parenchymal cells to improve the functioning of liver cells when co-cultured with them [37-39]. Thereby, LSCs niches may involve a combination of factors; however, a productive discussion might probably be helpful to build up the association among existing perceptions regarding LSCs and their niches.

INTRA-HEPATIC AND EXTRA-HEPATIC STEM CELLS AND LIVER REGENERATION

Though, the liver has remarkable regeneration ability but under certain extreme conditions like chronic liver disease, the organ has to be dependent on other sources for its re-

population for example, the intra-hepatic stem cells and ex-tra-hepatic stem cells. Intra-hepatic stem cells include mature (adult) hepatocytes and fetal hepatic progenitor cells while, extra-hepatic stem cells comprise of bone marrow stem cells, mesenchymal stem cells, and embryonic stem cells.

Adult Hepatocytes

According to the increasing literature, it has been shown that some adult hepatocytes have potential to proliferate 12-18 times to develop into functional progeny. To test their regenerative competency, researchers transplant hepatocyte (serial transplantation) from adult donors. No indication of altered liver architecture and function was detected in re-populated animals [24]. Researchers by adopting isolated hepatocyte cell transplantation model, further depicted that normal adult hepatocytes have proficiency to be serially transplanted through seven generations of mice, with indi-vidual transplanted cell undergo divisions with an average of more than 68 divisions and by applying retroviral marking, FAH+ hepatocytes can be amplified clonally and repopulate to FAH-/- mouse liver by serial passages [12, 40]. It has been reported that genetically modified hepatocytes that express seminal proliferative capability in hosts with liver injury have also been used experimentally [41]. In case of exces-sive bile duct injury, it has also been reported that hepato-cytes have potential to differentiate into cholangiocytes and form mature bile ducts, showing bipotential differentiation of hepatocytes [42].

Oval Cells

The hepatic progenitor cells in rats are termed as oval cells (OCs) which are minute proliferative cells with oval and large nuclei along the bile canaliculi. OCs possess het-erogeneous stemness potential i.e. they may differentiate into both hepatocytes and cholangiocytes [43].

Though OCs are particularly used for the rodent models however, in conditions of liver diseases like chronic intoxi-cation or viral infection which results in suppressed hepato-cytes proliferation, OCs-like cells emerge from peri-portal area (canal of hering, hepatocytes or cholangiocytes) and take the command to proliferate for the replacement of dam-aged hepatic cells [44, 45].

Though the exact mechanism for OCs activation is not completely understood, however, they are presumed to be activated by involvement of different cell types including, damaged hepatocytes, canal of hering, inflammatory and immune cells. Moreover, there are various factors involved in the activation of OCs [46] including cytokines like IL-6, leukemia inhibitory factor (LIF), and oncostatin M which further stimulate transcriptional pathways. After being acti-vated, different growth and/or differentiation factors have been recognized to promote the progression of OCs prolif-eration and differentiation (Fig. 2), including transforming growth factor- (TGF- ), transforming growth factor- (TGF- ) and hepatocyte growth factor (HGF) [46]. Moreo-ver, different inflammatory cytokines have been reported to modulate the OCs responses [47, 48]. Similarly, mesenchy-mal cells have been reported for signal induction in OCs by direct interaction [43]. Isolation, differentiation and in vivo transplantation of liver, OCs have been successfully


Fig. (2). Stimulation of oval cells and expression of typical growth factors and cytokines involved. During extreme liver conditions like

chronic liver toxicity or disease, oval cells also termed as facultative liver stem cells are known to emerge from peri-portal area in response to

stimulus like damaged hepatocytes and/or inflammatory cells. These oval cells become activated in presence of various cytokines released

and starts proliferating and differentiating into hepatocyte and cholangiocytes with the help of growth/differentiation factors to compensate

the loss.

performed over the last decade. Several studies have been published on putative liver progenitor cells isolation, charac-terization and differentiation of human liver [49]. Simple OCs are presumed as descendants of stem cells that are known to activate and proliferate as an amplifying transit compartment after stem cell activation [50]. OCs are known to express hepatocytes/cholangiocytes markers such as (al-pha-fetoprotein (AFP), albumin, chromogranin A and CK19 in rats, whereas EpCAM, NCAM, CK-19, CD133, CD44 and Claudin 3 in human [51].

Moreover, the canal of hering is known to contain minute undifferentiated epithelial cells that are reported to be in di-rect physical continuity with hepatocytes [52]. These epithe-lial cells show dual expression of fetal hepatocytes and bile ductular markers AFP and CK-19, and thus are recognized to represent OCs. Though much is recognized regarding OCs, but still little known about the exact mechanism for genera-tion of OCs and there is need for further understanding of various aspects at cellular and molecular level regarding OCs.

Fetal Hepatic Stem/Progenitor Cells

As discussed above, during development of fetal liver, the foregut endodermal cells give rise to hepatoblasts which grow and differentiate into liver cell linage (hepatocytes and cholangiocytes) (Fig. 1) which is considered as a hallmark of these stem/progenitor cells [14]. Numerous studies related to fetal liver stem/progenitor cells have shown their potential for liver transplantation because they are small, can rapidly proliferate and have ability to regenerate both hepatocytes and bile duct epithelial cells [53]. In xenotransplantation

studies, hepatic regenerating activity has been observed for human fetal hepatic stem/progenitor cells [54].

Bone Marrow Derived Liver Stem/Progenitor Cells

One of the very fascinating patches in the field of stem cell research is bone marrow derived liver stem cells (BM-LSCs). Various studies have been carried out using animal models and human to investigate the evidences for the con-tribution of bone marrow stem cells (BMSCs) in liver regen-eration. Though, in normal conditions, BMSCs may not help in repopulating the parenchymal cells, however, they may contribute in liver repair by providing growth factors. Moreover, it is difficult to select the optimal cells using BMSCs therapy for liver diseases. As BM has more than one stem cell population, so the BMSCs need to be clearly identi-fied as which type of BMSCs are accountable for liver re-generation is still a conflicting issue [55]. It has been be-lieved that although BMSCs might be able to engender OCs and hepatocytes, the phenomena of trans-differentiation oc-curs weakly and infrequently [50].

Researchers have designed a culture system for isolation and purification of BM-LSCs, where this system may yield hepatocyte like cells. Within this specially designed cultur-ing system and in an appropriate condition, the desired BM-LSCs could proliferate. BM-LSCs bring new hopes and open up new era in understanding the physiology of stemness po-tential to use BM-LSCs clinically for therapeutic purposes [1]. In recent years, to minimize the ethical problem related with ESCs, scientists have also focused on autologous BM-LSCs that have exceptional developmental potential in a proliferating culture system [56]. HSCs have been suggested


by Sell et al. in 2001 to be one of the probable sources for the regeneration of liver cells during liver injury [57]. How-ever, isolated hepatocyte progenitor cells from BM and liver have been known to be combination of both as they ex-pressed the common surface receptor and/or antigen (CD34, Thy-1, FLT and c-Kit) [58, 59]. Avital et al. [60] have re-ported the isolation of LSCs from BM using lack of 2

-

microglobulin ( 2m) and presence of Thymus cell antigen (Thy-1) as trackers for liver progenitor cells. However, ow-ing to the expression of hematopoietic cell antigens (e.g. Thy-1, CD34), a probable speculation for the derivation of OCs from BM also exist [61]. Unlike hepatocytes, where the derivation from BM is considered to be limited, the BMSCs are thought to contribute for liver non-parenchymal cells. The monocyte resident of liver which are known as kupffer have been recognized for their phagocytic function in dis-eased condition by releasing TNF- and IL-6 as inflamma-tory cytokines [55]. A bone marrow derived mesenchymal stem cell (BM-MSCs) population was studied that differenti-ated into hepatocyte-like cells expressing AFP, albumin markers (Fig. 3) and Cytochrome P450 activity [62]. Nu-merous investigational studies on murine transplantation have suggested BM derivation of Kupffer cells [63, 64] though few animal and patient studies have been conducted [65-67] on preliminary stages but there is still a need for wide-ranging understanding of BMSCs associated liver re-population.

Fig. (3). Bone Marrow derived-stem cells differentiation into

hepatocytes. Bone marrow has been known to comprise of two

types of major bone marrow stem cell (BMSCs) population includ-

ing hematopoietic stem cells (HSCs) and mesenchymal stem cell

(MSC). Studies have suggested HSCs and MSCs to participate in

replenishing the number of lost hepatocytes by differentiating into

hepatocyte-like cells and expressing hematopoietic cell specific

markers. However, the generation of oval cells (OCs) by bone mar-

row derived stem cell is still under debate.

Mesenchymal Stem Cells

Mesenchymal stem cells that are known as pluripotent cells are recognized to have long-term self-renewal ability deriving from cartilage, bone, other connective tissues, and nervous and adipose tissue [68]. MSCs are known to be de-rived from various tissues differentiating into a range of other cells [69]. MSCs were first isolated from BM as stro-mal cells [70]. Moreover, adipose tissue-derived mesenchy-mal stem cells (AT-MSCs) have been known to differentiate into hepatocytes [71, 72]. A sound engraftment of hepato-cyte-like cells which were resultant of AT-MSCs was ob-served in a xenogenetic transplantation model with 15 times higher serum albumin in pre-differentiated AT-MSCS trans-planted mice [73]. Several recent studies have reported nu-merous MSCs markers including CD44, CD73, CD90, CD105, and CD166 for BM-MSC. Besides BM-MSCs, markers for other MSCs are CD49e, CD51, CD54, CD59, CD71, and CD117 [74-78]. Interestingly, the extracellular matrix of umbilical cord (UC) is known to comprise of Wharton’s jelly cells (WJC) as a main component [79]. WJC has shown to secrete cytokines similar to BM-MSCs [80] and has been recognized to function for in vivo hema-topoietic stem cell engraftment [81]. WJC-MSCs have ex-pressed BM-MSCs like markers [82, 83] namely, CD10, CD13, CD29, CD44, CD49e, and CD166 [74, 84, 85]. Few have been designated WJ-MSCs as multipotent cells which can differentiate into various cell types [86]. A study showed well functioning of UCB after differentiation into hepato-cyte-like cells including AFP, albumin production and Cyto-chrome P450 activity [62]. Similarly, in an in vitro and in vivo study, authors have presented the importance of liver microenvironment on hepatic differentiation in which they observed in vivo loss of chimerical phenotype in BM-MSCs differentiated hepatocytes that was observed to be presenting in in vitro [87].

However, vigilance is still required regarding both the efficacy and side effects of the current proposed hepatic stem cell choices.

THERAPEUTIC APPLICATIONS OF LIVER

STEM/PROGENITOR CELLS

Though for the management and cure of many chronic liver diseases, liver transplant seems to be an eventual treat-ment, however, various recent investigations have paved a pathway for novel therapies of liver diseases. Such investiga-tions include progresses and challenges relating to the dis-covery, technical and clinical aspects of liver stem/progenitor cells which can provide a better alternate for liver treatment.

The major potential of these liver stem/progenitor cells is evident from their availability for cell transplantation, gene therapy, bioartificial devices and drug testing [22] as com-pared to the donor availability for organ transplant. We have already discussed it in earlier sections that ESCs, mature hepatocytes, HSCs/BM-LSCs, MSCs, fetal and/or adult liver progenitor/stem cells have the ability of self renewal and generating similar progeny with ability of self renewal and differentiation (Fig. 4). Here, we will discuss different stud-ies and investigations relating to diverse stem cells reported to be candidate for human clinical trials and their relevant challenges.


Fig. (4). Repopulation of liver cells by intra-hepatic and extra-hepatic stem cells. Normally, mature hepatocytes proliferate and divide to

balance the number of hepatocytes (top; shown in red), however in chronic liver toxicity or viral diseases, hepatocyte proliferation is sup-

pressed. In such conditions, oval cells become activated and divide to differentiate into hepatocytes (bottom; shown in green). Similarly, few

of the extra-hepatic sources are also recognized as a resource to provide stem cells including bone marrow stem cells (BSCs) and mesenchy-

mal stem cells (MSCs). These cells may differentiate into hepatocytes and participate in liver regeneration (right; shown in blue). Moreover,

according to therapeutic application, stem cell transplantation/liver assist devices can play vital role in contributing for liver regeneration as

suggested by ongoing researches using isolated adult/fetal hepatic stem/progenitor cells, embryonic stem cells and bone marrow derived-stem

cells (left; shown in yellow).

Liver Cellular Transplantation

The transplantation of liver cells has been now recog-nized as a novel and promising approach for the treatment of various liver diseases. The cells that have been considered for this type of cellular transplantation and differentiation to hepatic lineage include BM-LSCs, ESCs, and MSCs [88]. It has been suggested that the use of stem/progenitor cells de-rived hepatocytes might have more advantage over trans-plantation of mature hepatocytes [89]. The major advantages of cell therapy over organ transplant is that it can be cost effective, simply directed with efficient in vitro cellular growth and above all a solo donor liver may perhaps to be collectively utilized for the treatment of several patients with liver diseases [90]. For genetic liver defects, studies using hepatocyte transplantation have been conducted on animal models as well as on humans [91-94]. Contribution of donor hepatocytes up to 10% of total organ mass was observed when allogeneic hepatocytes were transplanted in patients with liver failure [95]. BM-LSCs have shown their efficiency by differentiating into hepatocytes in vitro and in vivo [96-98]. Recently, adult BM cells were transplanted into defi-cient in fumarylacetotate hydrolyse-deficient and type-1 ty-rosinemia mice which resulted in improved liver functioning [98]. Jang et al., have reported restoration of liver function

via conversion of HSCs into hepatocytes after a week of HSCs transplantation in liver-injured mice [99]. Clinical transplantation of hepatocytes, fetal hepatocytes, BMCs and MSCs has shown success in patients with fulminant liver failure [100-102] and liver cirrhosis [67, 103, 104]. The iso-lation of hepatic stem/progenitor cells from fetal and adult human livers has been carried out by means of human liver progenitor cell markers including Cell Adhesion Molecule (EpCAM) or Thy-1 [105, 106] and in future, the confront is still there to efficiently gain hepatocytes from non-damaged stem/progenitor cells.

There are few challenges that still have to be considered critical regarding liver cellular transplantations [107]. Though, pluripotent cells including ESCs and iPSCs have been known for their efficient replicative ability as compared to BMCs and MSCs but are also recognized to have high oncogenic potential. Similarly, while focusing over hepato-cyte derivation and differentiation from stem cells, the liver-specific functions also need to be well thought-out, i.e. the metabolic and detoxification capacity of differentiated hepa-tocytes, for example ammonia metabolism, bilirubin detoxi-fication, clotting factor synthesis. Route of liver cell trans-plantation could be another major concern to be confronted, however so far various routes of cell delivery to the livers


including catheter into the portal vein or hepatic/splenic ar-tery, have provided improved liver functioning [107]. Still there is a need to reconsider the routs for cellular transplanta-tion in a damaged liver keeping in mind the stressor factors like oxidative stress affecting the environment of diseased hepatocytes.

Liver Engineering/Liver Assist Devices

The purpose of bioengineered external devices is to en-gender biological functions within the body in the most probable similar way as the normal organ would. The bioar-

tificial liver devices have been also designed to perform the metabolic, detoxification synthetic properties of liver in an accurate manner in patients with acute or acute-on-chronic liver failure [108]. These devices are designed using hepato-

cytes and artificial supports which interface with patient’s circulation. The liver cell bioreactors either have hollow fi-ber or monolayer culture usually using porcine hepatocytes [109-111]. These bioreactors comprising of hepatocytes 10%

of liver weight allow blood to pump through them as like in dialysis [108]. As far as the standards for hepatocytes to be used in these devices is concerned, Fiegel et al. have flaw-lessly compiled criteria for the hepatocytes to accomplish the

purpose of these bioengineered devices, which include proper in vitro cell growth, genetic/immulogical handling of candidate cells, cell cryopreservation and genetic modifica-tion of recipient’s in ex vivo environment before re-

implantation [112]. These bioartificial liver assist devices have undergone various investigational and clinical studies. A bioartificial liver that was used for liver patients utilizing hepatoblastoma-derived HepG2A/C3A cell line was named

extracorporeal liver-assisted device (ELAD) system which has shown slight improvement from clinical point of view [113]. A phase I clinical trial with CellModule was also per-formed on liver patients utilizing human and porcine hepato-

cytes [114]. Another device, HepatAssist was used in a ran-domized, controlled multicenter study was conducted on patients with fulminant/subfulminant hepatic failure which showed an improved 30-day survival [115]. Recently,

adult/fetal hepatocytes were subjected to reversible immor-talization with the help of immortalization gene which helped in the growth of hepatocytes. These hepatocytes were then incorporated into bioartificial device after deleting the

immortalization genes [116]. Moreover, Kobayashi [117] proposed the abilities of human ES cell KhESC-1 cell line-differentiated hepatocytes which included albumin produc-tion (351ng / g cells), ammaonia and lidocaine metabolism

(7.8% and 23.6% respectively).

Though, as compared to the orthotopic liver transplanta-tion, the use of bioartificial liver assist devices and cellular transplantation seems to have advantage of being available preserved by cryopreservation [118] with appropriate freez-ing and thawing, however, these experimental procedures and cells used still need to undergo a lot of investigational studies to prove their ability used for surviving and maintain-ing liver functions being an external system.

Drug Screening

One of the unique application of stem cells is the drug screening by utilizing LSCs [119] though they require a

complete metabolic profile for screening of new drugs, as major drug withdrawals are expected to be correlated with liver toxicity [120].

ISOLATED LIVER STEM CELLS

In general, it is believed that liver transplantation is only

the effective treatment of end stage liver cirrhosis. However, molecular biologists proposed successful clinical trials based on the intra-hepatic liver cells transplantations that have ex-tensive potential to repopulate injured liver [121]. These

cells include hepatocytes, MSCs, OCs and iPSCs. Recently, bipotent epithelial colony-forming cells have been suggested to be isolated form mouse liver (fetal and adult) using Ep-CAM positive cells [122, 123], and by the activation of OCs,

EpCAM+

and TROP2+ cells contributed in the regeneration

of liver [123].

Isolation of Human Fetal Liver Stem Cells

Researchers have designed a system to isolate culture and identify human fetal LSCs in vitro in order to lay down the infrastructure/foundation of biological artificial liver sup-

porting system and hepatocyte transplantation [124]. They isolated fetal liver stem cells from the liver of dead human fetus and maintained the suspension in DMEM and incu-bated it. Immunohistochemistry was used to identify the

phenotypes of the cultured cells. By staining techniques, they found some cells were positive for CK19 and albumin. They concluded that in human fetal liver, stem cells exist and pos-sess potential to differentiate. Researchers have isolated,

cultured and passaged liver epithelial cells of human fetal with bipotent competency and numbers of studies have pro-claimed their consecutive differentiation into hepatocytes after transplantation into SCID mice [125]. Such studies in-

dicate that a human counterpart to the rodent fetal LSCs or progenitor cells still exists and mounting attainments paved these emerging grooves in the near future to ensure these cells will become available for liver repopulation.

Isolation of Human Adult LSCs

In 2006, an attempt was made to isolate and characterized

the population of human LSCs. They identified a pluripotent population in mature human liver that could provide a basis for clinical cell therapy strategies. Researchers isolated hu-man hepatocytes from surgical specimens of patients who

had undergone hepatectomies by collagenase dissection [126].

Mammalian Resident LSCs

Mammalian resident liver stem cell (RLSCs), a non-tumorigenic stem cell line has been obtained from fetal and neonatal murine livers. RLSCs possess potential to sponta-neously differentiate into hepatic epithelia and are reported

to derive from the murine fetal and neonatal livers. Al-though, it possesses potential to express -fetoprotein ( FP), a predominant marker for early hepatocyte but it fails to ex-press albumin. RLSCs are the useful tool that may enhance

our understandings of LSCs and might lead to a successful new therapeutic approach for liver diseases in the coming era [127].


CHALLENGES RELATED TO LSCs

Thus far, we have discussed the probable sources of LSCs, however, conversely some of these sources have been also identified as a cause of hepatocellular carcinoma [128] enlightening the fact that development of cancer might be a major challenge related to LSCs.

Tumorigenesis

Cancer stem cells (CSCs) are characterized as the cells that have self renewal, differentiation and tumor initiative characteristics [129]. Recently, it has been reported that in the cell population, tumor is inhabited and cells that acquire potential of tumor growth are known as CSCs. Worldwide, hepatocellular carcinoma (HCC) is the most abundant cancer among primary liver cancers [130]. It is believed that like other cancers, HCC develops due to 3-6 critical mutations in genes that are critical for cell renewal and cell growth. These critical mutations are vital for transformation of normal cells into cancerous cells. To date, a growing body of evidence has reported the existence of CSCs in breast cancer, brain, colon, lungs and prostate cancers [131, 132].

It is worth mentioning that oncomirs (miRNAs) have emerged as key regulators in the cellular process. Mounting evidence suggests about behaviors of miRNAs during differ-entiation of human embryonic stem cells (hESCSs) into a specialized cell type. hESCSs were induced to definitive endoderm (DE) cells and further differentiated to hepato-cytes [133]. Recently, it has been found that miR-512-3p, miR-512-5p and miR-520c-3p were enriched in hESCSs, miR-9, miR-205 and miR-375 in hESCS-derived DE cells [134]. Compelling evidence indicates that let-7 and miR-181 family members were up-regulated in HSCs. Moreover, sup-pression of let-7 enhanced the chemosensitivity of HSCs to sorafenib and doxorubicin whereas abrogation of miR-181 led to a reduction in invasive potential of HSCs [135]. It is interesting to note that miRNAs contribute in the neoplastic transformation of liver cancer stem cells (LCSCs) during hepatocarcinogenesis. In accordance with the assumption, miR-10b, miR-21, miR-470, miR-34c-3p and let-7i were identified as overexpressed in HCC cells compared to fetal liver cells [136].

On a similar note, it has lately been found that differenti-ated aggressive HBs overexpressed the miR-371-3 cluster with simultaneous down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster [137]. Consistent with the same con-cept miR-193b was considerably down-regulated in most of the HCC tissues however, re-establishment of miR-193b considerably suppressed the ability of hepatoma cells to form colonies in vitro and to develop tumors in nude mice [138]. Another intriguing mechanism that has recently been dis-mantled is cell-derived microvesicles (MVs). MVs after in-ternalization within target cells may deliver genetic informa-tion. Human liver resident stem cells were shown to release MVs shuttling functional mRNAs [139].

According to increasing information, it has been ac-claimed that microRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic trans-duction cascade and liver stem cell differentiation by target-ing SMADs [140]. The idealistic potential of regeneration in mammalian liver after intensive injury serves as an impres-

sive tool to understand the role of stem cells in tissue recon-struction as well as in tumorigenesis. There has been accu-mulating evidence supporting the fact that CD133+ stem / progenitor cells identified as part of CSCs population that are responsible for the tumor formation in brain, prostate, liver and other type of cancers [141]. CD133+ cells possess greater proliferative potential, efficient colony and tumor formation ability in vivo [61, 142].

Liver Cancer and Epigenetic Regulation of CSCs

Cancer is a genetically derived disease and is tightly regulated by number of epigenetic factors. CSCs are consid-ered as the germinal center of tumor evolution [142]. In the past few years, growing evidence also support the confirma-tion that CSCs are not only involve in the tumor initiation but also in the metastasis and relapses. Varieties of stem cell surface markers are used to identify CSCs. These markers detect surface protein and most widely used surface markers for CSCs are CD133, CD90 and EpCAM (epithelial cell ad-hesion molecule). Intensive research efforts have unraveled various molecular mechanism of hepato-tumorigenesis [51, 143, 144]. Number of molecular pathways that are linked to the stem cell self renewal, maintenance and pluripotency are identified including WNT/ -Catenin, Myc, P53, hedgehog, NOTCH, TGF- , EGF etc. Growing evidence supporting the fact that deregulation of these pathways leads to the progno-sis of liver cancer [145]. WNT signaling is found to be in-volved in the embryonic development and maintenance of proliferation, cell fate and survival. Disruption of WNT sig-naling due to genetic and epigenetic changes results in the wide range of diseases included liver and Colon cancer [146]. Proto-Oncogene “MYC” is stimulated by wide range of stimuli and involve in the regulation of 15% of all genes. Proto-Oncogene MYC play critical role in the apoptosis, proliferation, differentiation and maintenance of stem cell pluripotency. Structural modification and over-expression of MYC gene involve in wide range of cancers including hu-man and murine hepatocarcinogenesis [147].

Intensive research efforts have suggested that notch plays decisive role in apoptosis, differentiation, cell fate determi-nation and many other developmental processes. Any disrup-tion in notch signaling results in large number of cancers including liver cancer [148, 149].

CONCLUSIVE REMARKS AND FUTURE PERSPEC-

TIVES

In summary, to improve the repopulation of liver, re-searchers have focused on liver stem cell as alternative source for liver therapy along with immortalized human he-patocytes, stem cell derived hepatocytes and fetal hepato-cytes. To deal with the end stage liver complications, differ-ent alternative therapies for liver transplant have been carried out. Nowadays, scientists have focused on the concept of “regenerative medicines” that relies on the exclusive poten-tial of stem cells because of their eligibility for liver cell re-placement including embryonic and fetal stem cells, induce pluripotent cells, endogenous LSCs, annex stem cells and extra-hepatic stem cells. Primarily, it has to be figured out whether the animal studies can be practiced according to human situation. A major complication faced during human study is difficulty in isolating human LSCs and requires the


identification of suitable stem cell markers; moreover, the dilemma of immune rejection also needs to be addressed. Finally, understanding of rules for the basis of hepatic differ-entiation is critical for realistic advancement in the state of therapeutic stem-cell transplantation.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

Declared none.

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Received: July 30, 2013 Revised: September 27, 2013 Accepted: September 28, 2013

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