**1. Introduction**

The liver is a highly specialized detoxifying organ involved in: i) glucose homeostasis; ii) lipid homeostasis and ketone bodies production; iii) metabolism of amino acids. Most of the liver functions are carried out by the hepatocytes (about 70-75% of hepatic cells) that, together with cholangiocytes (10-5 % of hepatic cells), are of endodermal derivation and constitute the hepatic parenchyma.

The liver has a peculiar and fascinating ability: it is able to regenerate itself after loss of parenchyma for surgical resection or injuries caused by drugs, toxins or acute viral diseases. The ancient myth of Prometheus highlighted this capability: the Titan Prometheus was bound for ever to a rock as punishment by Zeus for his theft of the fire; each day a great eagle ate his liver and each night the liver was regenerated, only to be eaten again the next day.

The liver compensatory regeneration is a rapid and tightly orchestrated phenomenon efficiently ensuring the reacquisition of the original tissue mass and its functionality. Primarily, it involves the re-entry into cell cycle of parenchymal hepatocytes which are able to completely recover the original liver mass (Fausto, 2000). The liver anatomical and functional units reconstitution also requires non-parenchymal cells (endothelial cells, cholangiocytes, Kupffer cells, stellate cells). It is yet not clear if each cell histotype is involved in the proliferative process or if the regeneration requires the activity of a cell with multiple differentiation potential. Recently, the bipotentiality of the hepatocytes, able to divide giving rise to both hepatocytes and cholangiocytes, has been suggested. Furthermore, when injury is severe or the hepatocytes can no longer proliferate a progenitor cell population, normally a quiescent compartment is activated. A population of small portal cells named oval cells was first identified in 1978 by Shinozuka and colleagues (Shinozuka et al., 1978). Now as "oval cells" is indicated a heterogeneous population of bipotent transient amplifying cells, originating from the Canal of Hering (Dabeva & Shafritz, 1993). These cells are normally quiescent but, after injury, rapidly and extensively proliferate and differentiate in hepatocytes and cholangiocytes (Yovchev et al., 2008).

The observation that oval cells are a mixed precursor population suggests their differentiation from liver stem cells (Theise et al., 1999). Since the hepatocytes are able to

Hepatocytes and Progenitor – Stem Cells in Regeneration and Therapy 5

TGF- α is another growth factor relevant in liver regeneration (Tomiya et al., 2000). It belongs to the EGF family, of which all members (EGF, heparin binding EGF-like factor and amphiregulin) transduce trough the common receptor EGF receptor (EGFR) and exert overlapping functions (Fausto 2004). This factor acts in autocrine and paracrine fashions and

IL-6 induces mitotic signals in hepatocytes through the activation of STAT-3 (Cressman et al., 1996). The IL-6/STAT-3 signaling involves several proteins: the IL-6 receptor, gp130, receptor-associated Janus kinase (Jak) and STAT-3. The IL-6 receptor is in a complex with gp130, which, after recognition by IL-6, transmits the signal. Jak is responsible of gp130 and STAT-3 activation after IL-6 binding. The STAT-3 form released by gp130 dimerizes and translocates to the nucleus to activate the transcription. STAT3 controls cell cycle progression from G1 to S phase regulating the expression of cyclin D1. In fact, in the liverspecific STAT3-KO model mice, mitotic activity of hepatocytes after PH is reduced

The PIK/PDK1/Akt signaling pathways are activated by receptor tyrosine kinases or receptors coupled with G proteins by IL-6, TNF-α, HGF, EGF, TGF-α and others (Desmots et al., 2002) (Koniaris et al., 2003). An important downstream molecule of Akt for cell growth is mTOR (Fingar et al., 2002). The activation of this pathway coexists with STAT-3 signaling. In STAT-3-KO mice no significant differences were observed macroscopically in liver regeneration in comparison to control animals, reaching the liver of these mice after PH an equal size. This observation may be explained considering the increase in size of the hepatocytes. Increase in cell size corresponds to marked phosphorylation of Akt and its

The third phase in liver regeneration is the termination step. A stop signal is necessary to avoid an inappropriate liver functional size but the molecular pathways involved in this phenomenon are not yet clear. A key role is exerted by the cytokine TGF-β, secreted by hepatocytes and platelets, that inhibits DNA synthesis (Nishikawa et al., 1998). In fact, within 2-6 hours after PH, the insulin growth factor (IGF) binding protein-1 (IGFBP-1) is

When liver parenchyma damage is particularly serious and hepatocytes are no longer able to proliferate, liver regeneration can occur through the intervention of bipotent progenitor cells that can proliferate and differentiate into hepatocytes and bile duct cells. It was 1950 when Wilson and Leduc, studying the regeneration of rat liver after severe nutritional damage, observed for the first time these particular cells, located within or immediately adjacent to the Canal of Hering, and their differentiation into two histological types of liver epithelial cells (Wilson & Leduc, 1950). In 1956 Faber called these cells, which are found in the liver of mice treated with carcinogens (Farber 1956), "oval cells" for their morphology. The first characterization of oval cells has shown the simultaneous expression of bile ducts (CK-7, CK-19 and OV-6) and hepatocytes (alpha-fetoprotein and albumin) markers (Lazaro et al., 1998). Subsequent studies have shown the activation, during oval cell compartment proliferation, of stem cell genes such as c-kit (Fujio et al., 1994), CD34 (Omori et al., 1997)

downstream molecules p70 S6K, mTOR and GSK3beta (Haga et al., 2005).

produced to counteract its inhibitor effects (Ujike et al., 2000).

**3. Liver progenitor cells and regeneration** 

and LIF (Omori et al., 1996) .

its production and secretion are induced by HGF.

significantly (Li et al., 2002).

regenerate themself to compensate liver mass loss, the existence of a liver stem cell, able to drive regeneration in conditions of extreme toxicity affecting the same hepatocytes, has long been debated. Today, there is growing evidence that the liver stem cell exists and its isolation from the organ, its numerical expansion in vitro and its characterization are joint efforts in many laboratories around the world. The interest of the scientific community in the identification, isolation and manipulation of the hepatic stem cell also depends on the fact that the great hopes placed in the use of mature hepatocytes in cell transplantation protocols for the treatment of liver diseases have been disappointed. The basis of these unsatisfactory therapeutic approaches lie in the paradox, not yet resolved, of the inability of hepatocytes, which show *in vivo* a virtually unlimited proliferative potential, to grow *in vitro* to quantitatively and qualitatively amount suitable for cell transplantation in adults.

#### **2. Hepatocyte and regeneration**

Regeneration of the original liver mass after damage has been extensively studied in rodents after two-thirds partial hepatectomy (PH) (Bucher, 1963). Regeneration of the liver depends on both hyperplasia and hypertrophy of the hepatocytes, cells that in a normal adult liver exhibit a quiescent phenotype. Hypertrophy begins within hours after PH then hyperplasia follows (Taub, 2004). This occurs first in the periportal region of the liver lobule then spreads toward the pericentral region (Fausto & Campbell 2003).

The restoration of liver volume depends on three steps involving the hepatocytes: i) initiation, ii) proliferation and iii) termination phases.

The initiation step depends on the "priming" of parenchymal cells, mainly via the signaling pathways triggered by the cytokines IL-6 and TNF-α secreted by Kupffer cells, rendering the hepatocytes sensitive to growth factors and competent to replication.

After the G0/G1 transition in the initiation phase, the hepatocytes will enter into the cell cycle (Taub, 2004). Growth factors, primarily HGF, epidermal growth factor (EGF) and TGFα, are responsible of this second step of regeneration in which the hepatocytes both proliferate and grow in cell size, activating the IL-6/STAT-3 and the PI3K/PDK1/Akt pathways respectively. The first signaling cascade regulates the cyclin D1/p21 and also protects against cell death, for example by up-regulating FLIP, Bcl2 and Bcl-xL. The latter pathway regulates cell size via mammalian target of rapamycin (mTOR) (Fausto, 2000; Serandour et al., 2005; Pahlavan et al., 2006; Fujiyoshi & Ozaki 2011). Numerous growth factors (for example HGF, TGF-α, EGF, glucagon, insulin and cytokines like TNF, IL-1 and - 6 and somatostatin (SOM)) are implicated in the regeneration process.

The HGF is a potent growth factor mainly acting on hepatocytes in a paracrine manner binding to its specific trans-membrane receptor tyrosine kinase c-met. HGF is secreted as an inactive precursor and stored in the extracellular matrix (ECM), then activated by the fibrinolytic system (Kim et al., 1997). Plasmin and metalloproteinases (MMPs) degrade the ECM and release pro-HGF that, in turn, is cleaved into an activated form by the urokinasetype plasminogen activator (u-PA)(Kim et al., 1997). The HGF/met signaling is transduced to its downstream mediators, i.e. the Ras-Raf-MEK, ERK1/2 (Borowiak et al., 2004), PI3K/PDK1/Akt (Okano et al., 2003) and mTOR/S6 kinase pathways, resulting in cell cycle progression.

regenerate themself to compensate liver mass loss, the existence of a liver stem cell, able to drive regeneration in conditions of extreme toxicity affecting the same hepatocytes, has long been debated. Today, there is growing evidence that the liver stem cell exists and its isolation from the organ, its numerical expansion in vitro and its characterization are joint efforts in many laboratories around the world. The interest of the scientific community in the identification, isolation and manipulation of the hepatic stem cell also depends on the fact that the great hopes placed in the use of mature hepatocytes in cell transplantation protocols for the treatment of liver diseases have been disappointed. The basis of these unsatisfactory therapeutic approaches lie in the paradox, not yet resolved, of the inability of hepatocytes, which show *in vivo* a virtually unlimited proliferative potential, to grow *in vitro*

to quantitatively and qualitatively amount suitable for cell transplantation in adults.

spreads toward the pericentral region (Fausto & Campbell 2003).

the hepatocytes sensitive to growth factors and competent to replication.

6 and somatostatin (SOM)) are implicated in the regeneration process.

initiation, ii) proliferation and iii) termination phases.

Regeneration of the original liver mass after damage has been extensively studied in rodents after two-thirds partial hepatectomy (PH) (Bucher, 1963). Regeneration of the liver depends on both hyperplasia and hypertrophy of the hepatocytes, cells that in a normal adult liver exhibit a quiescent phenotype. Hypertrophy begins within hours after PH then hyperplasia follows (Taub, 2004). This occurs first in the periportal region of the liver lobule then

The restoration of liver volume depends on three steps involving the hepatocytes: i)

The initiation step depends on the "priming" of parenchymal cells, mainly via the signaling pathways triggered by the cytokines IL-6 and TNF-α secreted by Kupffer cells, rendering

After the G0/G1 transition in the initiation phase, the hepatocytes will enter into the cell cycle (Taub, 2004). Growth factors, primarily HGF, epidermal growth factor (EGF) and TGFα, are responsible of this second step of regeneration in which the hepatocytes both proliferate and grow in cell size, activating the IL-6/STAT-3 and the PI3K/PDK1/Akt pathways respectively. The first signaling cascade regulates the cyclin D1/p21 and also protects against cell death, for example by up-regulating FLIP, Bcl2 and Bcl-xL. The latter pathway regulates cell size via mammalian target of rapamycin (mTOR) (Fausto, 2000; Serandour et al., 2005; Pahlavan et al., 2006; Fujiyoshi & Ozaki 2011). Numerous growth factors (for example HGF, TGF-α, EGF, glucagon, insulin and cytokines like TNF, IL-1 and -

The HGF is a potent growth factor mainly acting on hepatocytes in a paracrine manner binding to its specific trans-membrane receptor tyrosine kinase c-met. HGF is secreted as an inactive precursor and stored in the extracellular matrix (ECM), then activated by the fibrinolytic system (Kim et al., 1997). Plasmin and metalloproteinases (MMPs) degrade the ECM and release pro-HGF that, in turn, is cleaved into an activated form by the urokinasetype plasminogen activator (u-PA)(Kim et al., 1997). The HGF/met signaling is transduced to its downstream mediators, i.e. the Ras-Raf-MEK, ERK1/2 (Borowiak et al., 2004), PI3K/PDK1/Akt (Okano et al., 2003) and mTOR/S6 kinase pathways, resulting in cell cycle

**2. Hepatocyte and regeneration** 

progression.

TGF- α is another growth factor relevant in liver regeneration (Tomiya et al., 2000). It belongs to the EGF family, of which all members (EGF, heparin binding EGF-like factor and amphiregulin) transduce trough the common receptor EGF receptor (EGFR) and exert overlapping functions (Fausto 2004). This factor acts in autocrine and paracrine fashions and its production and secretion are induced by HGF.

IL-6 induces mitotic signals in hepatocytes through the activation of STAT-3 (Cressman et al., 1996). The IL-6/STAT-3 signaling involves several proteins: the IL-6 receptor, gp130, receptor-associated Janus kinase (Jak) and STAT-3. The IL-6 receptor is in a complex with gp130, which, after recognition by IL-6, transmits the signal. Jak is responsible of gp130 and STAT-3 activation after IL-6 binding. The STAT-3 form released by gp130 dimerizes and translocates to the nucleus to activate the transcription. STAT3 controls cell cycle progression from G1 to S phase regulating the expression of cyclin D1. In fact, in the liverspecific STAT3-KO model mice, mitotic activity of hepatocytes after PH is reduced significantly (Li et al., 2002).

The PIK/PDK1/Akt signaling pathways are activated by receptor tyrosine kinases or receptors coupled with G proteins by IL-6, TNF-α, HGF, EGF, TGF-α and others (Desmots et al., 2002) (Koniaris et al., 2003). An important downstream molecule of Akt for cell growth is mTOR (Fingar et al., 2002). The activation of this pathway coexists with STAT-3 signaling. In STAT-3-KO mice no significant differences were observed macroscopically in liver regeneration in comparison to control animals, reaching the liver of these mice after PH an equal size. This observation may be explained considering the increase in size of the hepatocytes. Increase in cell size corresponds to marked phosphorylation of Akt and its downstream molecules p70 S6K, mTOR and GSK3beta (Haga et al., 2005).

The third phase in liver regeneration is the termination step. A stop signal is necessary to avoid an inappropriate liver functional size but the molecular pathways involved in this phenomenon are not yet clear. A key role is exerted by the cytokine TGF-β, secreted by hepatocytes and platelets, that inhibits DNA synthesis (Nishikawa et al., 1998). In fact, within 2-6 hours after PH, the insulin growth factor (IGF) binding protein-1 (IGFBP-1) is produced to counteract its inhibitor effects (Ujike et al., 2000).
