**5. Oval cells**

Oval cells were first described in rodents, emerging when the liver is exposed to certain toxins (for review, see (63)) (64). Termed "oval cells" because of their oval shaped nucleus, these small cells have a diameter of less than 10 μm. They are located near the portal triads and expand in the livers of animals exposed to oncogenic insults. The term "oval cells" frequently refers to liver stem cells or progenitors. However, oval cells can be distinguished from normal hepatic progenitors phenotypically and in their growth regulatory requirements (65). Several protocols have been shown to lead to the emergence of oval cells: administration of 2-acetylamino fluorine or dipin in combination with partial hepatectomy; administration of carbon tetrachloride, 3-methyl-diaminobenzidine, galactosamine, furane, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine; etluonine addition to a choline-deficient diet; or transgenic albumin-urokinase-type plasminogen-activator mice.

Oval cells were described as positive for several surface and intracellular markers (including hematopoietic and mesenchymal markers not found on normal epithelial hepatic stem cells) such as CD34 (66), CD117 (67), AFP, CK14, CK19 (68), GGT, OC.2, OV-6, and CD90 (69). CD90, however, was subsequently demonstrated to be expressed not by oval cells but by myofibroblasts (70).

Some primary liver tumors are suggested to emerge from oval cells (71).

mice. Similarly, Nierhoff *et al*. (35) demonstrated that fetal mouse liver epithelial cells positive for AFP or E-cadherin did not express hematopoietic stem cell markers CD34, CD117, Ter119, or CD45, but were positive for progenitor markers Sca-1 and Pancytokeratin. Both E-cadherin positive sorted as well as unsorted fetal liver cell fractions from wild type mice gave rise to liver parenchyma when transplanted into retrorsine treated

As described for human hepatoblasts above, mouse fetal liver hepatoblasts have been shown to express the surface marker Dlk-1 (35-37). Dlk-1 positive sorted mouse fetal liver cells can be cultured long-term and, when transplanted into the spleen, give rise to hepatocytes in the liver. Dabeva *et al*. (52) described the re-population potential of wild type fetal rat liver cells when transplanted into DPPIV-/- rat models. These models included knockouts that had undergone two-third partial hepatectomy and were either treated with retrorsine or not. In rats treated with retrorsine, which blocked proliferation of endogenous hepatocytes, mainly bipotential, transplanted progenitors were observed expressing AFP, albumin, and CK19. In non-treated rats, transplanted cells expressed mainly either

The positive expression of aldehyde dehydrogenase (ALDH) has been used as a feature to select progenitors from adult mouse liver (53). ALDH+ cells were shown to have stem cell characteristics and to express markers of human hepatic stem cells such as CD326, CK19,

Various hepatic progenitor cell lines have been developed from normal, genetically modified, or toxin treated rodents (54-62). Several of these lines were described as

Oval cells were first described in rodents, emerging when the liver is exposed to certain toxins (for review, see (63)) (64). Termed "oval cells" because of their oval shaped nucleus, these small cells have a diameter of less than 10 μm. They are located near the portal triads and expand in the livers of animals exposed to oncogenic insults. The term "oval cells" frequently refers to liver stem cells or progenitors. However, oval cells can be distinguished from normal hepatic progenitors phenotypically and in their growth regulatory requirements (65). Several protocols have been shown to lead to the emergence of oval cells: administration of 2-acetylamino fluorine or dipin in combination with partial hepatectomy; administration of carbon tetrachloride, 3-methyl-diaminobenzidine, galactosamine, furane, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine; etluonine addition to a choline-deficient diet;

Oval cells were described as positive for several surface and intracellular markers (including hematopoietic and mesenchymal markers not found on normal epithelial hepatic stem cells) such as CD34 (66), CD117 (67), AFP, CK14, CK19 (68), GGT, OC.2, OV-6, and CD90 (69). CD90, however, was subsequently demonstrated to be expressed not by oval cells but by

DPPIV-/- mice.

hepatocytic or biliary markers.

bipotential *in vitro* or when transplanted *in vivo*.

or transgenic albumin-urokinase-type plasminogen-activator mice.

Some primary liver tumors are suggested to emerge from oval cells (71).

CD133, and Sox9.

**5. Oval cells** 

myofibroblasts (70).

#### **6. Hepatic progenitors found in various mammalian species**

Few data have been published on hepatic progenitors from species other than human or rodent. In general, pigs are used as an animal model closely resembling human physiology and metabolic functions. This makes the pig model more favorable than the rodent model. However, this model is scarcely used due to obvious constraints in keeping animals. Kano *et al*. (72, 73) investigated hepatic progenitors isolated by culture selection from nonparenchymal liver cell suspensions of six-seven months old pigs. Cell clusters in culture were positive for the hepatic markers AFP, albumin, transferrin, CK18, CK7, and c-met, but did they not express biliary markers such as gamma-glutamyltransferase, CK19, and CK14, although they were positive for oval cell marker OV6. Duct-like structures emerged from clusters expressing biliary epithelial markers. Clonal cell growth could be established (74). Comparable cells could be obtained (75) by isolating small liver cells from pigs that had undergone partial hepatectomy. In addition to the hepatic markers albumin and AFP, these cells also expressed biliary marker CK19 and were positive for OV6. In culture, cells were positive for stem-cell factor, CD117, CD90, AFP, CK19, and OV6. Fetal porcine liver cells were used to establish colonies of pluripotent progenitors (76, 77).

#### **7. Extra-hepatic sources of potential liver progenitors**

Several extra-hepatic sources have been described to harbor progenitors able to differentiate into hepatic lineages *in vitro* and *in vivo*. It is widely debated whether cells of extra-hepatic origin are able to differentiate into hepatic cell types or if they fuse with the recipient's liver cells when transplanted. Tissue sources include bone marrow, adipose tissue, umbilical cord, and peripheral blood. Hepatic differentiation potentials of embryonic stem cells (ESC), placenta derived stem cells, or induced pluripotent stem cells (iPS cells) are not discussed here; further literature can be found in reviews (78-82).

Bone marrow cells or bone marrow derived hematopoietic stem cells have been suggested to be able to trans-differentiate into hepatic lineages. Petersen *et al*. performed initial experiments with cross-strain and cross-sex bone marrow and liver transplantations in rats (83). When male bone marrow was transplanted into female recipients and liver damage was induced, Y-chromosome positive cells could be detected in the female livers. Also, when male dipeptidyl peptidase (DPPIV) positive bone marrow was transplanted into female DPPIV negative recipients and liver damage was induced, DPPIV positive cells could be detected in the female livers. A further approach included transplantations of major histocompatibility complex class II L21-6 isozyme negative whole livers into positive enzyme expressing rats; after induction of liver damage, positive enzyme expressing cells could be detected. Alison *et al*. (84) investigated human female livers from patients who had received male bone marrow transplants. Y-chromosome positive cells that co-expressed CK8 were detected in the female livers. About 0.5 – 2% of all livers cells were Y-chromosome positive. Theise *et al*. described further *in vivo* experiments on the possible contribution of bone marrow cells towards hepatic lineages in mice (85) and humans (86). Whole bone marrow cells or CD34+lin- sorted cells from male mice were transplanted into female recipients; up to 2.2% (bone marrow) or about 0.7% (CD34+lin- ) Y-chromosome positive cells could be detected within the female livers. In human patients who had undergone cross-sex bone marrow transplantation, Y-chromosome positive cells could be observed in female livers. 4 – 43% of cholangiocytes and 4 – 38% of hepatocytes were positive for Y-

Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 53

for intracellular albumin and AFP expression; some cells demonstrated CK7 expression. Sun *et al*. (121) showed that human umbilical blood cells integrated into livers of rat chimeras, and these cells were positive for human hematopoietic, biliary, and hepatic proteins. Crema *et al*. isolated CD133+ cord blood cells (122). Transplantation into liver-damaged SCID mice resulted in clusters of human-derived cells expressing human leucocyte antigen-class I, HepPar1, and OV6 antigens. Within these clusters, human albumin, AFP, and CK19 could be detected. Human umbilical blood cells demonstrated *in vitro* hepatocyte-like differentiation and expression of hepatic proteins when transplanted in rodents with

Conclusively, it appears that extra-hepatic progenitors integrate into the liver only to a very minor percentage and only when severe liver damage is induced. The majority of these events appear to be due to fusion and not differentiation. The observed improvements of liver functions by mesenchymal cells could be attributed to their secretion of growth factors

Although there is still some debate about the detailed characteristics that identify hepatic progenitors, much progress has been achieved during recent years in defining, isolating, characterizing, and transplanting various types of progenitors. This is especially the case for hepatic progenitors isolated from human livers. Hepatic progenitors represent a population with potential advantages over total liver cell suspensions or hepatocytes for cell transplantation in patients (29, 128), for review see (129). Because of their high proliferation and differentiation potential a major advantage for transplantation of stem cells over total liver cell suspensions would be the requirement for less cell numbers to inject, which would decrease the risks associated with transplanting high cell numbers. In addition, because of their proliferation and differentiation potential, progenitors could be used in applications such as extracorporeal liver support systems (130, 131), and may be used as an alternative cell source in pharmacological screening models. Cultures of progenitors also provide an

[1] Michalopoulos GK. Liver regeneration: alternative epithelial pathways. Int J Biochem

[2] Michalopoulos GK. Liver regeneration after partial hepatectomy: critical analysis of

[4] Overturf K, al-Dhalimy M, Ou CN, Finegold M, Grompe M. Serial transplantation

[5] Schmelzer E, McClelland RE, Melhem A, Zhang L, Yao H, Wauthier E, et al. Hepatic

reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J

stem cell and the liver's maturational lineages: Implications for liver biology, gene expression, and cell therapies. In: Potten C, Clarke R, Wilson J, Renehan A, editors.

[3] Michalopoulos GK. Liver regeneration. J Cell Physiol. 2007 Nov;213(2):286-300.

Tissue stem cells. New York: Taylor and Francis; 2006. p. 161-214.

and cytokines and immunosuppressive properties (111, 125-127).

easy *in vitro* tool to study principles of developmental biology.

mechanistic dilemmas. Am J Pathol. Jan;176(1):2-13.

Cell Biol. Feb;43(2):173-9.

Pathol. 1997;151(5):1273-80.

induced liver damage (107, 123, 124).

**8. Conclusion** 

**9. References** 

chromosome. Lagasse *et al*. (87) intravenously injected adult wild type bone marrow cells in FAH–/– mice, an animal model of tyrosinemia type I. The mice were rescued and biochemical functions were regained. Only purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration from total bone marrow cells. However, subsequently published studies revealed that the majority of those liver cells, which were assumed to be donor derived differentiated bone marrow cells, are instead rather the product of donor cells fusing with host liver cells (88, 89). Other studies demonstrated bone marrow cells contributed nothing or very little to liver lineages *in vivo* (90-92). Jang *et al*. (93) and Harris *et al*. (94) could show, however, that a minor percentage (up to 0.1%) of bone marrow cells can contribute to liver cells *in vivo* without fusion. Most evidence to date indicates that only a minority of the observed trans-differentiation events is actually due to differentiation of bone marrow cells into liver lineages and the majority of observed trans-differentiated cells are indeed fusion events.

Similar to the findings of the above described *in vivo* studies, *in vitro* studies of the hepatic differentiation potential of hematopoietic stem cells produced contradicting findings (95-99). Overall, results from *in vitro* studies suggest that bone marrow hematopoietic stem cells can differentiate only barely, if at all, into hepatic lineages.

Mesenchymal stem cells (MSCs), which have similar characteristics, have been isolated from various tissue sources; MSCs from sources such as bone marrow (100-105), skin (106), umbilical cord (107, 108) and adipose tissues (109-115) have been analyzed for the potential to differentiate towards hepatic lineages *in vitro* and *in vivo*. MSC markers from various tissues show similar surface marker expression profiles, described first as classical MSC markers by Pittenger *et al* (116), which were CD29, CD44, CD71, CD90, CD106, CD120a, and CD124. Culture selected clonal bone marrow derived MSCs expressed mesenchymal cellspecific markers (e.g. CD13, CD29, CD44, and CD90), and were negative for hematopoietic markers such as CD3, CD14, CD34, and CD45 (100). When transplanted in SCID mice, nonfused human cells could be detected in the liver. Adipose tissue derived stem cells were described to differentiate into hepatic lineages (109-115). Adipose tissue derived MSCs were characterized to potentially express CD9, CD13, CD29, CD44, CD49d, CD54, CD73, CD90, CD105, CD146, CD166, osteopontin and osteonectin, and to be negative for hematopoietic and endothelial markers such as CD45, CD34 and CD31. Marker expressions and hepatic potential are further summarized in current reviews (117, 118). In general, most *in vivo* transplantation studies using MSCs did not exclude donor cell fusion with host cells. Only one study (Aurich *et al*. (112)) demonstrated the integration of non-fused human adipose MSCs in the livers of mice that had undergone combined toxin induced liver damage and hepatectomy.

Lee *et al.* (119) transplanted green fluorescent protein mouse gallbladder epithelial cells into non-fluorescent SCID mice that had undergone retrorsine treatment and either partial hepatectomy before transplantation or carbon tetrachloride treatment following transplantation. Within one to four months after transplantation, green fluorescent protein positive cells could be detected within the recipient mice. These cells expressed mostly biliary markers, but cells positive for hepatic markers could be detected as well.

Zhao *et al*. isolated hematopoietic stem cells from peripheral blood (120) and demonstrated their *in vitro* multilineage differentiation potential; treatment of cultures with HGF induced cells to acquire a round or oval-like flattened morphology. Most of the cells were positive for intracellular albumin and AFP expression; some cells demonstrated CK7 expression. Sun *et al*. (121) showed that human umbilical blood cells integrated into livers of rat chimeras, and these cells were positive for human hematopoietic, biliary, and hepatic proteins. Crema *et al*. isolated CD133+ cord blood cells (122). Transplantation into liver-damaged SCID mice resulted in clusters of human-derived cells expressing human leucocyte antigen-class I, HepPar1, and OV6 antigens. Within these clusters, human albumin, AFP, and CK19 could be detected. Human umbilical blood cells demonstrated *in vitro* hepatocyte-like differentiation and expression of hepatic proteins when transplanted in rodents with induced liver damage (107, 123, 124).

Conclusively, it appears that extra-hepatic progenitors integrate into the liver only to a very minor percentage and only when severe liver damage is induced. The majority of these events appear to be due to fusion and not differentiation. The observed improvements of liver functions by mesenchymal cells could be attributed to their secretion of growth factors and cytokines and immunosuppressive properties (111, 125-127).

#### **8. Conclusion**

52 Liver Regeneration

chromosome. Lagasse *et al*. (87) intravenously injected adult wild type bone marrow cells in FAH–/– mice, an animal model of tyrosinemia type I. The mice were rescued and biochemical functions were regained. Only purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration from total bone marrow cells. However, subsequently published studies revealed that the majority of those liver cells, which were assumed to be donor derived differentiated bone marrow cells, are instead rather the product of donor cells fusing with host liver cells (88, 89). Other studies demonstrated bone marrow cells contributed nothing or very little to liver lineages *in vivo* (90-92). Jang *et al*. (93) and Harris *et al*. (94) could show, however, that a minor percentage (up to 0.1%) of bone marrow cells can contribute to liver cells *in vivo* without fusion. Most evidence to date indicates that only a minority of the observed trans-differentiation events is actually due to differentiation of bone marrow cells into liver lineages and the majority of

Similar to the findings of the above described *in vivo* studies, *in vitro* studies of the hepatic differentiation potential of hematopoietic stem cells produced contradicting findings (95-99). Overall, results from *in vitro* studies suggest that bone marrow hematopoietic stem cells can

Mesenchymal stem cells (MSCs), which have similar characteristics, have been isolated from various tissue sources; MSCs from sources such as bone marrow (100-105), skin (106), umbilical cord (107, 108) and adipose tissues (109-115) have been analyzed for the potential to differentiate towards hepatic lineages *in vitro* and *in vivo*. MSC markers from various tissues show similar surface marker expression profiles, described first as classical MSC markers by Pittenger *et al* (116), which were CD29, CD44, CD71, CD90, CD106, CD120a, and CD124. Culture selected clonal bone marrow derived MSCs expressed mesenchymal cellspecific markers (e.g. CD13, CD29, CD44, and CD90), and were negative for hematopoietic markers such as CD3, CD14, CD34, and CD45 (100). When transplanted in SCID mice, nonfused human cells could be detected in the liver. Adipose tissue derived stem cells were described to differentiate into hepatic lineages (109-115). Adipose tissue derived MSCs were characterized to potentially express CD9, CD13, CD29, CD44, CD49d, CD54, CD73, CD90, CD105, CD146, CD166, osteopontin and osteonectin, and to be negative for hematopoietic and endothelial markers such as CD45, CD34 and CD31. Marker expressions and hepatic potential are further summarized in current reviews (117, 118). In general, most *in vivo* transplantation studies using MSCs did not exclude donor cell fusion with host cells. Only one study (Aurich *et al*. (112)) demonstrated the integration of non-fused human adipose MSCs in the livers of mice that had undergone combined toxin induced liver damage and

Lee *et al.* (119) transplanted green fluorescent protein mouse gallbladder epithelial cells into non-fluorescent SCID mice that had undergone retrorsine treatment and either partial hepatectomy before transplantation or carbon tetrachloride treatment following transplantation. Within one to four months after transplantation, green fluorescent protein positive cells could be detected within the recipient mice. These cells expressed mostly

Zhao *et al*. isolated hematopoietic stem cells from peripheral blood (120) and demonstrated their *in vitro* multilineage differentiation potential; treatment of cultures with HGF induced cells to acquire a round or oval-like flattened morphology. Most of the cells were positive

biliary markers, but cells positive for hepatic markers could be detected as well.

observed trans-differentiated cells are indeed fusion events.

differentiate only barely, if at all, into hepatic lineages.

hepatectomy.

Although there is still some debate about the detailed characteristics that identify hepatic progenitors, much progress has been achieved during recent years in defining, isolating, characterizing, and transplanting various types of progenitors. This is especially the case for hepatic progenitors isolated from human livers. Hepatic progenitors represent a population with potential advantages over total liver cell suspensions or hepatocytes for cell transplantation in patients (29, 128), for review see (129). Because of their high proliferation and differentiation potential a major advantage for transplantation of stem cells over total liver cell suspensions would be the requirement for less cell numbers to inject, which would decrease the risks associated with transplanting high cell numbers. In addition, because of their proliferation and differentiation potential, progenitors could be used in applications such as extracorporeal liver support systems (130, 131), and may be used as an alternative cell source in pharmacological screening models. Cultures of progenitors also provide an easy *in vitro* tool to study principles of developmental biology.

#### **9. References**


Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 55

[23] Malhi H, Irani AN, Gagandeep S, Gupta S. Isolation of human progenitor liver

[24] Sicklick JK, Li YX, Melhem A, Schmelzer E, Zdanowicz M, Huang J, et al. Hedgehog

[25] Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, Chiriboga L, et al. The canals of Hering and hepatic stem cells in humans. Hepatology. 1999;30(6):1425-33. [26] Roskams TA, Theise ND, Balabaud C, Bhagat G, Bhathal PS, Bioulac-Sage P, et al.

[27] Carpentier R, Suner RE, van Hul N, Kopp JL, Beaudry JB, Cordi S, et al. Embryonic

[28] Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S, Kuhara T, et al.

[29] Khan AA, Shaik MV, Parveen N, Rajendraprasad A, Aleem MA, Habeeb MA, et al.

[30] Sakamoto S, Yachi A, Anzai T, Wada T. AFP-producing cells in hepatitis and in liver

[31] Abelev GI. Alpha-fetoprotein in ontogenesis and its association with malignant tumors.

[32] Zhang L, Theise N, Chua M, Reid LM. The stem cell niche of human livers: symmetry between development and regeneration. Hepatology. 2008 Nov;48(5):1598-607. [33] Abelev GI, Eraiser TL. Cellular aspects of alpha-fetoprotein reexpression in tumors.

[34] Terrace JD, Currie IS, Hay DC, Masson NM, Anderson RA, Forbes SJ, et al. Progenitor

[35] Nierhoff D, Ogawa A, Oertel M, Chen YQ, Shafritz DA. Purification and

[36] Tanimizu N, Nishikawa M, Saito H, Tsujimura T, Miyajima A. Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci. 2003;116(Pt 9):1775-86. [37] Tanimizu N, Saito H, Mostov K, Miyajima A. Long-term culture of hepatic progenitors derived from mouse Dlk+ hepatoblasts. J Cell Sci. 2004 Dec 15;117(Pt 26):6425-34. [38] Tanimizu N, Miyajima A, Tanimizu N, Nishikawa M, Saito H, Tsujimura T, et al. Notch

[39] Germain L, Blouin MJ, Marceau N. Biliary epithelial and hepatocytic cell lineage

cell characterization and location in the developing human liver. Stem Cells Dev.

characterization of mouse fetal liver epithelial cells with high in vivo repopulation

signaling controls hepatoblast differentiation by altering the expression of liverenriched transcription factors. Isolation of hepatoblasts based on the expression of

relationships in embryonic rat liver as determined by the differential expression of

Gastrointest Liver Physiol. 2006;290(5):G859-70. Epub 2005 Dec 1.

ductular reactions in human livers. Hepatology. 2004;39(6):1739-45.

progenitor cells. Gastroenterology. Oct;141(4):1432-8, 8 e1-4.

exocrine pancreas and intestine. Nat Genet. Jan;43(1):34-41.

cirrhosis. Ann N Y Acad Sci. 1975;259:253-8.

Semin Cancer Biol. 1999 Apr;9(2):95-107.

capacity. Hepatology. 2005;42(1):130-9.

Dlk/Pref-1. J Cell Sci. 2004;117(Pt 15):3165-74.

Adv Cancer Res. 1971;14:295-358.

2010;19(4):409-18.

2007 Oct;16(5):771-8.

hepatocytes. J Cell Sci. 2002 Jul 1;115(Pt 13):2679-88.

epithelial cells with extensive replication capacity and differentiation into mature

signaling maintains resident hepatic progenitors throughout life. Am J Physiol

Nomenclature of the finer branches of the biliary tree: canals, ductules, and

ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver

Continuous cell supply from a Sox9-expressing progenitor zone in adult liver,

Human fetal liver-derived stem cell transplantation as supportive modality in the management of end-stage decompensated liver cirrhosis. Cell Transplant.


[6] Dan YY, Yeoh GC. Liver stem cells: a scientific and clinical perspective. J Gastroenterol

[7] Zaret K. Early liver differentiation: genetic potentiation and multilevel growth control.

[8] Zaret KS. Hepatocyte differentiation: from the endoderm and beyond. Curr Opin Genet

[9] Zaret KS. Regulatory phases of early liver development: paradigms of organogenesis.

[10] Kolterud A, Wandzioch E, Carlsson L. Lhx2 is expressed in the septum transversum

[12] Schmelzer E, Zhang L, Bruce A, Wauthier E, Ludlow J, Yao H, et al. Human Hepatic Stem Cells from Fetal and Postnatal Donors. J Exp Med. 2007;204(8):1973-87. [13] Shiojiri N, Koike T. Differentiation of biliary epithelial cells from the mouse hepatic endodermal cells cultured in vitro. Tohoku J Exp Med. 1997 Jan;181(1):1-8. [14] Shiojiri N, Mizuno T. Differentiation of functional hepatocytes and biliary epithelial

[15] Haruna Y, Saito K, Spaulding S, Nalesnik MA, Gerber MA. Identification of bipotential progenitor cells in human liver development. Hepatology. 1996 Mar;23(3):476-81. [16] Shiojiri N, Takeshita K, Yamasaki H, Iwata T. Suppression of C/EBP alpha expression

[17] Yamasaki H, Sada A, Iwata T, Niwa T, Tomizawa M, Xanthopoulos KG, et al.

[18] Schmelzer E, Wauthier E, Reid LM. The Phenotypes of Pluripotent Human Hepatic

[19] Dan YY, Riehle KJ, Lazaro C, Teoh N, Haque J, Campbell JS, et al. Isolation of

[20] Najimi M, Khuu DN, Lysy PA, Jazouli N, Abarca J, Sempoux C, et al. Adult-derived

[21] Khuu DN, Scheers I, Ehnert S, Jazouli N, Nyabi O, Buc-Calderon P, et al. In vitro

[22] Herrera MB, Bruno S, Buttiglieri S, Tetta C, Gatti S, Deregibus MC, et al. Isolation and

Progenitors. Stem Cells. 2006;24(8):1852-8. Epub 2006 Apr 20.

mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2. Gene Expr Patterns. 2004 Sep;4(5):521-8. [11] Shiojiri N. The origin of intrahepatic bile duct cells in the mouse. J Embryol Exp

cells from immature hepatocytes of the fetal mouse in vitro. Anat Embryol (Berl).

in biliary cell differentiation from hepatoblasts during mouse liver development. J

Suppression of C/EBPalpha expression in periportal hepatoblasts may stimulate biliary cell differentiation through increased Hnf6 and Hnf1b expression.

multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci U S A. 2006;103(26):9912-7.

human liver mesenchymal-like cells as a potential progenitor reservoir of

differentiated adult human liver progenitor cells display mature hepatic metabolic functions: a potential tool for in vitro pharmacotoxicological testing. Cell

characterization of a stem cell population from adult human liver. Stem Cells. 2006

Hepatol. 2008 May;23(5):687-98.

Dev. 2001;11(5):568-74.

Morphol. 1984 Feb;79:25-39.

1993 Mar;187(3):221-9.

Epub 2006 Jun 16.

Dec;24(12):2840-50.

Hepatol. 2004 Nov;41(5):790-8.

Transplant. 2011;20(2):287-302.

Development. 2006 Nov;133(21):4233-43.

hepatocytes? Cell Transplant. 2007;16(7):717-28.

Curr Opin Genet Dev. 1998;8(5):526-31.

Nat Rev Genet. 2002 Jul;3(7):499-512.


Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 57

[54] Strick-Marchand H, Weiss MC. Inducible differentiation and morphogenesis of

[55] Strick-Marchand H, Morosan S, Charneau P, Kremsdorf D, Weiss MC. Bipotential

[56] Rogler LE. Selective bipotential differentiation of mouse embryonic hepatoblasts in

[57] Richards WG, Yoder BK, Isfort RJ, Detilleux PG, Foster C, Neilsen N, et al. Isolation and

[58] Ott M, Rajvanshi P, Sokhi RP, Alpini G, Aragona E, Dabeva M, et al. Differentiation-

[59] Grisham JW. Cell types in long-term propagable cultures of rat liver. Ann N Y Acad Sci.

[60] Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells. Proc Soc Exp Biol Med. 1993;204(3):270-9. [61] Tsao MS, Smith JD, Nelson KG, Grisham JW. A diploid epithelial cell line from normal

[62] Lee LW, Tsao MS, Grisham JW, Smith GJ. Emergence of neoplastic transformants

[63] Newsome PN, Hussain MA, Theise ND. Hepatic oval cells: helping redefine a paradigm

[64] Farber E. Similarities in the sequence of early histological changes induced in the liver

[65] Oh SH, Hatch HM, Petersen BE. Hepatic oval 'stem' cell in liver regeneration. Semin

[66] Omori N, Omori M, Evarts RP, Teramoto T, Miller MJ, Hoang TN, et al. Partial cloning

[67] Fujio K, Evarts RP, Hu Z, Marsden ER, Thorgeirsson SS. Expression of stem cell factor

[68] Bisgaard HC, Nagy P, Ton PT, Hu Z, Thorgeirsson SS. Modulation of keratin 14 and

[69] Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology. 1998;27(2):433-45.

1):794-804.

1980;349:128-37.

1984;154(1):38-52.

2004;101(22):8360-5. Epub 2004 May 20.

vitro. Am J Pathol. 1997;150(2):591-602.

conditions. Am J Pathol. 1989;135(1):63-71.

Cell Dev Biol. 2002 Dec;13(6):405-9.

rat. Lab Invest. 1994;70(4):511-6.

the adult rat. Hepatology. 1997;26(3):720-7.

regeneration. J Cell Physiol. 1994 Jun;159(3):475-84.

in stem cell biology. Curr Top Dev Biol. 2004;61:1-28.

dimethylaminoazobenzene. Cancer Res. 1956;16(2):142-8.

TgN737Rpw mice. Am J Pathol. 1997;150(4):1189-97.

from the normal F344 rat liver. J Pathol. 1999;187(3):365-73.

bipotential liver cell lines from wild-type mouse embryos. Hepatology. 2002;36(4 Pt

mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes. Proc Natl Acad Sci U S A.

characterization of liver epithelial cell lines from wild-type and mutant

specific regulation of transgene expression in a diploid epithelial cell line derived

adult rat liver with phenotypic properties of 'oval' cells. Exp Cell Res.

spontaneously or after exposure to N-methyl-N'-nitro-N-nitrosoguanidine in populations of rat liver epithelial cells cultured under selective and nonselective

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

of rat CD34 cDNA and expression during stem cell-dependent liver regeneration in

and its receptor, c-kit, during liver regeneration from putative stem cells in adult

alpha-fetoprotein expression during hepatic oval cell proliferation and liver

cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components. Cancer Res. 1988;48(17):4909-18.


[40] Kubota H, Reid LM. Clonogenic hepatoblasts, common precursors for hepatocytic and

[41] Tateno C, Yoshizato K. Growth and differentiation in culture of clonogenic hepatocytes

[42] Mitaka T, Kojima T, Mizuguchi T, Mochizuki Y. Growth and maturation of small

[43] Mitaka T, Mikami M, Sattler GL, Pitot HC, Mochizuki Y. Small cell colonies appear in

[44] Mitaka T, Sato F, Mizuguchi T, Yokono T, Mochizuki Y. Reconstruction of hepatic

[45] Tateno C, Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo

[46] Suzuki A, Nakauchi H, Taniguchi H. In vitro production of functionally mature

[47] Suzuki A, Zheng YW, Fukao K, Nakauchi H, Taniguchi H. Liver repopulation by c-Met-

[48] Suzuki A, Zheng Y, Kondo R, Kusakabe M, Takada Y, Fukao K, et al. Flow-cytometric

[49] Suzuki A, Taniguchi H, Zheng YW, Takada Y, Fukunaga K, Seino K, et al. Clonal colony

[50] Suzuki A, Zheng Yw YW, Kaneko S, Onodera M, Fukao K, Nakauchi H, et al. Clonal

[51] Feng RQ, Du LY, Guo ZQ. In vitro cultivation and differentiation of fetal liver stem cells

[52] Dabeva MD, Petkov PM, Sandhu J, Oren R, Laconi E, Hurston E, et al. Proliferation and

[53] Dolle L, Best J, Empsen C, Mei J, Van Rossen E, Roelandt P, et al. Successful isolation of

antigen. Proc Natl Acad Sci U S A. 2000;97(22):12132-7.

epidermal growth factor. Hepatology. 1992;16(2):440-7.

Hepatogastroenterology. 2004 Mar-Apr;51(56):423-6.

conditioning medium. Transplant Proc. 2000;32(7):2328-30.

developing liver. J Cell Biol. 2002;156(1):173-84. Epub 2002 Jan 07.

liver. Hepatology. 2000;32(6):1230-9.

from mice. Cell Res. 2005;15(5):401-5.

Hepatology. 2012;55(2):540-52.

adult rat liver. Am J Pathol. 2000;156(6):2017-31.

Cancer Res. 1988;48(17):4909-18.

Pathol. 1996;149(5):1593-605.

1995;214(2):310-7.

1999;29(1):111-25.

1996;148(2):383-92.

2003;12(5):469-73.

cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components.

biliary lineages, are lacking classical major histocompatibility complex class I

that express both phenotypes of hepatocytes and biliary epithelial cells. Am J

hepatocytes isolated from adult rat liver. Biochem Biophys Res Commun.

the primary culture of adult rat hepatocytes in the presence of nicotinamide and

organoid by rat small hepatocytes and hepatic nonparenchymal cells. Hepatology.

multiple cell divisions and express normal parenchymal phenotypes. Am J Pathol.

hepatocytes from prospectively isolated hepatic stem cells. Cell Transplant.

positive stem/progenitor cells isolated from the developing rat liver.

separation and enrichment of hepatic progenitor cells in the developing mouse

formation of hepatic stem/progenitor cells enhanced by embryonic fibroblast

identification and characterization of self-renewing pluripotent stem cells in the

differentiation of fetal liver epithelial progenitor cells after transplantation into

liver progenitor cells by aldehyde dehydrogenase activity from naive mice.


Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 59

[87] Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, et al. Purified

[88] Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, et al. Cell

[89] Vig P, Russo FP, Edwards RJ, Tadrous PJ, Wright NA, Thomas HC, et al. The sources of

[90] Cantz T, Sharma AD, Jochheim-Richter A, Arseniev L, Klein C, Manns MP, et al.

[91] Menthena A, Deb N, Oertel M, Grozdanov PN, Sandhu J, Shah S, et al. Bone marrow

[92] Popp FC, Slowik P, Eggenhofer E, Renner P, Lang SA, Stoeltzing O, et al. No

[94] Harris RG, Herzog EL, Bruscia EM, Grove JE, Van Arnam JS, Krause DS. Lack of a

[95] Lian G, Wang C, Teng C, Zhang C, Du L, Zhong Q, et al. Failure of hepatocyte marker-

[96] Yamada Y, Nishimoto E, Mitsuya H, Yonemura Y. In vitro transdifferentiation of adult

[97] Avital I, Inderbitzin D, Aoki T, Tyan DB, Cohen AH, Ferraresso C, et al. Isolation,

[98] Crosby HA, Kelly DA, Strain AJ. Human hepatic stem-like cells isolated using c-kit or

[99] Miyazaki M, Akiyama I, Sakaguchi M, Nakashima E, Okada M, Kataoka K, et al.

[100] Tao XR, Li WL, Su J, Jin CX, Wang XM, Li JX, et al. Clonal mesenchymal stem cells

injured livers of SCID mice. J Cell Biochem. 2009 Oct 15;108(3):693-704. [101] Ayatollahi M, Soleimani M, Tabei SZ, Kabir Salmani M. Hepatogenic differentiation of

model of prolonged hepatic injury. Stem Cells. 2007 Mar;25(3):639-45. [93] Jang YY, Collector MI, Baylin SB, Diehl AM, Sharkis SJ. Hematopoietic stem cells

2000;6(11):1229-34.

2004;22(6):1049-61.

Epub 2004 May 09.

44.

2004 Jul 2;305(5680):90-3.

Cells. Dec 26;3(12):113-21.

vitro. Exp Hematol. 2006;34(3):348-58.

cells without fusion. Exp Hematol. 2006;34(1):97-106.

Biochem Biophys Res Commun. 2002;298(1):24-30.

cells. Biochem Biophys Res Commun. 2001;288(1):156-64.

Apr 24;422(6934):897-901.

Hepatology. 2006;43(2):316-24.

Cell Transplant. 2004;13(6):659-66.

hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med.

fusion is the principal source of bone-marrow-derived hepatocytes. Nature. 2003

parenchymal regeneration after chronic hepatocellular liver injury in mice.

Reevaluation of bone marrow-derived cells as a source for hepatocyte regeneration.

progenitors are not the source of expanding oval cells in injured liver. Stem Cells.

contribution of multipotent mesenchymal stromal cells to liver regeneration in a rat

convert into liver cells within days without fusion. Nat Cell Biol. 2004;6(6):532-9.

fusion requirement for development of bone marrow-derived epithelia. Science.

expressing hematopoietic progenitor cells to efficiently convert into hepatocytes in

bone marrow Sca-1+ cKit- cells cocultured with fetal liver cells into hepatic-like

characterization, and transplantation of bone marrow-derived hepatocyte stem

CD34 can differentiate into biliary epithelium. Gastroenterology. 2001;120(2):534-

Improved conditions to induce hepatocytes from rat bone marrow cells in culture.

derived from human bone marrow can differentiate into hepatocyte-like cells in

mesenchymal stem cells induced by insulin like growth factor-I. World J Stem


[70] Dezso K, Jelnes P, Laszlo V, Baghy K, Bodor C, Paku S, et al. Thy-1 is expressed in

[71] Faris RA, Monfils BA, Dunsford HA, Hixson DC. Antigenic relationship between oval

[73] Kano J, Tokiwa T, Zhou X, Kodama M. Colonial growth and differentiation of epithelial

[74] Tokiwa T, Yamazaki T, Ono M, Enosawa S, Tsukiyama T. Cloning and characterization

[75] He Z, Feng M. Activation, isolation, identification and culture of hepatic stem cells from

[76] Talbot NC, Pursel VG, Rexroad CE, Jr., Caperna TJ, Powell AM, Stone RT. Colony

[77] Talbot NC, Rexroad CE, Jr., Powell AM, Pursel VG, Caperna TJ, Ogg SL, et al. A

[78] Chun YS, Chaudhari P, Jang YY. Applications of patient-specific induced pluripotent

[79] Asgari S, Pournasr B, Salekdeh GH, Ghodsizadeh A, Ott M, Baharvand H. Induced pluripotent stem cells: a new era for hepatology. J Hepatol. Oct;53(4):738-51. [80] Chen X, Zeng F. Directed hepatic differentiation from embryonic stem cells. Protein

[81] Hannoun Z, Filippi C, Sullivan G, Hay DC, Iredale JP. Hepatic endoderm

[82] Miki T, Marongiu F, Ellis EC, Dorko K, Mitamura K, Ranade A, et al. Production of hepatocyte-like cells from human amnion. Methods Mol Biol. 2009;481:155-68. [83] Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al. Bone

[84] Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, et al. Hepatocytes

[85] Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, et al. Derivation of

[86] Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, et al. Liver

from non-hepatic adult stem cells. Nature. 2000;406(6793):257.

from bone marrow in humans. Hepatology. 2000;32(1):11-6.

porcine livers. Cell Transplant. 2008;17(1-2):179-86.

Vitro Cell Dev Biol Anim. 1994;30A(12):851-8.

for liver disease. Int J Biol Sci.6(7):796-805.

porcine liver tissues. Cell Prolif. 2011 Dec;44(6):558-66.

of the pig. In Vitro Cell Dev Biol Anim. 1994;30A(12):843-50.

"resistant hepatocyte" model system. Cancer Res. 1991;51(4):1308-17. [72] Kano J, Noguchi M, Kodama M, Tokiwa T. The in vitro differentiating capacity of

Am J Pathol. 2007 Nov;171(5):1529-37.

2000;156(6):2033-43.

Sep;13 Suppl:S62-9.

Cell. Mar;2(3):180-8.

Hepatology. 2000;31(1):235-40.

Sep;5(3):233-44.

70.

hepatic myofibroblasts and not oval cells in stem cell-mediated liver regeneration.

cells and a subpopulation of hepatic foci, nodules, and carcinomas induced by the

nonparenchymal epithelial cells derived from adult porcine livers. Am J Pathol.

cells derived from abattoir adult porcine livers. J Gastroenterol Hepatol. 1998

of liver progenitor cells from the scattered cell clusters in primary culture of

isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells. In

continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast

stem cells; focused on disease modeling, drug screening and therapeutic potentials

differentiation from human embryonic stem cells. Curr Stem Cell Res Ther.

marrow as a potential source of hepatic oval cells. Science. 1999;284(5417):1168-

hepatocytes from bone marrow cells in mice after radiation-induced myeloablation.


Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 61

[116] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al.

[117] Baer PC. Adipose-derived stem cells and their potential to differentiate into the

[118] Al Battah F, De Kock J, Vanhaecke T, Rogiers V, Goette M. Current status of human

[119] Lee SP, Savard CE, Kuver R. Gallbladder epithelial cells that engraft in mouse liver can differentiate into hepatocyte-like cells. Am J Pathol. 2009 Mar;174(3):842-53. [120] Zhao Y, Glesne D, Huberman E. A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci U S A. 2003;100(5):2426-31. [121] Sun Y, Xiao D, Zhang RS, Cui GH, Wang XH, Chen XG. Formation of human

[122] Crema A, Ledda M, De Carlo F, Fioretti D, Rinaldi M, Marchese R, et al. Cord blood

[123] Moon YJ, Lee MW, Yoon HH, Yang MS, Jang IK, Lee JE, et al. Hepatic differentiation

[124] Moon YJ, Yoon HH, Lee MW, Jang IK, Lee DH, Lee JH, et al. Multipotent progenitor

[126] Isoda K, Kojima M, Takeda M, Higashiyama S, Kawase M, Yagi K. Maintenance of

[127] Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al.

[128] Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetal hepatocyte

[129] Dhawan A, Puppi J, Hughes RD, Mitry RR. Human hepatocyte transplantation:

[130] Ring A, Gerlach J, Peters G, Pazin BJ, Minervini CF, Turner ME, et al. Hepatic

Perfusion Culture. Tissue Eng Part C Methods. 2010;16(5):835-45.

epithelial lineage. Stem Cells Dev. Oct;20(10):1805-16.

2;284(5411):143-7.

ScientificWorldJournal.11:1568-81.

2007 Jun 15;357(4):1160-5.

2008 Oct;32(10):1293-301.

Apr;129(1):118-29.

2004;97(5):343-6.

One. 2007;2(9):e941.

Oct 27;58(8):951-2.

May;7(5):288-98.

21.

Multilineage potential of adult human mesenchymal stem cells. Science. 1999 Apr

adipose-derived stem cells: differentiation into hepatocyte-like cells.

hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras. Biochem Biophys Res Commun.

CD133 cells define an OV6-positive population that can be differentiated in vitro into engraftable bipotent hepatic progenitors. Stem Cells Dev. Nov;20(11):2009-

of cord blood-derived multipotent progenitor cells (MPCs) in vitro. Cell Biol Int.

cells derived from human umbilical cord blood can differentiate into hepatocytelike cells in a liver injury rat model. Transplant Proc. 2009 Dec;41(10):4357-60. [125] Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C, et al.

Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br J Haematol. 2005

hepatocyte functions by coculture with bone marrow stromal cells. J Biosci Bioeng.

Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS

transplantation in patients with fulminant hepatic failure. Transplantation. 1994

current experience and future challenges. Nat Rev Gastroenterol Hepatol.

Maturation of Human Fetal Hepatocytes in Four-Compartment Three-Dimensional


[102] Ghaedi M, Soleimani M, Shabani I, Duan Y, Lotfi AS. Hepatic differentiation from

[103] Hwang S, Hong HN, Kim HS, Park SR, Won YJ, Choi ST, et al. Hepatogenic

[104] Lin N, Lin J, Bo L, Weidong P, Chen S, Xu R. Differentiation of bone marrow-derived

[105] Pournasr B, Mohamadnejad M, Bagheri M, Aghdami N, Shahsavani M, Malekzadeh R,

[106] De Kock J, Vanhaecke T, Biernaskie J, Rogiers V, Snykers S. Characterization and

[107] Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, et al.

[108] Campard D, Lysy PA, Najimi M, Sokal EM. Native umbilical cord matrix stem cells

[109] Seo MJ, Suh SY, Bae YC, Jung JS. Differentiation of human adipose stromal cells into

[110] Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, et al. Rapid

[112] Aurich H, Sgodda M, Kaltwasser P, Vetter M, Weise A, Liehr T, et al. Hepatocyte

[113] Talens-Visconti R, Bonora A, Jover R, Mirabet V, Carbonell F, Castell JV, et al.

[114] Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, et al. Adipose

[115] Okura H, Komoda H, Saga A, Kakuta-Yamamoto A, Hamada Y, Fumimoto Y, et al.

mesenchymal stem cells. Tissue Eng Part C Methods. Aug;16(4):761-70.

promotes hepatic integration in vivo. Gut. 2009 Apr;58(4):570-81.

potential for liver failure. J Gastroenterol Hepatol. 2009 Jan;24(1):70-7. [111] Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, et al. IFATS

induced Liver Cirrhosis. Cell Biol Int. 2012;36(3):279-88.

hepatocyte-like cells. Arch Iran Med. Jul;14(4):244-9.

Toxicol In Vitro. 2009 Dec;23(8):1522-7.

Gastroenterology. 2008 Mar;134(3):833-48.

Stem Cells. 2003;21(2):217-27.

4;328(1):258-64.

Oct;26(10):2705-12.

2006 Sep 28;12(36):5834-45.

Hepatology. 2007 Jul;46(1):219-28.

Mar;17(1):89-106.

Prolif. Oct;43(5):427-34.

human mesenchymal stem cells on a novel nanofiber scaffold. Cell Mol Biol Lett.

Differentiation of Mesenchymal Stem Cells in a Rat Model of Thioacetamide-

mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold. Cell

et al. In vitro differentiation of human bone marrow mesenchymal stem cells into

hepatic differentiation of skin-derived precursors from adult foreskin by sequential exposure to hepatogenic cytokines and growth factors reflecting liver development.

Human umbilical cord blood as a source of transplantable hepatic progenitor cells.

express hepatic markers and differentiate into hepatocyte-like cells.

hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun. 2005 Mar

hepatic fate specification of adipose-derived stem cells and their therapeutic

collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells. 2008

differentiation of mesenchymal stem cells from human adipose tissue in vitro

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World J Gastroenterol.

tissue-derived mesenchymal stem cells as a source of human hepatocytes.

Properties of hepatocyte-like cell clusters from human adipose tissue-derived


**1. Introduction** 

a lack of possible chromatin contamination.

**4** 

*1Italy 2UK* 

**Possible Roles of Nuclear** 

M. Viola-Magni1 and P.B. Gahan2

*2King's College London* 

**Lipids in Liver Regeneration** 

*1Perugia University, Enrico Puccinelli Foundation* 

Although no lipids were considered to be present inside the nuclear membrane (Berg, 1951), their presence in chromatin was first demonstrated cytochemically by Chayen et al (1957) in *Vicia faba* root apices and liver nuclei. Sphingomyelin was further demonstrated biochemically to represent some 7% of isolated calf thymus nucleohistone preparations (Chayen and Gahan, 1958), the presence of sphingomyelin being confirmed by X-ray diffraction studies (Wilkins M. H. F, personal communication). Nevertheless, the lipids and carbohydrate present in the nuclei were considered to be minor components, most of them being due to contamination during chromatin separation (Tata et al.1972). In contrast, some biochemical measurements showed the presence of neutral lipids (Song and Rebel 1987) and phospholipids in nuclei and chromosomes from a large variety of tissues (Chayen et al 1959, a, b, Gahan 1965a). The criticism linked to possible contamination cannot be applied to the cytochemical evidence that showed the presence of chromatin-associated phospholipid material in a broad range of tissues (Idelman 1957, 1958a,b, Chayen et al. 1959a,b, La Cour et al. 1958, Gahan 1965a,b, Cave and Gahan 1971, Gahan et al. 1974, Gahan et al. 1987, Viola-Magni et al. 1985a). In a combined autoradiographic and biochemical analysis, it was shown that H3 -ethalomine incorporated into *Vicia faba* root nuclei was localised at the level of chromatin and nucleoli rather than at the level of the nuclear membrane. Hepatocyte nuclei treated with Triton and hypotonic solutions liberate chromatin that contains 10% of the total nuclear lipids. The composition of fatty acids demonstrated an enrichment of palmitic acid and a reduction in arachidonic acid (Albi et al. 1994) thus supporting the idea that these lipids cannot be derived from the nuclear membrane. In addition, the chromatographic separation of phospholipids has demonstrated an enrichment of both sphingomyelin and phosphatidylserine with respect to the nuclear membrane composition (Albi et al. 1994). The data were also confirmed by studying the turnover of phospholipids at the level of the microsomes, nuclear membrane and chromatin from hepatocytes (Viola-Magni et al. 1986). In rats injected with radioactive phosphorus, the peak of incorporation was observed after 6 h in microsomes and nuclear membranes, but only after 9h in the chromatin. This confirmed

A clear demonstration was obtained by labelling the fatty acids of the nuclear membrane by radio-iodination. Hepatocyte nuclei were separated and then radio-iodinated; the chromatin

[131] Schmelzer E, Triolo F, Turner ME, Thompson RL, Zeilinger K, Reid LM, et al. Three-Dimensional Perfusion Bioreactor Culture Supports Differentiation of Human Fetal Liver Cells. Tissue Eng Part A. 2010;16(6):2007-16.
