**5. Hepatocyte transplantation in cell-based therapeutic**

Animal models in which transplanted cells show a selective advantage over resident hepatocytes have been used to study transplantation, proliferation and reconstitution potential of the hepatocytes. Liver animal models belong to three groups (Palmes & Spiegel 2004): i) hepatotoxin-induced models; ii) surgical models; iii) animal models of hereditary liver defects.

Normal adult hepatocytes can be serially transplanted and single hepatocyte can be clonally amplified, showing stem-like properties, and serially passaged to repopulate almost 70% of the liver of (Fah)-deficient mice (Overturf et al., 1999). Excellent results have been obtained by using transgenic *Rag2-/-/Il2rg-/-* mice (deficient for the recombinant activation gene-2 and the common γ-chain of the interleukin receptor) (Traggiai et al., 2004) or the *Alb-uPA(tg(+/-)*  mice (expressing the uroplasminogen activator (uPA) under the transcriptional control of the albumin promoter) (Sandgren et al., 1991)) or mice obtained by the crossing of the above reported genotypes (Haridass et al., 2009; Azuma et al., 2007).

Hepatocyte transplantation protocols in humans have been proposed as an alternative to orthotropic liver transplantation in patients and used for some metabolic disorders i.e. familial hypercholesterolemia, glycogen storage disease type 1a, urea cycle defects and congenital deficiency of coagulation factors (Quaglia et al., 2008). Currently, the liver transplantation is the treatment of choice for acute and chronic end-stage liver failure and for diseases refractory to other treatments; but the limited availability of donor organs is the major limiting factor in this therapeutic procedure. Although different techniques of implants using either complete liver, liver reduced or hyper-reduced "split liver" (liver for two) have tried to overcome the shortage of organs, liver transplantation remain an unsufficient approach to satisfy the needs of patients with liver disease.

In recent years, hepatocyte transplantation has emerged as a potential alternative or complementary procedure to liver transplantation, at least in certain circumstances. The application of this therapeutic modality is based on the concept that cell transplantation would replace the function of the affected organ, either temporarily, allowing the recovery of the organ functionality or the availability of a liver for the transplant, or permanently, preventing need for this last procedure.

The development of this therapeutic approach could provide a new opportunity for patients with liver disease, particularly for children suffering from some metabolic diseases, with certain advantages over liver transplantation. In fact it is a less invasive and risky procedure and it has a lower cost. There is also a greater availability of material to be transplanted and that could be used as a source of cells (organs considered "marginal", material resulting from organ reductions, from partial hepatectomy and cadaveric livers unsuitable for transplantation) and the possibility of using a donor to several recipients.

Despite these advantages, a number of critical issues are still unresolved: the rejection of transplanted hepatocytes, their correct localization and functionality and, mostly, cells availability at the right time. The latter remains a problem that would be definitively solved with the cultivation and the preservation of large scale culture of hepatocytes. Nevertheless, these cells in culture, contrary to what happens *in vivo* during liver regeneration, have a very low proliferative potential and quickly lose their differentiated characteristics.

Hepatocytes and Progenitor – Stem Cells in Regeneration and Therapy 13

Cardinale, V., Y Wang, et al. (2012). "The biliary tree-a reservoir of multipotent stem

Cicchini, C., D. Filippini, et al. (2006). "Snail controls differentiation of hepatocytes by

Colletti, M., C. Cicchini, et al. (2009). "Convergence of Wnt signaling on the HNF4alpha-

Conigliaro, A., M. Colletti, et al. (2008). "Isolation and characterization of a murine resident

Cressman, D. E., L. E. Greenbaum, et al. (1996). "Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice." Science 274(5291): 1379-1383. Dabeva, M. D. and D. A. Shafritz (1993). "Activation, proliferation, and differentiation of

Desmots, F., M. Rissel, et al. (2002). "Pro-inflammatory cytokines tumor necrosis factor alpha

Farber, E. (1956). "Similarities in the sequence of early histological changes induced in the

Fausto, N. (2004). "Liver regeneration and repair: hepatocytes, progenitor cells, and stem

Fausto, N. and J. S. Campbell (2003). "The role of hepatocytes and oval cells in liver

Fingar, D. C., S. Salama, et al. (2002). "Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E." Genes Dev 16(12): 1472-1487. Fujio, K., R. P. Evarts, et al. (1994). "Expression of stem cell factor and its receptor, c-kit,

investigation; a journal of technical methods and pathology 70(4): 511-516. Fujiyoshi, M. and M. Ozaki (2011). "Molecular mechanisms of liver regeneration and

Garibaldi, F., Cicchini, C., et al. (in press). "An epistatic mini-circuitry between the

Gebhardt, R. (1992). "Metabolic zonation of the liver: regulation and implications for liver

Gebhardt, R.& Reichen J. (1994). " Changes in distribution and activity of glutamine

repressing HNF4alpha expression." J Cell Physiol 209(1): 230-238.

liver stem cell." Cell death and differentiation 15(1): 123-133.

regeneration." Am J Pathol 143(6): 1606-1620.

carcinoma." Carcinogenesis 23(3): 435-445.

cells." Hepatology 39(6): 1477-1487.

Pancreat Sci 18(1): 13-22.

function". Pharmacol Ther; 53:275-354.

hyperammonemia". Hepatology;20:684-91

print]

dimethylaminoazobenzene." Cancer Res 16(2): 142-148. Fausto, N. (2000). "Liver regeneration." J Hepatol 32(1 Suppl): 19-31.

regeneration and repopulation." Mech Dev 120(1): 117-130.

28 february.

672.

cells".Nature reviews Gastroenterology&Hepatology, advance online publication

driven transcription in controlling liver zonation." Gastroenterology 137(2): 660-

progenitor cells into hepatocytes in the D-galactosamine model of liver

and interleukin-6 and survival factor epidermal growth factor positively regulate the murine GSTA4 enzyme in hepatocytes." J Biol Chem 277(20): 17892-17900. Dumble, M. L., E. J. Croager, et al. (2002). "Generation and characterization of p53 null

transformed hepatic progenitor cells: oval cells give rise to hepatocellular

liver of the rat by ethionine, 2-acetylamino-fluorene, and 3'-methyl-4-

during liver regeneration from putative stem cells in adult rat." Laboratory

protection for treatment of liver dysfunction and diseases." J Hepatobiliary

transcription factors Snail and HNF4alpha controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs". Cell Death and Differentiation 2011 Dec 2. doi: 10.1038/cdd.2011.175. [Epub ahead of

synthetase in carbon tetrachloride-induced cirrhosis in the rat: potential role in

This implies that cell therapy can be carried out only with freshly isolated cells, not expanded *in vitro*. The number of cells that can be achieved with this approach is usually not sufficient to colonize adult livers, while there is more chance of success in pediatric patients with metabolic diseases of genetic origin since they can be treated with a limited number of hepatocytes.

#### **6. Conclusion**

Liver stem cells may represent an important tool for the treatment of the liver diseases. They could be an alternative source of functional hepatocytes aimed at cell transplantation, tissue engineering and bio-artificial liver. Manipulation of stem cells will be more efficient since we know the factors controlling their biology. Only by dissecting the molecular events underlying the stemness, the differentiation choice and the maintenance of the differentiated phenotype can we control stem cell behavior for therapeutic purposes. The translation of *in vitro* studies in *in vivo* experimental models and, finally, in humans is one of the major challenges of experimental hepatology. Moreover, better understanding the mechanisms that control the proliferation of stem and progenitor cells will shed new light on the molecular and cellular basis of liver cancer.

#### **7. References**


This implies that cell therapy can be carried out only with freshly isolated cells, not expanded *in vitro*. The number of cells that can be achieved with this approach is usually not sufficient to colonize adult livers, while there is more chance of success in pediatric patients with metabolic diseases of genetic origin since they can be treated with a limited number of

Liver stem cells may represent an important tool for the treatment of the liver diseases. They could be an alternative source of functional hepatocytes aimed at cell transplantation, tissue engineering and bio-artificial liver. Manipulation of stem cells will be more efficient since we know the factors controlling their biology. Only by dissecting the molecular events underlying the stemness, the differentiation choice and the maintenance of the differentiated phenotype can we control stem cell behavior for therapeutic purposes. The translation of *in vitro* studies in *in vivo* experimental models and, finally, in humans is one of the major challenges of experimental hepatology. Moreover, better understanding the mechanisms that control the proliferation of stem and progenitor cells will shed new light on the

Allain, J. E., I. Dagher, et al. (2002). "Immortalization of a primate bipotent epithelial liver

Amicone, L., F. M. Spagnoli, et al. (1997). "Transgenic expression in the liver of truncated

Azuma, H., N. Paulk, et al. (2007). "Robust expansion of human hepatocytes in Fah-/-

Benhamouche, S., Decaens, T., et al. (2006). "Apc tumor suppressor gene is the "zonation-

Blau, H. M., T. R. Brazelton, et al. (2001). "The evolving concept of a stem cell: entity or

Borowiak, M., A. N. Garratt, et al. (2004). "Met provides essential signals for liver

Braun, L., M. Goyette, et al. (1987). "Growth in culture and tumorigenicity after transfection

Bucher, N. L. (1963). "Regeneration of Mammalian Liver." International review of cytology

Burdon, T., A. Smith, et al. (2002). "Signalling, cell cycle and pluripotency in embryonic stem

Cardinale, V., Y. Wang, et al. (2011). "Multipotent stem/progenitor cells in human biliary

with the ras oncogene of liver epithelial cells from carcinogen-treated rats." Cancer

tree give rise to hepatocytes, cholangiocytes, and pancreatic islets" Hepatology

/Rag2-/-/Il2rg-/- mice." Nature biotechnology 25(8): 903-910.

regeneration." Proc Natl Acad Sci U S A 101(29): 10608-10613.

keeper" of mouse liver". Dev Cell;10:759-70.

function?" Cell 105(7): 829-841.

research 47(15): 4116-4124.

cells." Trends Cell Biol 12(9): 432-438.

15: 245-300.

54:2159-2172.

stem cell." Proceedings of the National Academy of Sciences of the United States of

Met blocks apoptosis and permits immortalization of hepatocytes." EMBO J 16(3):

hepatocytes.

**6. Conclusion** 

**7. References** 

495-503.

molecular and cellular basis of liver cancer.

America 99(6): 3639-3644.


Hepatocytes and Progenitor – Stem Cells in Regeneration and Therapy 15

Omori, N., R. P. Evarts, et al. (1996). "Expression of leukemia inhibitory factor and its

Overturf, K., M. Al-Dhalimy, et al. (1999). "The repopulation potential of hepatocyte

Pahlavan, P. S., R. E. Feldmann, Jr., et al. (2006). "Prometheus' challenge: molecular, cellular and systemic aspects of liver regeneration." J Surg Res 134(2): 238-251. Palmes, D. and H. U. Spiegel (2004). "Animal models of liver regeneration." Biomaterials

Pelletier, L., S. Rebouissou, et al. (2011). "HNF1alpha inhibition triggers epithelialmesenchymal transition in human liver cancer cell lines." BMC cancer 11(1): 427. Prindull, G. and D. Zipori (2004). "Environmental guidance of normal and tumor cell

Quaglia, A., S. C. Lehec, et al. (2008). "Liver after hepatocyte transplantation for liver-based metabolic disorders in children." Cell transplantation 17(12): 1403-1414. Reddy, G. P., C. I. McAuliffe, et al. (2002). "Cytokine receptor repertoire and cytokine

Sandgren, E. P., R. D. Palmiter, et al. (1991). "Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene." Cell 66(2): 245-256. Santangelo, L., A. Marchetti, et al. (2011). "The stable repression of mesenchymal program is

Sells, M. A., S. L. Katyal, et al. (1981). "Isolation of oval cells and transitional cells from the

Serandour, A. L., P. Loyer, et al. (2005). "TNFalpha-mediated extracellular matrix

Shinozuka, H., B. Lombardi, et al. (1978). "Early histological and functional alterations of

Schmelzer, E., L. Zhang, et al. (2007). "Human hepatic stem cells from fetal and postnatal

Spagnoli, F. M., L. Amicone, et al. (1998). "Identification of a bipotential precursor cell in

Stanulovic ,V.S., Kyrmizi, I., et al. (2007) "Hepatic HNF4alpha deficiency induces periportal

Strick-Marchand, H., S. Morosan, et al. (2004). "Bipotential mouse embryonic liver stem cell

journal of technical methods and pathology 75(1): 15-24.

phase progression." Exp Hematol 30(7): 792-800.

pathology 155(6): 2135-2143.

25(9): 1601-1611.

2899.

Hepatology.

41(3): 478-486.

38(4): 1092-1098.

donors". J Exp Med 204:1973-1987

Cell Biol 143(4): 1101-1112.

of America 101(22): 8360-8365.

Hepatology;45:433-44

Institute 66(2): 355-362.

receptor during liver regeneration in the adult rat." Laboratory investigation; a

populations differing in size and prior mitotic expansion." The American journal of

plasticity: epithelial mesenchymal transitions as a paradigm." Blood 103(8): 2892-

responsiveness of Ho(dull)/Rh(dull) stem cells with differing potentials for G1/S

required for hepatocyte identity: A novel role for hepatocyte nuclear factor 4alpha."

livers of rats fed the carcinogen DL-ethionine." Journal of the National Cancer

remodeling is required for multiple division cycles in rat hepatocytes." Hepatology

ethionine liver carcinogenesis in rats fed a choline-deficient diet." Cancer research

hepatic cell lines derived from transgenic mice expressing cyto-Met in the liver." J

expression of glutamine synthetase and other pericentral enzymes".

lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes." Proceedings of the National Academy of Sciences of the United States


Grompe, M. (2003). "The role of bone marrow stem cells in liver regeneration." Seminars in

Haga, S., W. Ogawa, et al. (2005). "Compensatory recovery of liver mass by Akt-mediated

Hailfinger, S., M. Jaworski, et al. (2006). "Zonal gene expression in murine liver: lessons from

Haridass, D., Q. Yuan, et al. (2009). "Repopulation efficiencies of adult hepatocytes, fetal

Hay, E. D. (1995). "An overview of epithelio-mesenchymal transformation." Acta Anat

Herrera, M. B., S. Bruno, et al. (2006). "Isolation and characterization of a stem cell

Hixson, D. C., R. A. Faris, et al. (1990). "An antigenic portrait of the liver during

Kim, T. H., W. M. Mars, et al. (1997). "Extracellular matrix remodeling at the early stages of

Kinugasa, A. & Thurman, R.G. (1986)."Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule". Biochem J;236:425-30. Koniaris, L. G., I. H. McKillop, et al. (2003). "Liver regeneration." J Am Coll Surg 197(4): 634-

Kosman, D., et al. (1991) "Establishment of the mesoderm-neuroectoderm boundary in the

Lazaro, C. A., J. A. Rhim, et al. (1998). "Generation of hepatocytes from oval cell precursors

Li, W., X. Liang, et al. (2002). "STAT3 contributes to the mitogenic response of hepatocytes

McConnell, S. K. and C. E. Kaznowski (1991). "Cell cycle dependence of laminar determination in developing neocortex." Science 254(5029): 282-285. Menthena, A., N. Deb, et al. (2004). "Bone marrow progenitors are not the source of

Najimi, M., D. N. Khuu, et al. (2007). "Adult-derived human liver mesenchymal-like cells as

Nishikawa, Y., M. Wang, et al. (1998). "Changes in TGF-beta receptors of rat hepatocytes

Okano, J., G. Shiota, et al. (2003). "Hepatocyte growth factor exerts a proliferative effect on

Omori, M., R. P. Evarts, et al. (1997). "Expression of alpha-fetoprotein and stem cell factor/ckit system in bile duct ligated young rats." Hepatology 25(5): 1115-1122.

a potential progenitor reservoir of hepatocytes?" Cell transplantation 16(7): 717-728.

during primary culture and liver regeneration: increased expression of TGF-beta receptors associated with increased sensitivity to TGF-beta-mediated growth

oval cells through the PI3K/AKT signaling pathway." Biochem Biophys Res

during liver regeneration." J Biol Chem 277(32): 28411-28417.

expanding oval cells in injured liver." Stem Cells 22(6): 1049-1061.

population from adult human liver." Stem Cells 24(12): 2840-2850.

liver regeneration in the rat." Hepatology 26(4): 896-904.

hepatocellular hypertrophy in liver-specific STAT3-deficient mice." J Hepatol 43(5):

liver progenitor cells, and embryonic stem cell-derived hepatic cells in albuminpromoter-enhancer urokinase-type plasminogen activator mice." The American

carcinogenesis." Pathobiology : journal of immunopathology, molecular and

liver disease 23(4): 363-372.

tumors." Hepatology 43(3): 407-414.

journal of pathology 175(4): 1483-1492.

Drosophila embryo". Science 254, 118-122.

inhibition." J Cell Physiol 176(3): 612-623.

Commun 309(2): 298-304.

in culture." Cancer research 58(23): 5514-5522.

799-807.

659.

(Basel) 154(1): 8-20.

cellular biology 58(2): 65-77.


**2** 

*1Curtin University,* 

*Australia* 

**Liver Progenitor Cells, Cancer Stem Cells** 

Janina E.E. Tirnitz-Parker1,2, George C.T. Yeoh2 and John K. Olynyk1,2

*2Western Australian Institute for Medical Research, University of Western Australia* 

There is great interest in the biology of liver progenitor cells (LPCs) because of their stem cell-like ability to regenerate the liver when the hepatocyte pool is exhausted. Barely detectable in healthy tissue, they emerge upon chronic insult in periportal regions, proliferate and migrate to injury sites in the parenchyma and eventually differentiate into hepatocytes and cholangiocytes to restore liver mass, morphology and function. The increasing worldwide shortage of livers for orthotopic transplantation means LPCs have assumed more prominence as candidates for cell therapy as an alternative therapeutic approach for the treatment of various liver diseases. However, an LPC response is usually seen in pre-cancerous liver pathologies and their high proliferation potential makes them possible transformation targets; associations that overshadow their restorative capability. This mandates that we continue to investigate the factors that govern their activation, proliferation and especially their differentiation into mature, functional cells to effectively

Tissue regeneration and maintenance in healthy intestine and skin is achieved within days and weeks respectively. In contrast healthy liver has a very slow cell turnover rate and the vast majority of hepatocytes is considered to be in the quiescent, non-proliferative G0 phase of the cell cycle. It has been estimated that at any one time only 1 in 20,000 to 40,000 hepatocytes is undergoing mitotic cell division with an average life span of 200 to 300 days

The mechanisms by which hepatic cells are replaced in healthy liver are controversial. An early model, the "streaming liver" hypothesis is based on the metabolic zonation and differential gene expression patterns of periportal compared to pericentral hepatocytes. Periportal cells were proposed to proliferate and migrate ("stream") towards the central area with maturation during the journey and terminal differentiation achieved when the cells reached the central zone (Zajicek *et al.*, 1985; Arber *et al.*, 1988; Sigal *et al.*, 1992). However there is no convincing evidence for a periportal to pericentral differentiation gradient and while hepatocytes in opposing lobular areas are responsible for different

direct transplanted cells towards regeneration and not tumorigenicity.

**2. Normal liver tissue turnover** 

(Bucher & Malt, 1971).

**1. Introduction** 

**and Hepatocellular Carcinoma** 

