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OCR for page 34
Colloquium
Little evidence of bone marrow-derived hepatocytes
in the replacement of injured liver
Yoshiyuki Kanazawa* and Inder M. Vermat
Laboratory of Genetics, The Salk Institute, La Jolla, CA 92037
We have tested the ability of bone marrow (BM) cells (BMCs) to
form hepatocytes in liver injury models. We used three models:
(/D carbon tetrachloride (CCI4) treatment, (i/D albumin-urokinase
transgenic mouse [TgN(Alb1Plau)], and (ii/0 hepatitis B transgenic
mouse lTgN(Alb1HBV)]. As a nonselective liver injury model, irra-
diated C57BL/6 (B6) mice were transplanted with BMCs from
GFP transgenic mouse 1TgN(ActbEGFP)] or ,(3-galactosidase trans-
genic mouse lTgN(MtnLacZ)] followed by the administration
of CCI4. Irradiated TgN(Alb1HBV) and TgN(Alb1Plau) were also
transplanted with BMCs from TgN(ActbEGFP) or TgN(MtnLacZ).
Approximately 1.5 x 106 hepatocytes per liver were analyzed
for GFP-positive cells, and the whole livers were inspected for
,8-galactosidase expression. No GFP-positive hepatocytes and no
gross blue staining of the livers with 5-bromo-4-chloro-3-indolyl
,`3-D-galactoside in any of the 18 recipient mice analyzed were
detected. The livers from female animals with gender-mismatched
BM transplantation were also tested with Y chromosome fluores-
cent in situ hybridization analysis to detect donor-derived cells. A
total of five isolated hepatocytes were positive for Y chromosome
in 4.1 x 105 hepatocytes analyzed. Our results demonstrate that
there is little or no contribution of BMCs to the replacement of
injured livers in these models. We conclude that BM-derived cells
cannot generally lead to a cure of liver damage.
There have been a number of reports that show the potential
of adult rodent bone marrow (BM) cells (BMCs) to
transdifferentiate into a variety of cell types (1-7), including
hepatocytes (8-10~. One of the best examples for the trans-
differentiation has been the ability of BM-derived hapatocytes
to repopulate the liver of mice with fumarylacetoacetate
hydrolase knockout mouse (FAH-/-) and correct the liver
disease (10), although recent reports have shown that this
correction of liver disease is caused by fusion of donor BM to
recipient hepatocytes rather than transdifferentiation (11, 12~.
However, it is not fully elucidated whether or not the signif-
icant level of the contribution of BM-derived hepatocytes in
the FAH- /- model, either in the form of transdifferentiation
or cell fusion, can be generalized in other liver injury models.
Here we report our results with nonselective liver injury model
and two selective liver injury models {carbon tetrachloride
(CCl4), albumin-urokinase transgenic mouse tTgN(AlblPlau)]
(13), and hepatitis B transgenic mouse [TgN(AlblHBV)]
(14~), to examine how general is the phenomenon of the
repopulation by BM-derived hepatocytes. It is of considerable
clinical importance to know the level of contribution of
BM-derived hepatocytes in a slowly progressive chronic liver
disease model like TgN(AlblHBV), because chronic viral
hepatitis is a leading cause of cirrhosis and hepatocellular
carcinoma worldwide (15, 16~.
Materials and Methods
Animals. TgN(AlblHBV) and GFP transgenic mice
[TgN(ActbEGFP)] (17) were kindly provided by F. V. Chisari
(The Scripps Research Institute) and Masaru Okabe (Osaka
11850-11853 1 PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1
University, Osaka), respectively. I3-Galactosidase transgenic
mouse [TgN(MtnLacZ)] (13) and TgN(AlblPlau) were pur-
chased from The Jackson Laboratory, and the latter was
back-crossed to C57BL/6J (B6) (The Jackson Laboratory).
Animal study protocols were approved by The Salk Institute
Animal Care and Use Committee. Donor mice used in these
studies were generally 7-8 weeks old, and recipient animals
were 8-12 weeks old except for neonatal transplantation.
Biochemical analysis of serum from animal was performed by
ANILYTICS (Anilytics, Gaithersburg, MD).
BM Transplantation. BMCs were isolated from the femurs and
tibias of TgN(ActbEGFP) or TgN(MtnLacZ). Erythrolysis was
done by treating the cells with buffered ammonium chloride
(StemCell Technologies, Vancouver). BM mononuclear cells
were obtained by Histopaque-1077 (Sigma) density gradient
centrifugation. Adult recipient mice were injected via the tail
vein with these cells either with preparative irradiation (1,200
cG) or without irradiation.
Neonatal BM Transplantation. After whole-body irradiation with
200-400 cG, neonatal recipient mice (1-5 days of age) were
injected with BMCs through the superficial temporal vein by
using hypothermic anesthesia.
CCI4 Liver Injury. B6 mice were i.p. injected with CC14 (0.02-1.0
ml/kg of animal weight, twice a week, total of eight times).
Choline-Deficient, Ethionine-Supplemented Diet. Mice were admin-
istered a diet consisting of a 1:1 mixture of choline-deficient
chow (ICN) and normal chow and drinking water supplemented
with 0.15% (wt/vol) Dr-ethionine (ICN) for 2 weeks (18~.
Tissue Preparation. Transplanted animals were anesthetized and
perfused intracardially with 4% paraformaldehyde/PBS. Per-
fused livers were dissected and further fixed in 4% paraformal-
dehyde at 4°C overnight. In some cases, animals were killed,
and thin liver slices (2-3 mm) were removed immediately.
Portions of the liver slices were snap-frozen, and the other slices
were fixed in 4% paraformaldehyde at 4°C overnight. Fixed liver
slices were washed with PBS, cryoprotected by incubation in
This paper results from the Arthur M. Sackier Colioqulum of the Nationai Acaclemy of
Sciences, "Regenerative Meclicine," heicl October 18-22, 2002, at the Arnoicl ancl Mabei
Beckman Center of the Nationai Academies of Science ancl Engineering in Irvine, CA.
Abbreviations: BM, bone marrow; BMC, BM ceil; CC4, carbon tetrachioricle; TgN(Aib] Piau),
albumin-urokinase transgenic mouse; TgN(Aib] HBV), hepatitis B transgenic mouse;
TgN(AcibEGFP), GFP transgenic mouse; TgN(MinEacZ), ,0-gaiactosiclase transgenic mouse;
B6, C57BE/61.
*Present aciciress: Osaka University Gracluate Schooi of Meclicine, Suita, Osaka 565 0871,
Japan.
iTo whom corresponclence shouicl be aciciressecl. E-maii: verma@?saik.eclu.
2003 by The Nationai Acaclemy of Sciences of the USA
www.pnas.org/cgi/cloi/10. ~ 073/pnas. ~ 834198100
OCR for page 35
Table 1. Summary of experimental data
Donor- Donor- Ychromosome-
derived Weeks Weeks from derived positive
cells in from BM the end hepatocytes* hepatocyte nuclei/
No. of Type and no. peripheral transplantation of CCI4 to or nodules hepatocytes
Liverinjury mice of donor ceils blood,% to analysts analysis observed analyzed
Transplantation ~ CCI4 liver injury
0.02 ml/kg x 8times 1 GFP(1 x 106 BMMNC) 95 (12w)
0.02 ml/kg x 8 times 1 LacZ (1 x 106 BMMNC) 92 (4 w)
0.02 ml/kg x 8 times 1 GFP (1 x 106 BMMNC) 96 (12 w)
0.02 ml/kg x 8 times 1 LacZ (1 x 106 BMMNC) 91 (4 w)
CCIA liver iniurv ~ transplantation (without Drecarative irradiation)
.
12
12
16
16
~ , . . . . .
0.02 ml/kg x 8 times 2 GFP (1 x 105 BMC)
0.02 ml/kg x 8 times 2 LacZ (1 x 105 BMC)
0.2 ml/kg x 8 times 2 GFP (1 x 107 BMC)
1.0 ml/ka x 8 times 2 GFP (1 x 107 BMC)
1 (neonate) GFP (1 x 106 BMC)
GFP (1 x 107 BMC)
GFP (1 x 107 BMC)
LacZ (5 x 106 BMC)
GFP (1 x 106 BMC)
GFP (1 x 106 BMC)
.. .... , . ~ . . _ .... _
TgN(Alb1 Plau)
TgN(Alb1 HBV)
TgN(Alb1 HBV)
TgN(Alb1 HBV)
TgN(Alb1HBV) + CDEt 1 (neonate)
TgN(Alb1HBV) + CDEi 1 (neonate)
0 (4 w)
0 (4 w)
ND
0 (2 w), ND
17 (13 w)
44 (11 w)
97 (7 w)
~100 (7 w)
30 (9 w)
18 (9 w)
4
2, 4
15
13
23
32
47
47
4
4
8
8
4
4
4
2, 4
o
o
o
o
o
o
o
o
o
o
o
o
o
o
1/130,000
2/60,000
1 /140,000
1 /80,000
ND, not done; BMMNC, BM mononuclear cells.
*Sixty sections containing 1.5 x 106 hepatocytes on average were analyzed per liver.
iCDE, choline-deficient, ethionine-supplemented diet.
increasing concentration of cold sucrose (10%, 15%, and 20%;
total 12-24 h), and quick-frozen in cryo-embedding compound
(Microm International, Walldorf, Germany).
Histology and Immunofluoreseence Analysis. For intrinsic GFP
analysis, 10-,um frozen sections were cut from several distinct
lobes and mounted with VECTASHIELD with 4',6-diamidino-
2-phenylindole (DAPI) (Vector Laboratories). Immunohisto-
chemical stainings with anti-mouse albumin (Biogenesis,
Brentwood, NH) or anti-GFP (Novus Biologicals, Littleton,
CO) were performed on 10-,u m frozen sections by using
standard protocol. Appropriate Cy3-conjugated secondary
reagents (Jackson ImmunoResearch) were used for the detec-
tion and mounted with VECTASHIELD with DAPI. Immu-
nof luorescence analysis was performed by using a DeltaVision
restoration microscopy system (Applied Precision, Issaquah,
WA) consisting of an Olympus IX70 microscope equipped
with HBO100 epiillumination source. Excitation wavelengths
were 490 nm for GFP, 555 nm for Cy3, and 360 nm for DAPI.
Fluorescent emission was collected at 528, 617, and 457 nm
respectively. In the analysis of GFP-positive cells, autofluo-
rescent cells were excluded by examining red emission (617
nm) and green emission (528 nary). For detection of h-galac-
tosidase, the whole lobes of livers were fixed in 0.2% para-
formaldehyde/0.05% glutaraldehyde at 4°C overnicht and
stained with 5-bromo-4-chloro-3-indolyl I3-D-galactoside
(Sigma). For some samples, a small portion of the livers were
removed and snap-frozen for fluorescent in situ hybridization
before the fixation of the rest of the whole livers.
Fluorescent in Situ Hybridization. Cryostat sections, 6 ,um in thick-
ness, were fixed three times, 10 min each, in Carnoy's fixative.
Serial ethanol dehydration was done, and the slides were air-
dried at room temperature. Sections were denatured at 65°C for
1.5 min in prewarmed 70% formamide and 2x SSC solution, pH
7.0 and quenched in ice-cold 70% ethanol for 4 min. Dehydration
by serial ethanol washing was done again. The mouse Y chro-
mosome probe labeled with Cy3 (STAR FISH, Cambio, Cam-
bridge, U.K.) was denatured at 65°C for 10 min and applied on
Kanazawa and Verma
the sections at 37°C. The sections were covered with parafilm
and incubated overnight in a hydrated slide box at 37°C. Then,
the sections were washed according to the manufacturer's in-
structions and mounted with VECTASHIELD with 4',6-
diamidino-2-phenylindole (Vector Laboratories). Fluorescence
analysis was performed by using a DeltaVision restoration
microscopy system (Applied Precision) as described above. In
the analysis of Cy3 signal, autofluorescent nonspecific signals
were excluded by examining green emission (528 nm) and red
emission (617 nary).
Calculations of Section Size and Cell Numbers. At least three images
of liver sections were analyzed to determine the average
number of hepatocytes per unit area for each animal. The
surface area of sections was measured by scanning glass slides
along with a size standard and by analyzing the scanned images
with METHAMORPH 6.1 (Universal Imaging, Downingtown, PA)
software.
Results and Discussion
The first model we tested was the CCl4-induced liver injury
model. Recently, it has been suggested to be effective in
inducing hepatocytic differentiation of BMC (11~. Four fe-
male B6 mice were lethally irradiated and transplanted with
male TgN(ActbEGFP) or TgN(MTnLacZ) BM mononuclear
cells (1 x 106~. TgN(ActbEGFP) is known to express GFP
constitutively in most cells including hepatocytes, and
TgN(MTnLacZ) allows inducible expression of ,6-galactosi-
dase in hepatocytes by administration of heavy metal ions.
Donor engraftment (79-92%) was confirmed 4 weeks post-
transplantation, and the animals were administered CC14 (0.02
ml/kg animal weight, twice a week for 4 weeks). The existence
of liver injury was confirmed by the elevated serum alanine
transaminase levels (146-217 units/liter). Four or 8 weeks
after the last administration of CCl4, the livers of the recipients
were checked for donor-derived cells (Table 1~. In the liver
sections (50 sections per liver for GFP fluorescence and 10
sections per liver for GFP immunohistochemistry), GFP-
positive cells were located in sinusoids or associated with other
PNAS | September30, 2003 | vol. JoO | suppl. ~ | 11851
OCR for page 36
.~
I B
. ,~
I _
1 ~
1 _
1 _
_
_
__
, ~ _
Fig. 1. (A) GFP-positive nonhepatocytes observed in a liver section from
CCI4-treated mouse. Bromo-4-chloro-3-indolyl ,B-D-galactoside (X-gal) stain-
ing of the whole liver from TgN(MtnLacZ) as a positive control (B) and B6
transplanted with TgN(MtnLacZ) BM mononuclear cells and administered CCI4
(C). No X-gal positive nodule was observed in the B6 liver. (D) Y chromosome-
positive hepatocyte nucleus (arrow) from a mouse in CCI4 liver injury model.
(Scale bars: 50 ~m, A; 30 ,um, D.)
larger vessels. These cells showed several different types of
morphology, including globular, elongated, or star-shaped
cells, and were readily distinguishable from hepatocytes that
are large polyhedral cells with round nuclei (Fig. 1A). Addi-
tionally, the GFP-positive cells were negative for the hapato-
cyte marker albumin by immunostaining. In animals trans-
planted with TgN(MTnLacZ), the whole livers was tested for
,B-galactosidase expression after induction with cadmium;
however, no gross blue staining was detected (Fig. 1 B and C),
suggesting that no distinct nodules consisted of donor BM-
derived hepatocytes.
The livers from these four animals were also tested for Y
chromosome-positive hepatocytes, because Mezey et al. (19)
suggested a discrepancy between transgenic expression tagging
and DNA markers might occur. In 40 liver sections (10 sections
for each animal), a total of five isolated hepatocytes were
positive for Y chromosome (Table 1 and Fig. ID). A nonirra-
diated protocol was also tested, in which eight B6 mice were first
administered CC14 eight times, then the protocol was followed by
transplantation of TgN(ActbEGFP) or TgN(MTnLacZ) BMC
(Table 1~. Protocols differing in the number of transplanted cells
and the dosage of CC14 administered were tested. Not surpris-
ingly there was no donor engraftment in the nonradiated ani-
mals. Four weeks after transplantation, the liver of each animal
was analyzed for donor-derived GFP- or 13-galactosidase-positive
hepatocytes. No donor-derived hepatocytes were observed in
any of the recipients.
The TgN(AlblPlau), in which regeneration stimulus for hepa-
tocytes is present for 6-8 weeks after birth (13), was tested as a
selective liver injury model. An irradiated neonate
TgN(AlblPlau) was transplanted with GFP-positive BMCs (1 x
106~. After having confirmed the donor cell engraftment (17%),
the recipient liver was assessed for donor-derived cells at 15
11852 1 www.pnas.org/cgi/doi/10.1073/pnas.1834198100
weeks of age. There were no GFP-positive hepatocytes observed
in 60 sections of the liver (Table 14.
Another selective liver injury model we tested- is
TgN(AlblHBV), which produces toxic quantities of hepatitis B
surface antigen within the hepatocytes. Two TgN(AlblHBV)
were lethally irradiated and transplanted with GFP-marked
BMCs (1 x 107) from TgN(ActbEGFP). After having con-
firmed the donor cell engraftment (44% and 97%) and the
existence of mild liver injury by slightly elevated serum alanine
transaminase levels (121 and 143 units/liter), the livers of the
recipient mice were analyzed for GFP-positive cells at 13 and
23 weeks posttransplantation. However, none of the liver
sections contained GFP-positive donor-derived hepatocytes
(Table 1~.
One female TgN(AlblHBV) was transplanted with 5 x 106
BMCs derived from male TgN(MTnLacZ). Seven weeks post-
transplantation, ~100% of the recipient peripheral blood cells
were positive for the Y chromosome and elevated serum alanine
transaminase level (185 units/liter) was confirmed. Thirty-two
weeks after transplantation, the whole liver was examined for
I3-galactosidase expression after induction with cadmium. How-
ever, nodules originating from donor-derived hepatocytes were
not observed (Table 14.
We next transplanted GFP-positive BMCs (1 x 10`') into two
irradiated neonatal TgN(AlblHBV) for long-term follow-up.
These mice were administered a choline-deficient, ethionine-
supplemented diet for 2 weeks at 5 months posttransplantation.
This diet is known to induce proliferation of an intrahepatic
progenitor population, "oval cells," of which potential source is
thought to be BM (8~. These mice were kept for 6 more months
to allow the induced oval cells to differentiate further. At 47
weeks posttransplantation, the recipient livers were tested for
donor-derived hepatocytes. In 60 sections from each mouse
(50 for GFP fluorescence and 10 for GFP immunohistochem-
istry), there were no GFP-positive hepatocytes in either of the
recipients.
It is possible that, in this model, primary hepatocyte trans-
plantation will be as unsuccessful to replace injured liver as
BM transplantation, probably because the liver injury level is
not so severe and the recipient hepatocytes could repair the
injury. However, the situation is different between BM trans-
plantation and hepatocyte transplantation. In the BM recon-
stituted model, donor BM-derived hematological cells are
continuously supplied, circulate in the recipient liver, and have
a lifelong chance to compete with endogenous defective
hepatocytes. Our data show that even with this continuous
chance BMCs failed to repopulate the diseased liver in the
TgN(AlblHBV) model.
Thus, analysis of 18 mice in the nonselective and even in
selective liver injury models revealed only five isolated hepa-
tocytes that might have been derived from donor BMCs,
although it still remains to be elucidated whether these cells
arise from spontaneous fusion events (12, 20) or transdiffer-
entiation. We conclude from these data (Table 1) that the
contribution of BM-derived hepatocytes to the replacement of
injured livers may be very low except for a certain limited
experimental condition (10, 12, 204. We do not believe that
BM-derived hepatocytes can generally lead to a cure of liver
disease.
We thank F. V. Chisari and M. Okabe for transgenic animals, F. H. Gage
and B. Spencer for critical reading of the manuscript, and R. G. Summers
for technical assistance. Y.K. was partially supported by the Japan
Research Foundation for Clinical Pharmacology. I.M.V. is an American
Cancer Society Professor of Molecular Biology. He is supported in part
by grants from the National Institutes of Health, the Larry L. Hillblom
Foundation, Inc., the Lebensfeld Foundation, the Wayne and Gladys
Valley Foundation, and the H. N. and Frances C. Berger Foundation.
Kanazawa and Verma
OCR for page 37
1. Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A.,
Cossu, G. & Mavilio, F. (1998) Science 279,1528-1530.
Gussoni, E., Soneoka, Y., Strickland, C. D., Buzney, E. A., Khan, M. K., Flint,
A. F., Kunkel, L. M. & Mulligan, R. C. (1999) Nature 401, 390-394.
Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S. M., Li, B., Pickel,
J., McKay, R., Nadal-Ginard, B., Bodine, D. M., et al. (2001) Nature 410,
701-705.
4. Orlic, D., Kajstura, J., Chimenti, S., Limana, F., Jakoniuk, I., Quaini, F.,
Nadal-Ginard, B., Bodine, D. M., Leri, A. & Anversa, P. (2001) Proc. Natl.
Acad. Sci. USA 98, 10344-10349.
. Jackson, K. A., Majka, S. M., Wang, H., Pocius, J., Hartley, C. J., Majesky,
M. W., Entman, M. L., Michael, L. H., Hirschi, K. K. & Goodell, M. A. (2001)
"~\ '"t""'' 9-
J. Clin. Invest. 107, 1395-1402.
.. Mezey, E., Chandross, K. J., Harta, G., Maki, R. A. & McKercher, S. R. (2000)
Science 290, 1779-1782.
,. Brazelton, T. R., Rossi, F. M., Keshet, G. I. & Blau, H. M. (2000) Science 290,
1775-1779.
8. Petersen, B. E., Bowen, W. C., Patrene, K. D., Mars, W. M., Sullivan, A. K.,
Murase, N., Boggs, S. S., Greenberger, J. S. & Goff, J. P. (1999) Science 284,
1 168-1 170.
Theise, N. D., Badve, S., Saxena, R., Henegariu, O., Sell, S., Crawford, J. M.
& Krause, D. S. (2000) Hepatology 31, 235-240.
Kanazawa and Verma
10. Lagasse, E., Connors, H., Al-Dhalimy, M., Reitsma, M., Dohse, M., Osborne,
L., Wang, X., Finegold, M., Weissman, I. L. & Grompe, M. (2000) Nat. Med.
6, 1229-1234.
11. Wang, X., Ge, S., McNamara, G., Hao, Q. L., Crooks, G. M. & Nolta, J. A.
(2003) Blood 101, 4201-4208.
12. Vassilopoulos, G., Wang, P. R. & Russell, D. W. (2003) Nature 422, 901-904.
Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D. & Brinster, R. L.
(1994) Science 263, 1149-1152.
14. Chisari, F. V., Klopchin, K., Moriyama, T., Pasquinelli, C., Dunsford, H. A.,
Sell, S., Pinkert, C. A., Brinster, R. L. & Palmiter, R. D. (1989) Cell 59,
1 145-1156.
15. Esteban, R. (2002) Sem~n. Live, Dis. 22, Suppl. 1, 1-6.
16. Walker, M. P., Appleby, T. C., Zhong, W., Lau, J. Y. & Hong, Z. (2003)
Antiviral Chem. Chemother. 14, 1-21.
17. Ikawa, M., Yamada, S., Nakanishi, T. & Okabe, M. (1998) FEBS Lett. 430, 83-87.
18. Akhurst, B., Croager, E. J., Farley-Roche, C. A., Ong, J. K., Dumble, M. L.,
Knight, B. & Yeoh, G. C. (2001) Hepatology 34, 519-522.
19. Mezey, E., Nagy, A., Szalayova, I., Key, S., Bratincsak, A., Baffi, J. & Shahar,
T. (2003) Science 299, 1184.
20. Wang, X., Willenbring, H., Akkari, Y., Torimaru, Y., Foster, M., Al-Dhalimy,
M., Lagasse, E., Finegold, M., Olson, S. & Grompe, M. (2003) Nature 422,
897-901.
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11853
Representative terms from entire chapter:
transgenic mouse
