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GASTROENTEROLOGY 2007;132:1077–1087

ASIC–LIVER, PANCREAS, AND BILIARYRACT

one Marrow–Derived Hepatic Oval Cells Differentiate Intoepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Inducediver Regeneration

EH–HOON OH,* RAFAL P. WITEK,* SI–HYUN BAE,* DONGHANG ZHENG,* YOUNGMI JUNG,* ANNA C. PISCAGLIA,*nd BRYON E. PETERSEN,*,‡

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Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, and Program for Stem Cell Biology, University of Floridahands Cancer Center, Gainesville, Florida

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ackground & Aims: The ability of the bone marrowells to differentiate into liver, pancreas, and otherissues led to the speculation that these cells might behe source of adult stem cells found in these organs.he present study analyzed whether the bone marrowells are a source of hepatic oval cells involved in rativer regeneration induced by 2-acetylaminofluorene2-AAF) and 70% partial hepatectomy (PHx).ethods: Three groups of mutant F344 dipeptidyl

eptidase IV– deficient (DPPIV�) rats were requiredor the study. Groups A and B received the mitoticnhibitor monocrotaline, followed by male F344DPPIV�) bone marrow transplantation. Next, group

received PHx only, while group B received the-AAF/PHx required for the oval cell activation. Theast group C was used to analyze the effects of mono-rotaline on transplanted bone marrow cells. Theseats underwent transplantation with bone marrowells and were then treated with monocrotaline. Sub-equently, the animals were treated with 2-AAF/PHx.esults: In group A, DPPIV� hepatocytes were found

n the liver. Group B showed that approximately 20%f the oval cell population expressed both donorarker (DPPIV) and �-fetoprotein, and some differ-

ntiated into hepatocytes. In contrast, animals inroup C failed to significantly induce oval cells withhe donor DPPIV antigen. In addition, X/Y-chromo-ome analysis revealed that fusion was not contribut-ng to differentiation of donor-derived oval cells.onclusions: Our results suggest that under certainhysiologic conditions, a portion of hepatic stemells might arise from the bone marrow and canifferentiate into hepatocytes.

n the absence of resident cell proliferation, organ re-generation would require the presence of a progenitor

ell population, which is characterized by the ability to

roliferate, self-renew, and differentiate into phenotypicells of the organ. Reports of progenitor cells have led tohe isolation and purification of hematopoietic stemells,1 neural stem cells,2 and hepatic oval cells.3 In addi-ion, recent advances have documented that bone mar-ow cells can differentiate into muscle,4 heart,5 liver,6 –9

ancreas,10 and lung cells,11,12 thus describing a poolrom which exogenous cells could be derived to partici-ate in organ repair or regeneration. Furthermore, initro studies reveal that bone marrow cells per se expressepatic oval cell markers,13 differentiate into phenotypicepatocytes,14,15 and are able to differentiate into beta-

ike cells that produce and secrete insulin.16

Hepatic oval cells participate in liver regeneration un-er certain physiologic conditions and are implicated inepatic carcinogenesis.17,18 They are believed to have thebility to clonally expand and possess a bipotential ca-acity, which allows them to differentiate into both hepa-ocytes and bile duct epithelial cells.17 Furthermore, he-atic oval cells in culture may be induced to differentiate

nto pancreatic-like cells.19,20 Hepatic oval cells can beistinguished from hepatocytes using specific markers,uch as oval cell marker (OV6), �-fetoprotein (AFP), andytokeratin-19 (CK19).21 In addition, it has been shownhat oval cells can be isolated using Thy-1, which is anown hematopoietic stem cell marker.22

Studies with adult stem cells have shown their capa-ility to differentiate into other specific cell types. Thehenomenon of direct differentiation has been very con-roversial, because cell fusion has been reported as an al-

Abbreviations used in this paper: 2-AAF, 2-acetylaminofluorene;FP, �-fetoprotein; CK19, cytokeratin-19; DAB, 3,3=-diaminobenzidine

etrahydrochloride; DPPIV, dipeptidyl peptidase IV; NPC, nonparenchy-al cell; PHx, partial hepatectomy.

© 2007 by the AGA Institute0016-5085/07/$32.00

doi:10.1053/j.gastro.2007.01.001

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1078 OH ET AL GASTROENTEROLOGY Vol. 132, No. 3

ernative mechanism responsible for cell fate changes.23,24

owever, many reports have supported the concept thatdult stem cells directly differentiate into several cellypes without evidence of fusion.15,25–28 In addition toell fusion, a recent report has suggested that bone mar-ow progenitor cells are not the source of oval cells innjured livers,29 adding even more controversy to adulttem cell research.

The liver has a robust capacity to regenerate afternjury. This regeneration process is mediated either byepatocyte proliferation30 or by activation and differen-iation of hepatic stem cells.21 To determine whether livertem cells originate from bone marrow cells, proliferationf endogenous hepatocytes or resident liver hepatic stemells (also called oval cells) needs to be inhibited bydministration of a mitotic inhibitor to stimulate activa-ion of donor bone marrow– derived cells. Retrorsine and

onocrotaline are mitotic inhibitors that have beenhown to specifically inhibit hepatocyte proliferation.31

owever, they also exert mitotic inhibitory activity in theung and bone marrow cells.32 Therefore, treatment withither retrorsine or monocrotaline has to be administeredn a carefully timed fashion if the role of bone marrow inhe liver regeneration is to be analyzed.

To shed light on the controversy on the origin of ovalells, the potential of bone marrow cells to become he-atic progenitor cells was examined. Using monocrotal-

ne treatment before bone marrow cell transplantationthus protecting the newly transplanted cells from itsnhibitory effect), this study shows that hepatic oval cells,s well as hepatocytes, can be derived from the donorone marrow. In addition, bone marrow– derived ovalells were isolated and subsequently transplanted intoecondary recipients, which showed that these bone mar-ow– derived oval cells were capable of differentiatingnto hepatocytes.

Materials and MethodsAnimalsDipeptidyl peptidase IV– deficient (DPPIV�) F344

reeding rats were originally obtained from the Albertinstein College of Medicine via a generous gift from Dranjeev Gupta. These animals were in-house bred andaintained on standard laboratory chow and daily 12-

our light/dark cycles. Female DPPIV� rats were used atpproximately 8 –10 weeks of age (150 –170 g). Normalale DPPIV� F344 rats (age 8 –10 weeks, 200 –250 g)ere purchased from Charles River Laboratories (Wil-ington, MA) and used as donor animals. All animalork was conducted under protocols approved by theniversity of Florida Animal Care and Usage Committee.

Treatment With MonocrotalineTreatment with monocrotaline was conducted ac-

ording to a previously reported procedure.31 Briefly, the p

nitial monocrotaline treatment for rats that later under-ent irradiation and bone marrow transplantation

groups A and B) consisted of 2 intraperitoneal injectionsf 30 and 10 mg/kg performed 2 weeks apart. The secondose had to be lowered to 10 mg/kg as to preventortality that resulted from the additive effect of

sing multiple carcinogens (ie, monocrotaline and-acetylaminofluorene [2-AAF]). Two weeks after the last

njection, the animals underwent transplantation withhe bone marrow cells. Group C first underwent trans-lantation with bone marrow cells and 4 weeks latereceived monocrotaline in the same doses as describedreviously. The animals that underwent oval cell trans-lantation (secondary recipients) were treated with 2 in-

ections of 30 mg/kg, 2 weeks apart. Because the second-ry recipients were not treated with any additionalarcinogens, the previously optimized dose of monocro-aline was used.31 After the final injection, all rats wereoused for 2 more weeks before further studies wereonducted (Figure 2A).

Isolation and Transplantation of BoneMarrow CellsDPPIV� female rats were exposed to 900 rad total

ody gamma radiation (137 Cesium, JL Shepherd Mark I)dministered in 2 doses of 450 rad each, 3 hours apart.one marrow cells were isolated from the long bones ofPPIV� F344 male rats. The cells were passed through a

30-�m cell strainer, collected by centrifugation at 220gor 5 minutes, and finally resuspended in Iscove’s media.ubsequently, 5 � 107 freshly isolated bone marrow cellsere transplanted via tail vain injection immediately fol-

owing the second dose of radiation.

Isolation and Transplantation of Oval CellsDonor oval cells for the secondary recipient trans-

lantation were isolated from female DPPIV� rats thatad been treated with monocrotaline, underwent bonearrow cell transplantation, and were placed on the

-AAF/partial hepatectomy (PHx) protocol (group B; Fig-re 2A). Isolation of oval cells was performed using atandard 2-step collagenase perfusion.33 Cells were gra-ient centrifuged at 55g to separate the larger hepatocyteraction from the nonparenchymal cell (NPC) fraction,hich was later collected at 220g The NPC fraction of

ells was incubated with Thy-1 fluorescein isothiocya-ate– conjugated antibody and then with anti–fluores-ein isothiocyanate microbeads. After incubation, ovalells were positively selected using magnetic cell sorting.ell viability was determined to be �90% as establishedy Trypan blue dye exclusion. After isolation, oval cellsere resuspended in Iscove’s media at the appropriate con-

entration to give approximately 1 � 107 viable cells/mL.Transplantation of oval cells into monocrotaline-

reated female DPPIV� rats was performed following a

rocedure similar to that described by Witek et al.31

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March 2007 2-AAF/PHX–INDUCED LIVER REGENERATION 1079

riefly, rats were subjected to 70% PHx under generalnesthesia, at which time 200 �L of oval cell suspensionapproximately 2 � 106 viable cells) was injected intohe spleen using a 25-gauge needle. To aid in attainingperative hemostasis, an absorbable hemostat (Medicalnc, Arlington, TX) was applied at the splenic injectionite. Postsurgery, animals were placed back in generalousing until they were killed at 2-week intervals.rom each animal, samples of liver tissue were col-

ected and subsequently placed in O.C.T. medium forrozen sectioning or frozen in liquid nitrogen for DNAnd RNA isolation.

Immunohistochemistry and Enzyme AssayAll histochemical stainings were performed ac-

ording to previously described protocols. The DPPIVtaining procedure was performed as described by Da-eva et al.34 Double immunofluorescence staining forPPIV and OV6 or AFP was detected using the cyto-

hemical method described by Oh et al.14 Immunostain-ng for CD45 (Becton Dickinson Bioscience, San Jose,A), CK19 (Dakocytomation, Carpinteria, CA), AFP (Da-ocytomation), OV6 (gift from Dr Steward Sell), andD26 (anti-DPPIV; Becton Dickinson Bioscience) waserformed on cytospin NPCs and on frozen liver sec-ions. Briefly, slides were blocked with peroxidase andvidin/biotin (Vector Laboratories, Burlingame, CA), af-er which they were incubated with primary antibody for

hour, followed by secondary antibody for 30 minutes.etection was performed using Vector ABC kit (Vectoraboratories) and 3,3=-diaminobenzidine tetrahydrochlo-ide (DAB) reagent (Dakocytomation). The samples werehen photographed using an Olympus microscope andptronics digital camera (Olympus, Melville, NY).

DNA Polymerase Chain Reaction andIn Situ HybridizationPolymerase chain reaction analysis for the Y chro-

osome was performed on DNA extracted from trans-lanted female animals using primers for the sry gene.6

Cytospin slides were fixed for 15 minutes each in 4%araformaldehyde. The rat Y chromosome–specific sryene probes were prepared following the description byetersen et al.6 The rat X-chromosome probes were de-igned using the X-specific sequence of the rat ameloge-in gene, which is flanked by the following sequences:=-ACA CCC TTC AGC CTC ATC A-3= (sense) and 5=-AG AAC AGT GGA GGC AGA G-3= (antisense). Finalrobes were digoxigenin labeled (Roche, Indianapolis,N) for in situ hybridization and ARES DNA labeledInvitrogen, Carlsbad, CA) for fluorescence in situ hybrid-zation, denaturated at 80oC for 5 minutes, and appliedo sections at 52oC. The sections were coverslipped andealed with rubber cement for incubation overnight in aydrated slide box at 52oC. The next day, the coverslips

ere carefully removed in preheated 2� standard saline c

itrate buffer, pH 7.0, at 65oC. The sections were washedwice in 50% formamide in 5� standard saline citrateuffer for 5 minutes each at room temperature and werehen gently washed twice in preheated 0.1� standardaline citrate buffer for 5 minutes each at 65oC. For initu hybridization, color development was performedt room temperature in buffer (Tris 100 mmol/L, NaCl00 mmol/L, and MgCl2 50 mmol/L, pH 9.5) contain-

ng NBT and BCIP (Roche). Sections were counter-tained with nuclear fast red (Vector Laboratories)nd mounted in Cytoseal (Richard-Allan Scientific,alamazoo, MI).

ResultsCharacterization of Oval CellsIn the standard 2-AAF/PHx hepatic oval cell acti-

ation model described by Evarts et al,18,35 there is mas-ive proliferation of oval cells in the periportal regionFigure 1A). In H&E-stained liver sections, the popula-ion of oval cells appears as dark blue areas due to theirarge nuclei and small amount of cytoplasm. Oval cellsan be further confirmed by labeling with OV6 and CK19ile ductular markers (Figure 1B and C). An additional

mmunomarker widely utilized in oval cell labeling,hy-1, can be used for their isolation. Thy-1 antibody inonjunction with magnetic cell sorting can isolate ovalells with yields averaging 3 � 106 cells per animal (days–11 post-PHx). Approximately 3.4% � 2.4% of Thy-1

solated hepatic oval cells stain for CD45, a lymphocyticarker (Figure 1D). Nevertheless, the Thy-1 isolated cell

opulation contains cells staining for both OV6 (81.6% �.8%) and CK19 (93.9% � 2.5%) (Figure 1E and F). Theseesults confirm that the 2-AAF/PHx model can be effec-ively used for activation of oval cells in periportal re-ions during liver regeneration and that using Thy-1ntigen sorting is an efficient means of isolating ovalells.

To test the hypothesis that oval cells originate fromone marrow cells, an experimental model was developedFigure 2A). In the first stage of the experiment, all

PPIV� female animals in both groups A and B werereated with monocrotaline and then underwent trans-lantation with DPPIV� male bone marrow cells. Theecond stage of the experiment consisted of 2 parts. Therst part used 70% PHx alone (group A in Figure 2A), andhe second part used 70% PHx in conjunction with 2-AAFgroup B in Figure 2A). The third stage of the experimenttilized transplantation of Thy-1 isolated oval cells fromroup B to secondary recipient animals (Figure 2A).

In the third group of animals (group C), the bonearrow cell transplantation occurred 4 weeks beforeonocrotaline treatment. The animals were subse-

uently placed on the 2-AAF/PHx protocol for oval cellctivation (group C in Figure 2A). This group served as a

omparison model to determine the effects of timing

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1080 OH ET AL GASTROENTEROLOGY Vol. 132, No. 3

onocrotaline administration on transplanted bonearrow cells and their ability to differentiate to oval cells.

Induction of Hepatocytes and Oval CellsFrom Bone Marrow–Transplanted AnimalsDNA polymerase chain reaction for SRY per-

ormed 2 weeks posttransplantation was used to deter-ine whether the bone marrow transplantation was suc-

essful.6 Figure 2B shows the presence of the sry gene inhe blood of all animals that underwent transplantationith male bone marrow cells.Liver tissue analysis for DPPIV marker, conducted on

he recipient animals that underwent 70% PHx followedy 4 weeks of recovery, revealed scattered patches ofPPIV� hepatocytes (Figure 2C). The size of these clus-

ers ranged from a single cell to multiple cells. In addi- c

ion to DPPIV histochemical staining of the bile canalic-lar membrane, which is characteristic of adult matureepatocytes36 (Figure 2D, black arrow), the same sectionsontained cells with diffused cytoplasmic staining (Fig-re 2D, white arrowheads). The DPPIV� cells are morpho-

ogically similar to hepatic oval cells based on their sizend low cytoplasm-to-nucleus ratio. These results indi-ated that bone marrow– derived cells were able to differ-ntiate into hepatocytes and that not all donor-derivedells became hepatic cells.

Activation of Hepatic Oval Cells From theBone MarrowThe group B female recipient rats in stage 2 of the

xperiment were treated with 2-AAF/PHx to activate oval

Figure 1. Activation of hepaticoval cells in the liver portal re-gions following 2-AAF/PHx (day9). (A) H&E staining of liver tis-sue. Note large clusters ofsmall and dark blue cells in theperiportal regions representingactivated oval cells. Immuno-histochemical detection (DABbrown) of (B) OV6 and (C) CK19expressed by hepatic oval cells.(D) CD45, (E) OV6, and (F) CK19staining (DAB brown) of Thy-1magnetically sorted cells. PT,portal triad. (Original magnifica-tion: A–C, 200�; D–F, 400�.)

ell proliferation. DPPIV morphologic analysis of tissue

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March 2007 2-AAF/PHX–INDUCED LIVER REGENERATION 1081

ollected 2 weeks post-PHx revealed normal oval cellctivation with DPPIV� cells mixed in the liver lobuleFigure 3A). Additional staining of serial sections showedhat DPPIV� cells also labeled for OV6 (Figure 3B). Fur-hermore, double immunohistochemistry for DPPIVgreen) and OV6 (red) was performed to verify these cellss donor-derived oval cells (Figure 3C). The nonfraction-ted NPCs were further analyzed for the presence of

igure 2. Experimental design for the induction of hepatocytes and herom bone marrow cells (n � 8); group B, activation of hepatic oval ce

arrow–derived oval cells into secondary recipients (n � 7); group Conocrotaline and then underwent transplantation with bone marrow cone marrow transplantation; HOC, hepatic oval cells; Tx, transplantatiofficiency of bone marrow transplantation. Numbers indicate animal idDPPIV�, red/orange) found at 4 weeks after PHx in animals in group Aresence of scattered DPPIV� hepatocytes (black arrow) and smallxpressed at the canalicular membrane of hepatocytes and can be detOriginal magnification: B–E, 400�.)

epatic oval cell markers. Figure 3D shows that approx- d

mately 20% of isolated NPCs colocalized donor markerPPIV (green) with hepatic oval cell marker AFP (red).his means that from a total number of cells isolated

1162 cells), 224 cells were positive for both markersDPPIV and AFP). Figure 3E–H shows DPPIV (14.7% �.7%), AFP (45.3% � 4.4%), CK19 (33.2% � 7.7%), andV6 (42.3% � 5.1%) marker expression, respectively,ithin the NPC fraction. These results indicate that the

oval cells from the bone marrow. (A) Group A, induction of hepatocytesm bone marrow (n � 8); secondary recipient, transplantation of boneivation of hepatic oval cells from animals that were first treated withpposite of group B, n � 8). MCT, monocrotaline; IR, irradiation; BMTx,) DNA polymerase chain reaction amplification of male sry gene to verifyation. (C) Regenerated section containing donor-derived hepatocytese 2A). (D) A representative section from the same animals showing the� cells appearing in the portal region (white arrowheads). DPPIV isby an enzymatic reaction that stains positive cells in red/orange color.

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1082 OH ET AL GASTROENTEROLOGY Vol. 132, No. 3

he normal markers characteristic of liver resident ovalells.

Bone Marrow–Derived Hepatic Oval CellsTransplanted Into Monocrotaline-TreatedSecondary RecipientsThy-1/magnetic cell sorted isolated oval cells from

he NPC fraction collected from group B were used forransplantation to female DPPIV� rats that were pre-reated with monocrotaline (Figure 2A, secondary recip-ent). At 8 weeks posttransplantation, liver and spleenissues were collected and analyzed. Polymerase chaineaction for the sry gene showed the presence of donor-erived cells in the liver but not in the spleen (Fig-re 4B). DPPIV staining on liver sections revealed amall number (�0.5%) of scattered clusters of contig-ous donor-derived hepatocytes (Figure 4A1–A4).In addition, we examined sry gene expression in recip-

ent livers (Figure 4B). We found that animals that un- a

erwent transplantation with bone marrow– derived he-atic oval cells were expressing the sry gene in the liver.hese results indicated that hepatic oval cells can origi-ate from donor cells and are capable of differentiating

nto phenotypic hepatocytes.

Activation of Hepatic Oval Cells in AnimalsFirst Transplanted With Bone Marrow Cellsand Then Treated With Monocrotaline(Group C)At 7 and 14 days after 2-AAF/PHx, animals in

roup C showed oval cell activation in the periportalegion of the liver (Figure 5A). At those time points, liverections show occasional small clusters of DPPIV� cellsFigure 5B and C). In comparison, sections of liver col-ected at the same time points (days 7 and 14 after-AAF/PHx) from animals in group B showed frequently

arge clusters of DPPIV� cells (Figure 5D and E). In

Figure 3. Characterization andisolation of bone marrow–de-rived oval cells from 2-AAF/PHx–treated animals in group B.(A–C) Liver tissue from 2-AAF/PHx hepatic oval cell inductionmodel (group B) collected at 2weeks after PHx. (A) OV6�-stained and (B) DPPIV�-stainedserial sections showing clustersof overlapping cells on both ofthe stains. Black arrowheadspoint to the same hepatocyteson each figure, and the area ofoverlapping stains is indicatedby a red circle. (C) Double im-munofluorescence for DPPIV(green) and OV6 (red). Overlap-ping stains are distinguished byyellow. (D–H) Isolated NPCsfrom animals in group B. (D)Double immunofluorescence forDPPIV (green) and AFP (red).White arrowheads point to thecells colocalizing both of themarkers (yellow). Staining ofNPCs for (E) DPPIV and hepaticoval cell markers: (F) AFP, (G)CK19, and (H) OV6. (Original mag-nification: A–C and E–H: 200�.)

ddition, double immunofluorescent staining revealed

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hat oval cells in the group C liver express AFP at day 14ost-PHx; however, these cells did not stain for DPPIV

Figure 5F). The results obtained from animals in groupare consistent with a previously published report byenthena et al.29 The results also indicate that mitotic

uppression by pyrrolizidine alkaloids (ie, monocrotaliner retrorsine) leads to a strong inhibition of bone marrowells and subsequent oval cell induction after 2-AAF/PHxreatment.

X- and Y-Chromosome Analysis of BoneMarrow–Derived Hepatic Oval CellsThe X/Y-chromosome expression in hepatic oval

ells isolated from group B was assessed by in situ hy-

igure 4. Secondary transplantation of bone marrow–derived hepaticval cells into monocrotaline-treated DPPIV� recipients. (A1–A3) Serialections of representative cluster of hepatocytes found in animals thatnderwent transplantation (white arrowheads). Note that the cells arerowing in all directions, which is indicative of clonal expansion of trans-lanted cells (DPPIV staining; red/orange). Black arrowheads indicateortal triad. (A4) Representative group of donor-derived hepatocytes

ound in the animals that underwent transplantation. (B) DNA polymer-se chain reaction amplification of sry gene showing the presence ofale markers in representative livers of animals that underwent

ransplantation.

ridization. Figure 6A and B show X chromosome in the p

ucleus of isolated hepatocytes (white arrowheads) andval cells (black arrowhead). Approximately 55% of ovalells contain one X chromosome, suggesting a 1:1 ratioith Y chromosome (bone marrow origin). Figure 6C

hows detection of Y chromosome in oval cells (blackrrowhead), suggesting their bone marrow origin, andecipient female hepatocytes (white arrowhead). Figure 6Dhows analysis of X-chromosome expression in hepato-ytes from 2-AAF/PHx–treated animals (control). Normalloidy and tetraploidy are detected in these hepatocytes.igure 6E shows analysis of X-chromosome expression inroup B liver cells, where 99% or more of the cells showhe presence of either one or 2 X chromosomes (normalloidy). Only 3 cells (out of 1400 cells analyzed) wereound with 3 X chromosomes, suggesting possibility ofusion (XX-XY) or staining artifacts. These results indi-ate that a population of the hepatic stem cells can ariserom the bone marrow, and it appears that fusion is notignificantly involved in this process.

To further support the technique used for in situybridization, double fluorescence in situ hybridizationnalysis for X and Y chromosome was performed. FigureF and G show X and Y chromosome in the nucleus of

solated female and male bone marrow cells, respectively.igure 6H and I show double fluorescence in situ hybrid-

zation for X and Y chromosome in isolated oval cellssame as Figure 6B and C). As shown in these figures,

and Y are clearly visible, suggesting their male bonearrow origin.

DiscussionIn recent years, the possibility of using organ- or

issue-specific adult stem cells has been examined in bothelds of gene and cell transplantation therapy. These cellsould represent a renewable source of cells for patientherapy and could be used to study developmental mech-nisms and pathways. Within the field of hepatology,iver oval cells represent this cell source. Initially, hepaticval cells were believed to originate in the liver withinhe canals of Hering17; however, in the past few years, theontroversial possibility of their extrahepatic origin be-an to surface. Petersen et al6 reported that the bonearrow cells were a potential source of hepatic oval cells.owever, a recent report suggested that bone marrowrogenitors are not the source of expanding oval cells in

njured livers.29

To clarify the controversy surrounding an extrahepaticource of liver stem cells, we examined the timing effectf monocrotaline exposure and oval cell activation fromhe bone marrow. In the current study, using a combi-ation of monocrotaline treatment along with bone mar-ow transplantation followed by 2-AAF/PHx oval cellctivation (group B), approximately 20% of oval cells werebserved coexpressing donor bone marrow DPPIV andFP (Figure 3D). Additionally, bone marrow– derived he-

atic oval cells continuously differentiated into hepato-

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1084 OH ET AL GASTROENTEROLOGY Vol. 132, No. 3

ytes (group A). However, when the timing of thereatments was reversed and the mitotic inhibitor

onocrotaline was used after the bone marrow trans-lantation (group C), hepatic oval cells induced after-AAF/PHx did not originate from donor bone marrowells (Figure 5).

In addition, secondary transplantation of isolatedone marrow– derived oval cells from animals in group Bhowed that these cells are indeed oval cells retaining theonor characteristics (DPPIV and SRY) and were able toifferentiate into hepatocytes (Figure 4).To induce hepatic oval cells from bone marrow, 2

equirements need to be fulfilled. First, the liver injuryas to be sufficient to activate the oval cell compartmentithin the liver. The 2-AAF/PHx induces liver injury that

s known to produce a high number of oval cells at days m

–11 post-PHx,18,35 which fulfills the first requirement.he second requirement is inhibition of proliferation ofll resident cell types that could influence and promoteiver regeneration. The pyrrolizidine alkaloid family ofepatotoxic chemicals, which includes monocrotalinend retrorsine, can be used to fulfill this requirement.n general, pyrrolizidine alkaloids have been reportedo possess potent antimitotic and hepatotoxic activi-y.32,37 They are metabolically converted by the P450ystem to produce reactive, highly toxic dehydroalka-oid intermediates that form adducts with DNA, re-ulting in mitotic arrest in rapidly dividing cells. How-ver, because P450 is not only restricted to theepatocytes, pyrrolizidine alkaloids inhibit prolifera-ion of other cell types, including pulmonary and bone

Figure 5. Comparison of he-patic oval cell induction be-tween group B and group C. (A)OV6 staining (DAB brown) inliver tissue collected at day 14after PHx from the animals ingroup C. (B and C) Liver tissuescollected from animals in groupC at days 7 and 14 after 2-AAF/PHx, respectively. (D and E)Liver tissue collected fromgroup B at days 7 and 14 after2-AAF/PHx, respectively. Notethe difference in staining inten-sity between groups B and Ccompared at the same timepoints. Black arrowheads indi-cate the observed DPPIV stain-ing (red/orange). (F) Double im-munofluorescence staining ofhepatic oval cells for expressionof AFP (red) and DPPIV (green)in the portal region. (Original mag-nification: A–E, 400�; F, 600�.)

arrow cells.32

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March 2007 2-AAF/PHX–INDUCED LIVER REGENERATION 1085

In the current experiments, monocrotaline was in-ected before bone marrow transplantation to minimizets activity on transplanted cells (Figure 2A; groups And B). This timing is very critical because it gives arowth advantage to the donor cells. This could be veri-ed by the animals in group C, which received monocro-aline after bone marrow transplantation. As shown inigure 5B and C, group C liver tissue showed significantly

ess staining than corresponding group B liver sectionsFigure 5D and E). The results obtained from animals inroup C are comparable to the results obtained by Men-hena et al,29 and the timing of pyrrolizidine alkaloidnjection may explain their inability to find bone mar-ow– derived oval cells in retrorsine-treated livers.

The final question as to what is the origin of hepatic

igure 6. In situ hybridizationor X and Y chromosome inone marrow–derived hepato-ytes and oval cells. X-chromo-ome detection in (A) hepato-ytes and (B) oval cells isolatedrom animals in group B. (C) De-ection of Y chromosome inone marrow–derived oval cells

black arrowhead) and femaleecipient hepatocytes (white ar-owhead). (D) Normal ploidyblack arrowhead) and tet-aploidy (white arrowhead) inhe 2-AAF/PHx–treated hepato-ytes analyzed for X chromo-ome. (E) Quantitative analysisf X chromosome in isolatedval cells from group B. (F–I)luorescence in situ hybridiza-ion analysis of X and Y chromo-omes in bone marrow–derivedepatic oval cells. (F and G) Xhromosome and Y chromo-ome in the nucleus of isolatedemale and male bone marrowells, respectively. (H and I)epresentative X- and Y-chro-osome staining in nuclei ofone marrow–derived hepaticval cells. X, X chromosome; Y,chromosome. (Original mag-

ification: A–C, 1000�.)

val cells could be answered by looking at the phenotypic d

haracteristics of oval cells and the possibility that theval cell population is heterogeneous38 and exists as 2ifferent populations. One population of cells is endog-nously present in the liver and can be located in theanals of Hering.39,40 Because the liver has been function-ng as a hematopoietic organ during the first stages ofevelopment, it is possible that some of the stem cellsecome quiescent and are retained in the liver during itsrogression to maturity, hence the presence of hemato-oietic markers on isolated oval cells. This population ofells would arise if hepatocyte proliferation were compro-ised (2-AAF/PHx treatment) and would be sufficient to

llow liver regeneration. Other oval cell activation meth-ds (CCl4 and D-galactosamine in rats, 3,5-diethoxycar-onyl-1, 4-dihydro-collidine [DDC] in mice) would pro-

uce similar results because they only affect hepatocyte

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1086 OH ET AL GASTROENTEROLOGY Vol. 132, No. 3

ivision and have no effect on liver resident oval cells. Forhis reason, previous reports suggested that hematopoi-tic cells have little contribution to oval cell origin41 or inepatocyte formation.42 However, if the activation ofesident oval cells were blocked, oval cells would home tohe liver from the bone marrow as a second population ofval cells. Under this condition, bone marrow– derivedval cells would allow liver regeneration without the

nfluence of resident oval cells.To assess whether bone marrow cells differentiated to

ecome oval cells, we looked for fusion events. Bonearrow– derived hepatic oval cells and hepatocytes have

nly one Y chromosome in the nucleus. These resultsndicate that these cells originated from the donor maleells and directly differentiated into hepatocytes. Addi-ionally, normal recipient cells contain 2 X chromosomeser cell (females) as shown in Figure 6A, and male donorells contain only one X chromosome (Figure 6B, blackrrowhead). If fusion of these 2 cells occurred, 3 or more

chromosomes would reflect it. Due to the fact thatepatocytes show different ploidy (2N, 4N, 8N, and 16N),ny odd counts of X-chromosome number would repre-ent fusion. Based on the data collected, there were not aignificant number of cells containing a fusion genotypeonly 3 cells out of 1400 cells counted; Figure 6E), thusuggesting that bone marrow– derived hepatocytes wereredominantly formed through direct differentiation.his result was further supported by data obtained fromouble fluorescence in situ hybridization analysis shown

n Figure 6F–I. In this procedure, only cells with normalloidy were observed. Additionally, given the fact thatesident cells were under the influence of monocrotaline,usion with donor cells would possibly render the newlystablished cells unable to replicate and form clusters.

The results reported by Wang et al41 demonstrate fu-ion as the mechanism by which the liver repairs itself.lso, results reported by Michalopoulos et al43 showed

hat under certain physiologic conditions, hepatocytesave the ability to trans-differentiate into cholangiocytes.hese data, in conjunction with the present studies, show

hat changing the timing of one event can cause drasticifferences in outcomes and subsequent interpretation ofhe mechanisms driving liver repair. The literature is fullf controversy where contradictory results have been ob-ained, indicating that bone marrow stem cells lack anyvidence of plasticity and describing single stem cell ex-eriments indicating huge plasticity capability of theseame cells. It is well known that the body has multiple

echanisms of repair, and perhaps during the repairrocess all of these pathways are invoked. However, ulti-ately one pathway becomes selected as the most effi-

ient and thus becomes the avenue of repair. Occam’sazor states that the explanation of any phenomenonhould make as few assumptions as possible, eliminatinghose that make no difference in the observable predic-

ions of the hypothesis or theory. So, when given 2

qually valid explanations for a phenomenon, one shouldmbrace the less complicated formulation. This can bearaphrased as “All things being equal, the simplest so-

ution tends to be the best one.”Due to the complexity of the systems we are working

n, it is not necessarily evident to us which pathway is theimplest or most efficient in terms of the complex inter-ctions of the body. Clearly, more data are needed tohow whether or not all oval cells originate from bone

arrow; however, the findings of this study suggest thatpopulation of oval cells could represent progeny of

one marrow– derived stem cells. Efforts should be madeo isolate a highly enriched fraction of bone marrow–erived oval cells from the liver and characterize themore closely. A better understanding of the bone marrow

o liver pathway could lead to development of clinicallyseful protocols for the treatment of hepatic disorders.

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Received December 20, 2005. Accepted November 30, 2006.Address requests for reprints to: Bryon E. Petersen, PhD, Depart-ent of Pathology, Immunology and Laboratory Medicine, Room641 MSB, University of Florida College of Medicine, PO Box 100275,ainesville, Florida 32610-0275. e-mail: petersen@pathology.ufl.edu;

ax: (352) 392-6249.Supported by National Institutes of Health grants DK058614 and

K065096 and a Howard Hughes Start-up Grant (to B.E.P.).S.–H.O. and R.P.W. contributed equally to this study.B.E.P. is an inventor of the patent(s) related to this technology anday benefit from royalties paid to University of Florida related to its

ommercialization.