hepatic stem cells: from bone marrow cells to hepatocytes

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Biochemical and Biophysical Research Communications 281, 1–5 (2001)

doi:10.1006/bbrc.2001.4270, available online at http://www.idealibrary.com on

REAKTHROUGHS AND VIEWS

epatic Stem Cells: From Bone Marrow Cellso Hepatocytes

oshihiro Mitaka1

epartment of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine,huo-Ku, S-1, W-17, Sapporo 060-8556, Japan

eceived December 27, 2000

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Hematopoiesis and the hepatic environment arenown to have a close relationship at the time of he-atic development and systemic diseases. Recently,ransplanted cells isolated from bone marrow of ro-ents and humans have been shown to differentiate

nto oval cells, which are considered to be hepatictem cells, and hepatocytes in the liver. Then, purifiedematopoietic stem cells were shown to have the abil-

ty to replace original liver cells in mice with heredi-ary tyrosinemia. In this review the interactions be-ween hepatic stem cells are summarized and aypothesis of hepatic differentiation will be proposed.2001 Academic Press

Key Words: hepatic stem cell; hematopoietic stemell; oval cell; small hepatocyte; differentiation;aturation.

Are there stem cells in the livers in adult animals?his question has been debated for about half a cen-ury. Although many researchers around the worldave tried to identify and characterize such cells inhe liver, convincing results have not yet been re-orted. Some candidates for hepatic stem or progeni-or cells such as oval cells and small hepatocytesave been proposed. However, if the stem cells areefined as undifferentiated, multipotent cells, and therogenitor cells as nonhepatocyte epithelial cellsenerating a dual lineage (biliary and hepatocytic)ith hepatocyte markers, it can be said that noure population of either type of cell has been isolated

Abbreviations used: HSC, hematopoietic stem cell; 2AAF,-acetylaminofluorene; PH, partial hepatectomy; DPPIV, dipeptidyleptidase IV; ECM, extracellular matrix; NPC, nonparenchymal cell;NF, hepatocyte nuclear factor.1 Fax: 181-11-615-3099. E-mail: [email protected].

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rom normal, injured or preneoplastic adult liver. Now,owever, it appears that we close to answering thebove question, owing to the great advances of hema-opoietic cell research in the 1990s, especially identifi-ation of surface markers of hematopoietic stem cells,he development of cell sorting technology, and clini-al experience with bone marrow transplants. Thisrogress has indeed contributed to the successful iden-ification of certain types of hepatic stem cells. Pe-ersen et al. (1) first proved that bone marrow cellsransplanted into lethally irradiated rats migrated tohe liver and then differentiated into oval cells andepatocytes. Thereafter, similar results were reportedor mice (2) and human patients (3, 4). Very recently,agasse et al. (5) showed that purified hematopoietictem cells (HSCs) could differentiate into hepatocytesnd replace parts of the liver in mice with hereditaryyrosinemia with new mature hepatocytes from thetem cells.It seems obvious from historical studies about he-

atic development that most hepatocytes and bile ductells in the adult liver in animals originally derive fromepatoblasts resident in the fetal liver. Thus, hepato-lasts are considered to be bipotential stem/progenitorells of epithelial cells in the liver. However, we canever find cells morphologically and phenotypicallyimilar to hepatoblasts in the normal adult liver, evenn the regenerating liver. Thus, we have to return tohe primary question: “Are there stem cells in the livern adult animals?”

Now most cell types related to the hepatic lineagerom bone marrow cells to mature hepatocytes mayppear on the stage. Therefore, the next steps of he-atic stem cell research are to elucidate the mecha-isms of differentiation/maturation of each cell and tolarify the roles of the various cells in the hepaticineage.

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 281, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANDIDATES OF HEPATIC STEM CELLS

In certain conditions in the liver treated with hepa-otoxins and hepatocarcinogens, or infected with he-atic virus, epithelial cells different from mature hepa-ocytes have been histologically found and designateds follows.

a) Oval Cells

When liver damage is so severe that hepatocytes areargely killed or that their proliferation is prevented byxposure to hepatotoxins or carcinogens, liver progen-tor cells, so-called oval cells, appear in the periportalreas of liver lobules. Oval cells are small cells withcant cytoplasm and ovoid nuclei, and they are thoughto have the ability to proliferate clonogenically and aipotential capacity to differentiate into both hepato-ytes and bile duct cells. To induce oval cells in the rativer, the combination of 2-acetylaminofluorene (2AAF)reatment and two-thirds partial hepatectomy (PH)/Cl4 is often used. The continuous administration of a

ow concentration of 2AAF suppress the proliferation ofepatocytes and at the middle point of the treatmentusually after 7 to 10 days) the animals are subjected tomassive loss of hepatocytes by administration of CCl4

r PH. The lack of response of hepatocytes to growthignals results in the rapid growth of oval cells. Theval cells initially appear next to biliary ductules andhen some migrate into the hepatic parenchyma. Therigin of oval cells have been questioned, althoughost researchers tend to believe that the cells may

ome from the canals of Hering, which is a transitionalone between the periportal hepatocytes and the bili-ry cells lining the smallest terminal bile ducts. Recenttudies clearly showed that one of the original cells waserived from bone marrow cells. Furthermore, detailedracing examination revealed that oval cells firsthange into basophilic small hepatocytes and then dif-erentiate into mature hepatocytes.

b) Bone Marrow-Derived Cells

The accumulation of data concerning oval cells hasevealed that a number of surface makers are sharedetween hematopoietic stem cells and oval cells, in-luding c-kit, CD34, and Thy-1 in rodents, and c-kit andD34 in humans. In 1999 Petersen et al. (1) firsthowed that a population of rat bone marrow cellsight give rise to oval cells in the liver and have the

otential to further differentiate into hepatocytesnd/or bile duct cells. To prove their results, they usedhree different protocols combined with 2-AAF/CCl4

reatment: (1) Bone marrow cells obtained from maleats were transplanted into lethally irradiated femaleecipients and the fate of Y chromosome-positive cells

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n the livers of the recipients was investigated; (2)arrow from dipeptidyl peptidase IV (DPPIV)-positiveale rats was transplanted into DPPIV-deficient fe-ale ones; and (3) whole liver transplantation withewis rats that expressed the L21-6 antigen as recip-

ents was performed using Brown-Norway rats that didot express the antigen as allogeneic donors to confirmhat an extrahepatic source could repopulate the trans-lanted liver. A similar approach using a mouse mar-ow transplant model without overt liver damage wasarried out and some hepatocytes were shown to ex-ress the markers of transplanted cells (2). Then, in auman female liver receiving bone marrow transplan-ation from a male donor and in male patients afteriver transplantation from a female donor, some hepa-ocytes in the recipients possessed Y-chromosomes inheir nuclei (3, 4). In these studies, the number of cellshich underwent the transition appeared to be rela-

ively small. However, very recently, Laggasse et al. (5)eported that only a small number of purified HSCsould rescue the fumarylacetoacetate hydrolase (FAH)-eficient mouse (a model animal of hereditary tyrosine-ia type I), which show a progressive liver failure. Atmonths after transplantation, more than 30% of

epatocytes were replaced with donor-derived cells.These results remind us of a familiar association

etween hematopoiesis and the liver. Embryonic HSCsrst appear in the aorta–gonad–mesonephros region,nd migrate into the fetal liver. Most hematopoieticells leave the liver to the bone marrow and spleenefore birth. However, some HSCs can still persist inhe rodent liver into adulthood (6). In addition, thedult liver can again become a site for hematopoiesishen patients suffer from some diseases, for example,yeloproliferative disorders such as myelofibrosis and

evere structural damage to the marrow secondary tonfiltration by tumor cells. To my knowledge, hemato-oietic stem cells, which have the potential to differen-iate into hepatocytes and/or bile duct cells, may notifferentiate into hepatocytes or bile duct cells in anyther tissues or organs except the liver. The microen-ironment in which the liver provides the stem cellsay be important for hepatic differentiation even in

dulthood, as well as in the fetus.

c) Small Hepatocytes

Small hepatocytes have been identified as proliferat-ng cells with hepatic characteristics. We first found

remarkable increase of small mononucleate cellsithin primary hepatocytes cultured in medium sup-lemented with 10 mM nicotinamide and EGF (7). Amall hepatocyte can proliferate and form a colony. Therowth speed is slow and 5 to 6 divisions occur within0 days. The population of small hepatocytes in thedult rat liver is estimated to be 1.5–2.0% of hepato-

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ytes, and the number of the cells decreases with age.he cells can also be isolated from the human liver andheir clonal expansion has been demonstrated in cul-ure. Although the cells can continue growing withoutosing hepatic characteristics for several months, themmortalization of the cells is so difficult that cell linesave not yet been established. On the other hand,lusters of small hepatocytes in vivo are observed inodents with severe liver diseases resulting from treat-ent with hepatotoxins such as dipin, D-galactos-

mine, retrorsine and so on. Small hepatocytes alsoppear in human livers suffering from acute viral hep-titis and that have recovered from fulminant hepati-is. Furthermore, pathologists often observe small celllusters in liver specimens from long-term chronic hep-titis and liver cirrhosis. Although the morphologicalnd histological appearance of the “small cells” ob-erved in in vitro and in vivo may be very similar, theirquivalence has not been proved. As specific antibodieso small hepatocytes have not been found, the preciserigin or location within the liver cannot be definedither.

IFFERENTIATION/MATURATIONF HEPATIC STEM CELLS

The relationships between the cells of hepatic lin-age mentioned above are shown in Fig. 1. In the lateeriod of the fetal liver, hepatoblasts differentiate intomall hepatocytes and the cells located in the peripor-al region may differentiate into bile duct cells. At theommitment of either differentiation of the liver cells,ematopoietic cells may play an important role be-ause oncostatin M produced by CD45-positive hema-opoietic cells in the fetal liver can induce the differ-ntiation of hepatoblasts, from fetal to postnatalhenotypes, and make the microenvironment unfavor-ble for hematopoiesis (8). Therefore, most hematopoi-tic cells leave the liver for the bone marrow andpleen. However, some HSCs still remain in the liver6). On the other hand, the cells in the canals of Heringre known to be c-kit1 and/or CD341 (9). These resultsnvite the speculation that some HSCs resident in orirculating to the liver may integrate into the canals ofering and differentiate into hepatocytes or bile duct

ells. Most hepatic cells may be small hepatocytes inhe neonatal period and then actively divide with liverrowth.Small hepatocytes in vitro can maintain the ability of

apid proliferation unless hepatic nonparenchymalells (NPCs) such as stellate cells grow and attach tohem (10). When NPCs rapidly proliferate and invadender colonies of proliferating small hepatocytes, mat-ration of small hepatocytes is stimulated with accu-ulation of an extracellular matrix (ECM). The cells

nlarge their cytoplasm that is rich in mitochondria,

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ough endoplasmic reticulum, peroxisomes and glyco-en. Some cells possess two nuclei. Thereafter, alter-tion of the cellular morphology is attributed to theeconstruction of hepatic tissues, which may mimicepatic plate formation (Fig. 2). Between the cells bileanaliculi are formed, and these move actively. On thether hand, proliferating small hepatocytes expressepatocyte nuclear factor 4 (HNF4), whereas the ma-ured large hepatocytes express CCAAT/enhancerinding protein a and HNF6 in their nuclei as well asNF4 (unpublished observation). Changes of cell

hape may result in the sequential expression of liver-nriched transcription factors. Although it is unclearhich ECMs can induce the maturation, these resultslso fuel speculation that, in pre- and postnatal peri-ds, the arrangement of plates in liver lobules may beerformed by the invasion of stellate and sinusoidalndothelial cells. Plates several thick become 1 or 2ells thick accompanying the maturation of hepaticells. When NPCs invade and secrete ECMs with form-ng sinusoids, hepatic cells are becoming mature. Indulthood, most dead hepatocytes may be compensatedor by new hepatocytes formed when neighboring ma-ure ones divide, and dead bile duct cells may be re-laced the same way as hepatocytes. Some cells in theanals of Hering may contribute to the supplementa-ion of hepatocytes and bile duct cells. When the cellsn the canals of Hering die, the loss may be compen-ated for by circulating or resident HSCs. This mayxplain why a very small number of bone marrow-erived hepatocytes in liver plates was observed inale patients without lethal irradiation or any liver

amage, to whom a female liver was transplanted.At the time of an emergency of hepatic functions in

he adult mammal liver, responses to the growth sig-als of each cell may be dependent on the degree of theamage: if the growth responses of mature hepatocytesre suppressed and/or the continuous loss of hepato-ytes finally consumes the mature hepatocytes that canroliferate, the growth of small hepatocytes is acti-ated and their clonal expansion occurs. In the case ofevere damage to liver cells accompanying hepatic dys-unction, when, for example, most hepatocytes areead and/or exposed to specific hepatotoxins, oval cellsapidly proliferate and differentiate into small hepato-ytes and then mature hepatocytes to compensate forhe loss. In this case, bone marrow cells will become aajor source of oval cells.

UTURE PROSPECTS

Although the mechanisms of fetal hepatic develop-ent have gradually been clarified, we do not have

nough clues as to how the differentiation and/or mat-ration of hepatic stem cells occur in adulthood, fromSCs to oval/canal of Hering’s cells or bile duct cells

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nd from oval/canal of Hering’s cells to hepatocytes orile duct cells. We now know that, when hepatic stemells are transplanted to the conditioned liver thatossesses a genetic alteration, or to which a certainreatment has already been applied, the cells can bentegrated into hepatic plates several months later.he cells then change their phenotype to that of ma-ure and/or bile duct cells, and the liver sometimeseplaces original hepatocytes with the descendants ofransplanted ones. Although the entrance and the exitre so clear, the inside of the liver is a black box. Tolarify the mechanisms of hepatic differentiation/aturation, we must establish new culture systems inhich the proliferation and differentiation/maturationf hepatic stem cells can be freely manipulated and thenteraction between hepatic stem cells and other typesf liver cells such as stellate cells, sinusoidal endothe-ial cells, and Kupffer cells, can be investigated. Suchnvestigations using hepatic stem cells will contributeo the construction of an artificial liver and the forma-ion of hepatic tissues ex vivo as well as to cell trans-lantation therapy.

FIG. 1. Illustration of the interaction between hepatic stem cells. Thrrows represent emergent routes. Dotted arrows are other routes for whicells, which were isolated from murine fetal liver cells and express hepatic a

ick blue arrows show the major stream of hepatic development and thin redh convincing results are not yet available. H-CFU-C (c-kit2/CD452/TER1192)nd bile ductular markers, were recently introduced as hepatic stem cells (11).

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FIG. 2. A small hepatocyte colony possessing piled-up cells onhe colony at day 84. The piled-up cells proliferate like formingepatic plates, which consist of mature hepatocytes.

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showed that the livers of mice with hereditary tyrosinemia can

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CKNOWLEDGMENTS

I am grateful to Drs. Shinichiro Ikeda, Shinichi Sugimoto, Keisukearada, Fumihiko Sato, Toru Mizuguchi, and Yohichi Mochizuki forelping with my work. I also thank Ms. M. Kuwano, Ms. Y. Taka-ashi, and Mr. H. Itoh for technical assistance, and thank Mr. K.arrymore for help with the manuscript.

EFERENCES

1. Petersen, B. E., Bowen,W. C., Patrene, K. D., Mars, W. M.,Sullivan, A. K., Murase, N., Boggs, S. S., Greenberger, J. S., andGoff, J. P. (1999) Science 284, 1168–1170. [This is the first paperreporting that bone marrow-derived cells can differentiate intooval cells and hepatocytes.]

2. Theise, N. D., Badve, S., Saxena, R., Henegariu, O., Sell, S.,Crawford, J. M., and Krause, D. S. (2000) Hepatology 31, 235–240.

3. Alison, M. R., Poulsom, R., Effer, R., Dhillon, A. P., Quaglia, A.,Jacob, J., Novelli, M., Prentice, G., Williamson, J., and Wright,N. A. (2000) Nature 406, 257.

4. Theise, N. D., Nimmakayalu, M., Gardner, R., Illei, P. B., Mor-gan, G., Teperman, L., Henegariu, O., and Krause, D. S. (2000)Hepatology 32, 11–16. [Note: The first four papers showed thathuman and murine bone marrow-derived cells could turn intohepatocytes.]

5. Lagasse, E., Connors, H., Al-Dhalimy, M., Reitsma, M., Dohse,M., Osborne, L., Wang, X., Finegold, M., Weissman, I. L., andGrompe M. (2000) Nature Med. 6, 1229–1234. [This paper first

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be replaced with transplanted purified hematopoietic stem cells.]6. Taniguchi, H., Toyoshima, T., Fukao, K., and Nakauchi, H.

(1996) Nature Med. 2, 198–203. [This paper showed that hema-topoietic stem cells exist in the adult murine liver.]

7. Mitaka, T., Mikami, M., Sattler, G. L., Pitot, H. C., and Mochi-zuki, Y. (1992) Hepatology 16, 440–447. [This paper showed theexistence of a population that could clonally expand in primaryhepatocyte cultures and the cells were designated small hepato-cytes.]

8. Kinoshita, T., Sekiguchi, T., Xu, M.-J., Ito, Y., Kamiya, A., Tsuji,K., Nakahata, T., and Miyajima, A. (1999) Proc. Natl. Acad. Sci.USA 96, 7265–7270. [This paper showed that oncostatin M couldinduce hepatic differentiation of hepatoblasts in the murineliver.]

9. Theise, N. D., Saxena, R., Ormann, B. C., Thung, S. N., Yee, H.,Chiriboga, L., Kumar, A., and Crawford, J. M. (1999) Hepatology30, 1425–1433. [This paper showed that cells in canals of Heringexpressed c-kit and CD34.]

0. Mitaka, T., Sato, F., Mizuguchi, T., Yokono, T., and Mochizuki,Y. (1999) Hepatology 29, 111–125. [This was first paper report-ing that small hepatocytes can differentiate into mature ones byinteracting with hepatic NPCs and that NPCs may play animportant role in the hepatic morphogenesis by secreting ECM.]

1. Taniguchi, H., Suzuki, A., Zheng, Y., Kondo, R., Takada, Y.,Fukunaga, K., Seino, K., Yuzawa, K., Otsu, M., Fukao, K., andNakauchi, H. (2000) Transplant. Proc. 32, 249–25. [Anothercandidate for hepatic stem cells in the murine fetal liver wasrecently introduced.]

hepatic stem cells: from bone marrow cells to hepatocytes

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