Note: Descriptions are shown in the official language in which they were submitted.
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BONE MARROW TRANSPLANTATION
FOR HEPATIC REGENERATION AND REPAIR
SPECIFICATION
1. INTRODUCTION
The present invention relates to methods and compositions for
stimulating liver regeneration in subjects with liver disorders. Specifically,
the
methods and compositions of the invention provide for the transplantation of
bone
marrow cells into a recipient host in amounts sufficient to result in the
production of
hepatocytes, bile ductal cells and oval cells during liver regeneration. The
invention is
based in the observation that bone-marrow derived cells, can participate in
the
production of hepatocytes, bile ductal cells and oval cells during liver
regeneration.
The present invention further provides methods for deriving enriched
populations of hepatic oval cells, considered to be hepatic stem cells,
utilizing
antibodies that recognizes the Thy-1 cell surface antigen expressed on the
surface of
hepatic oval cells. The enriched populations of hepatic oval cells can be
transplanted
into a host for stimulating liver regeneration in subjects with liver
disorders. The
present invention, by enabling methods for the transplantation of bone marrow
cells
and/or oval cells for stimulation of liver regeneration provides a safer
alternative to
whole liver transplantation in subjects having liver disorders including, but
not limited
to, cirrhosis of the liver, alcohol induced hepatitis, chronic hepatitis,
primary
sclerosing cholangitis and alpha,-antitrypsin deficiency.
2. BACKGROUND OF INVENTION
The origin of the hepatic oval cell has been a topic of considerable
interest and controversy for the past several decades. Because oval cells
proliferate
when hepatocytes are prevented from proliferating in response to liver damage,
these
cells have been considered to be hepatic stem cells, or the intermediate
progeny of a
hepatic stem cell. The prevailing opinion is that oval cells originate either
from cells
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present in the canals of Herring ( Grisham, J.W. and Thorgiersson, S.S. in
Stem Cells,
C.S. Potter, Ed. (Academic Press, San Diego, CA 1997) pp. 233-282) or from
blast-
like cells located next to bile ducts (Novikoff, P.M. et al., 1996, Am J.
Pathol.
148:1473 ). Oval cells are not easily detected in normal livers. In certain
pathological
conditions, however, in which an inhibition of hepatocyte proliferation is
followed by
severe hepatic injury, oval cells are readily apparent due to their active
proliferation.
In recognized experimental animal models, hepatocyte proliferation is
generally
suppressed by exposure of the animal to 2-acetylaminofluorene (2-AAF) and,
subsequently, hepatic injury is usually induced by partial hepatectomy (PHx)
or by
administration of carbon tetrachloride (CCl4) (Evarts, R.P., et al., 1989,
Cancer Res.
49:1541; Petersen, B.E. et al., 1998, Hepatology 27:1030). Oval cells have
been
observed in organs other than the liver, such as the pancreas of rats fed a
copper-
deficient diet (Bartles, J.R. et al., 1991, J Cell Science 98:45; Rao, M.S.
and Reddy,
J.K., 1995, Seminars in Cell Biology 6:151).
Throughout life, hepatic and hematopoietic cells intermingle and
appear to be interdependent. During fetal life, hematopoietic stem cells (HSC)
move
out of the yolk sac and take up residency in the liver, and until the time of
birth, the
liver functions as a hematopoietic organ (Baker, J.E. et al., 1969, J Cell
Physiol 74:51;
Moore, M.S. et al., 1970, Br J Haematol 18:279). This function ceases in the
neonate,
but under certain conditions it can be reactivated in the form of extra-
medullary
hematopoiesis (Tsamanda, A.C., 1995, Modern Pathol 8:671). The adult liver has
been shown to harbor a significant number of HSC (Hayes, E.F. et al., 1975, J
Cell
Physiol 86:213), and it has been shown that the bone marrow of lethally
irradiated
animals can be reconstituted by whole liver transplantation (Murase N et al.,
1996
Transplantation 61:1).
Hematopoietic activity and erythropoietic cells have been shown to
reappear in the liver during liver regeneration following a partial
hepatectomy
(Naugton, B.A. et al., 1982, Exp Hematol 10:451: Barbera-Guillem, E. et al.,
1989
Hepatology 9:29; Sakamoto, T., et al, 1992, Reg Immunol 4:1). It has also been
shown that cultured rat hepatocytes can produce granulocyte-macrophage colony
stimulating factor, a known hematopoietic cytokine (Sakamoto, T. et al., 1991,
Reg
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Immunol 3:260). Recently, oval cells were found to express CD34, Thy-1 and c-
kit
mRNAs and proteins (Petersen, B. et al., 1998, Hepatology 27:433; Omori, N. et
al,
1997, Hepatology 26:720; Fujio, K. et al., 1994, Lab Invest 70:511). These
antigens
are known to be expressed on HSC. In addition, oval cells express flt-3
receptor
mRNA, which in humans and mice has been reported to be restricted to
populations of
hematopoietic progenitor cells (Omori, M. et al., 1997, Am J Pathol 150:1179).
The
adult mammalian liver can therefore be considered a potential hematopoietic
organ.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for
stimulating liver regeneration in subjects with liver disorders. The
compositions and
methods of the invention provide for the transplantation of bone marrow cells
into a
recipient in amounts sufficient to result in the production of hepatocytes,
bile ductal
cells and oval cells during liver regeneration. The invention is based on the
observation that bone-marrow derived cells, can participate in the production
of
hepatocytes, bile ductal cells and oval cells during liver regeneration.
The present invention further provides methods for deriving enriched
populations of hepatic oval cells, considered to be hepatic stem cells,
utilizing
antibodies that recognizes the Thy-1 cell surface antigen expressed on the
surface of
hepatic oval cells. The enriched populations of hepatic oval cells may also be
utilized
in methods directed to regeneration of liver tissue.
The present invention also provides compositions for use in stimulating
liver regeneration comprising bone marrow cells and/or hepatic oval cells in a
pharmaceutical acceptable carrier. The compositions of the invention may be
utilized
for treatment of subjects with liver disorders where the stimulation of liver
regeneration is desired. Such disorders include cirrhosis of the liver,
alcohol-induced
hepatitis, chronic hepatitis, primary sclerosing cholangitis, alpha-
antitrypsin
deficiency and liver cancer.
In an embodiment of the invention, the bone marrow derived stem cells
and/or hepatic oval cells may be genetically engineered, prior to
transplantation, to
enable them to produce a wide range of proteins, including but not limited to,
growth
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factors, cytokines, or biologically active molecules, such as hormones. In
this way,
any new liver tissue derived from the transplanted stem cells or hepatic oval
cells will
produce the desired biologically active protein.
The invention further relates to the in vitro attachment of stem cells or
hepatic oval cells to a matrix prior to transplantation for the purpose of
increasing the
viability and growth of the transplanted cells. In addition, the matrix may be
composed of additional materials including other types of cells or
biologically active
molecules.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1. PCR analysis of DNA from female rats transplanted with
bone marrow from the femurs of male donors rats. The data presented here show
that
the transplanted animals tested positive for the Y chromosome in both the
Thyl.l+ and
Thyl.l- sub-populations of non-parenchyma) cells (NPC) at both days tested.
The Thy-
1- fraction shows strong signal probably due to the presence of hematopoietic
cells.
After successful BMTx, presumably the mature (differentiated) male
hematopoietic
cells could be found in the NPC population. The mature hematopoietic (i.e.
Kupffer
cells, monocytes, etc.) cells will be negative for Thy-1, but positive for the
Y
chromosome. Note the Day 13 fraction of hepatocytes are now expressing the Y
chromosome PCR product. This is the time point when oval cells begin to make
the
transition to hepatocytes. As noted in the text, the Day 9 hepatocyte fraction
was
negative for the Y chromosome. This would be expected in the prevailing view
of the
timing events in the cascade of oval cell proliferation and differentiation.
The control
female DNA as well as the non transplanted female DNA is negative for Y
chromosome expression. The ~i-actin product reveals that the DNA was present
and
intact.
FIGURE 2 A-C. Photomicrographs of in situ hybridizations of the Y
chromosome sry gene performed on frozen liver sections. The arrows indicate
positive reaction in the nucleus of hepatocytes. A, untreated control normal
male rat
(positive control); B, female treated with BMTx and the 2-AFF/CC 14 protocol
and
sacrificed at day 13 following hepatic injury; and C, untreated female
(negative
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control). It should be noted that the color reaction-time for the untreated
female was
45 minute, whereas the color reaction-time for both the male and the BMTx/2-
AAF/CC14 treated female was only 5 minutes. The magnification of all the
photomicrographs is 1000X.
5 FIGURE 3 A-F. DPPIV activity in frozen liver sections. A, untreated
DPPIV+ rat used as a positive control. A diffuse decoration of the bile
ductular site of
hepatocytes is evident (orange color). B, untreated DPPIV- rat showing a
complete
absence of DPPIV activity (negative control). C-F are four different BMTx
(male
DPPIV+ donor) DPPIV- rat (recipient) exposed to the 2-AAF/CC14 protocol for
oval
cell induction and sacrificed at day 11 or day 13 following hepatic injury. A
positive
reaction is evident not only between hepatocytes from all four animals, but
also a on a
few oval or transitional cells (D and E). The DPPIV staining can appear as a
line
(open arrowheads) or as a dot (closed arrowheads) depending the plane of the
section
through the bile cannilcuar region. In all cases there are clusters of cells
(2-5)
expressing the DPPIV marker. Hepatocytes that are from donor origins are
denoted
by *. Original magnifications, 200x.
FIGURE 4 A-B. Sections from two Brown-Norway rat livers,
transplanted into Lewis recipient rats, sacrificed 11 days (A) and 13 days (B)
after
CC 14 administration in the oval cell induction protocol. The sections were
immunostained with an L21-6 mAb. Positive oval cells (arrow heads) and
positive
cells in ductal structures (arrows) can be seen. Original magnifications, A,
100x, and
B, 200x.
FIGURE 5 A-D. Frozen liver section showing double
immunofluorscence staining of a periportal region in a Brown-Norway liver
transplanted into a Lewis recipient rat (A). Green fluorescence: anti L21-6, a
recipient
marker; red fluorescence: anti OC-2, an oval cell and a mature ductal cell
marker.
When the two antibodies are in close proximity, the light waves mix and the
emitted
fluorescence is yellow. Oval cells co-expressing the two markers are evident
(arrows). Other cells can be seen that express only L21-6 (presumably
inflammatory
cells), or only OC.2 (oval cells that could have been derived from, L21-6
negative
precursor-cells resident in the Brown-Norway liver). B-D shows a frozen
section of a
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Brown-Norway liver transplanted into a Lewis recipient rat and put on the 2-
AAF/CCl4 protocol, day 13 after CC14 exposure. Centered in the photomicrograph
is
a ductal structure (B-D), the same type of structure seen in Figure 4A. Double
immunofluorscence staining was performed using anti-L21-6 (B) and anti-OC-2
(D).
Merged images of the L21-6 and OC-2 immunofluorescence staining are shown in
the
middle photomicrograph (C). Cells expressing both antigens are yellow (C). As
a
point of origin, * indicates the center of the bile duct. The origin of the
cells within
this duct is extrahepatic because they are positive for L21-6, which makes
them
recipient derived cells. Arrows in B-D indicate the same cell in all three
photomicrographs. Bars represent 10 ,um in length.
FIGURE 6. Time line of events for activation of oval cell
proliferation. The presence of 2-AAF is necessary to suppress hepatocyte
proliferation and to allow extended proliferation of oval cells. The diagram
represents
the different stages of oval cell proliferation.
FIGURE 7 A-B. Liver section obtained from a rat on day 11 on the 2-
AAF/CCl4 protocol. Sections were stained with hematoxylin-eosin. Centered in
the
photomicrograph is a portal triad (PT). The small oval cells (arrows) can be
seen
between the larger hepatocytes. (Original magnification [A] X100; [B] X200.)
FIGURE 8 A-D. Immunohistochemistry for BrdU incorporation on
liver sections obtained from animals on the 2-AAF/CCl4 protocol at various
time
points post-hepatic injury. (A) Day 9; (B) day 11; (C) day 13; and (D)
positive control
for BrdU at the 24-hour Phx time point. The peak of proliferation occurs at
day 9,
with a drastic drop-off of labeling on subsequent days. The day 13 time point
is also
represented in Fig. 8. HV, hepatic vein. (Original magnification [A-D] X200.)
FIGURE 9 A-B. Frozen liver sections obtained from normal rat liver.
Histological appearance of liver section stained with (A) Thyl.l antibody and
(B) OC.2 antibody. Normal rat liver was used as a negative control for
immunostaining procedures. Thyl.l expression cannot be detected in a normal
adult
liver, and OC.2 can be detected only on the ductular epithelium in the portal
triads.
The portal triad region (PT) and central vein region (CV) are designated in
the lobule.
(Original magnification X40.)
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FIGURE 10 A-F. Frozen serial sections from rat liver exposed to the
2-AAF/CC14 protocol (day 21). (A and D) Immunohistochemical expression of CK-
19. (B and E) expression of OC.2. (C and F) Expression of Thy 1.1 staining as
can be
seen at lower magnification (A-C), CK-19, OC.2, and Thyl.l exhibit nearly the
same
staining pattern. At higher magnification (D-F), the individual oval cells
stain
positive for each antibody. Open arrows in (A-C) indicate the reference point
at
which the photomicrographs of higher magnifications were taken. Solid arrows
in (D-
F) show individual oval cells positively stained for the appropriate antibody.
It should
be noted that the ductular epithelium is now staining positive for Thy-1. In
normal
liver, this is not the case. This same result can be seen in Fig. 6 as well.
(Original
magnification [A-C] X40; [D-F] X200.)
FIGURE 11 A-F. Frozen serial sections from rat liver exposed to 2-
AAF/PHx protocol (day 13). (A and D) Immunohistochemical expression of CK-19.
(B and E) Expression of OC.2. (C and F) Expression of Thy 1.1 staining. At
lower
magnification (A-C), CK-19, OC.2, and Thyl.l exhibit nearly the same staining
pattern. At higher magnification (D-F), the individual oval cells stain
positive for
each antibody. Open arrows in (A-C) indicate the reference point at which the
photomicrographs of higher magnifications were taken. Solid arrows in (D-F)
show
individual oval cells positively stained for the appropriate antibody. This
figure
illustrates that, in this model, there are no inflammatory cells to confound
the issue as
to which cell type is expressing Thy-1. Staining patterns are alike for all
three
markers, and this pattern is similar to what is seen in the 2-AAF/CC14 model.
(Original magnification [A-C] X40; [D-F] X200.)
FIGURE 12 A-D. Profiles of NPC fraction obtained from perfused rat
liver exposed to the 2-AAF/CC14 protocol. Cells were labeled with Thyl .1-FITC
antibody and sorted by flow cytometry. Cells were separated into two factions:
(A)
right gate, Thy-1+-labeled cells and (B) left gate, Thy-1- cells. Histograms
revealed
that 95% to 97% purity could be obtained for Thy-1.1+ cells, while 99% purity
could
be achieved for negative cells. (C) Sorted Thyl.l+ cells were stained with PI,
and
flow cytometric cell cycle analysis was performed. Analysis revealed that 94%
Thyl.l+ cells are in the GO/G1 (resting) stage of the cell cycle. (D) Thy-1-
labeled
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cells (dark solid line) that are in the GO/Gl stage of the cell cycle (dashed
line).
Approximately 90% of the resting cells are Thy-1+.
FIGURE 13 A-B. Immunohistochemistry of liver section obtained
from a day 13 2-AAF/CC14 rat. (A) BrdU staining of oval cells in the
proliferative
state. Very few cells are positively stained, which may represent the small
peak of
cells in the G2/M phase seen in Fig. 7C and 7D. The ductular formation (BD) is
devoid of any BrdU staining. (B) Liver section stained for PCNA also shows
little or
no PCNA staining. This corroborates the BrdU staining. Both of these stains
document that the majority of oval cells taken at this time point are not in a
proliferative state. ~ and D) The corresponding positive controls for BrdU and
PCNA
staining on rat liver sections from the 24-hour Phx time point. Arrows
indicate
individual oval cells. (Original magnification [A-D] X100.)
FIGURE 14 A-H. Cytocentrifuged preparation of Thyl.l+ sorted cells
from 2-AAF/CCl4-treated rats. Thy-1.1+ were stained with oval cell-specific
antibodies. (A) Hematoxylin-eosin stain. (B) A representative of negative
control
(omission of primary antibody). (C-F) AFP, CK-19, GGT, and OV6 staining,
respectively. The majority of the Thy-1+ cells were positive for oval cell-
specific
markers. (G) A representative of cells stained for desmin. All
photomicrographs are
at 100X magnification. (H) Dual staining of oval cells. AFP-Texas Red (red)
and
Thyl.l--FITC (green) showing both markers on the same cells. Where the two
antibodies are in close proximity to each other, the wavelengths mix and the
resulting
fluorescence is yellow. Photomicrograph for (H) was obtained using confocal
microscopy.
FIGURE 15 A-E. Cytocentrifuged preparation of Thyl.l- sorted cells
from 2-AAF/CCl4-treated rats. The Thy-1- cells were also stained with oval
cell
antibodies. (A) Hematoxylin-eosin stain. (B and D) Cells stained for AFP and
GGT,
respectively. The corresponding negative controls are shown in (C) and (E).
All
photomicrographs are at 200X magnification.
FIGURE 16 A-F. (16A) Normal Rat Spleen. DPPIV positive cells
with stain with a reddish/burn orange color. Notice that the white pulp region
of the
spleen is devoid of stain. lOx objective. (16B) Normal Rat Spleen. DPPIV
positive
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cells with stain with a reddish/burn orange color. Notice that the white pulp
region of
the spleen is devoid of stain. 20x objective. (16C) Experimental DPPIV
deficient rat
transplanted with DPPIV positive bone marrow. Spleen 60 days post BMTx. Notice
the reddish/burn orange staining present throughout the spleen. The white pulp
region
is now showing a large number of cells positive for DPPIV. 4x objective. (16D)
Experimental DPPIV deficient rat transplanted with DPPIV positive bone marrow.
Spleen 60 days post BMTx. Notice the reddish/burn orange staining present
throughout the spleen. The white pulp region is now showing a large number of
cells
positive for DPPIV. lOx objective.
FIGURE 17A-D. (17A) Normal Rat pancreas. DPPIV positive cells
with stain with a reddish/burn orange color. lOx objective. (17B) Normal Rat
pancreas. DPPIV positive cells with stain with a reddish/burn orange color.
20x
objective. (17C) DPPIV deficient rat pancreas. No staining is visible
throughout the
section. lOx objective. (17D) DPPIV deficient rat pancreas. No staining is
visible
throughout the section. 20x objective. (17E) Experimental DPPIV deficient rat
transplanted with DPPIV positive bone marrow. Pancreas 60 days post BMTx.
Notice there are a few reddish/burn orange cells staining positive in the
pancreas. l Ox
objective. (17F) Experimental DPPIV deficient rat transplanted with DPPIV
positive
bone marrow. Pancreas 60 days post BMTx. Notice there are a few reddish/burn
orange positive cells present in the pancreas. 20x objective.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods and compositions for
stimulating liver regeneration in a subject having a liver disorder. The
invention
provides methods and compositions for transplanting of bone marrow cells into
a
recipient host in amounts sufficient to result in the production of
hepatocytes, bile
ductal cells and oval cells during liver regeneration.
In a specific embodiment of the invention, bone marrow cells are
administered to a subject in need of new liver tissue. The bone marrow cells
can be
injected into the recipient, wherein the bone marrow cells will migrate to the
liver,
undergo proliferation, and differentiation leading to the production of new
liver tissue
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containing hepatocytes, bile ductal cells and oval cells. Alternatively, the
bone
marrow cells may be transplanted directly into the liver where the cells will
proliferate
and differentiate to form new liver tissue.
The compositions of the invention comprise bone marrow cells in a
5 pharmaceutically acceptable carrier for administration into a recipient host
in need of
new liver tissue. The bone marrow cells may also be genetically engineered to
enable
them to produce a wide range of functionally active proteins, such as for
example,
growth factors, cytokines and hormones. The compositions of the invention also
comprise bone marrow cells on a support matrix for transplantation into the
liver. The
10 matrix may further comprise growth factors capable of stimulating the
proliferation
and/or differentiation of hepatic stem cells or other types of cells.
The invention further relates to methods for enriching for populations
of hepatic oval cells, a hepatic stem cell, using the Thy-1 cell surface
antigen as an
antibody tag. Once purified, the oval cells may be transplanted into a
recipient in need
of new liver tissue. The hepatic oval cells may be transplanted directly into
the liver
of the recipient where the hepatic oval cells will undergo proliferation, and
differentiation leading to the production of new liver tissue containing
hepatocytes,
bile ductual cells and oval cells. Alternatively, the cells may be injected
into the
portal vein where the cells will go directly to the liver. In yet another
embodiment of
the invention, the hepatic oval cells may be injected into the spleen followed
by
migration to the liver.
5.1. SOURCES OF BONE MARROW CELLS
Bone marrow cells may be obtained from a variety of different donor
sources. In a preferred embodiment, autologous bone marrow is obtained from
the
subject who is to receive the bone marrow cells. This approach is especially
advantageous since the immunological rejection of foreign tissue and/or a
graft versus
host response is avoided. In yet another preferred embodiment of the
invention,
allogenic bone marrow may be obtained from donors who are genetically related
to
the recipient and share the same transplantation antigens on the surface of
their blood
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cells. Alternatively, if a related donor is unavailable, bone marrow from
antigenically
matched (identified through a national registry) donors may be used.
Bone marrow cells can be obtained from the donor by standard bone
marrow aspiration techniques known in the art. For example, bone marrow cells
can
be removed from the donor by placing a hollow needle into the marrow space and
withdrawing a quantity of marrow cells by aspiration. Alternatively,
peripheral stem
cells can be obtained from a donor, for example, by standard phlebotomy or
apheresis
techniques. For convenience, the following embodiments of the invention are
described for bone marrow cells, although it should be understood that
peripheral stem
cells may be used as equivalent to bone marrow cells.
Before administration into the recipient, bone marrow cell populations
maybe enriched for stem cells by selecting for cells that express stem cell
surface
antigens such as Thy-l, CD34, Flt-3 ligand and c-kit, in combination with
purification
techniques such as immuno-magnetic bead purification, affinity chromatography
and
fluorescence activated cell sorting. In addition, where the possibility of a
graft versus
host response exists, the stem cells to be administered to the recipient can
be T-cell
depleted to prevent the development of a graft versus host response. The cell
population maybe depleted of T-cells by one of many methods known to one
skilled in
the art (e.g., Blazer et al., 1985, Experimental Hematology 13:123-128).
Prior to transplantation into the recipient host, the bone marrow cells
may be stimulated with a number of different growth factors that can regulate
tissue
regeneration by affecting cell proliferation, differentiation and gene
expression. Such
growth factors include those capable of stimulating the proliferation and/or
differentiation of bone marrow cells and hepatic progenitor cells. For
example,
epidermal growth factor (EGF), transforming growth factor a (TGF-a) or
hepatocyte
growth factor/scatter factor (HGF/SF) may be utilized. The bone marrow cells
may be
stimulated in vitro prior to transplantation into the recipient subject.
Alternatively, the
stem cells may be stimulated in vivo by injecting the recipient with such
growth
factors following transplantation.
The present methods and compositions can also employ bone marrow
cells genetically engineered to enable them to produce a wide range of
functionally
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active biologically active proteins, including but not limited to growth
factors,
cytokines, hormones, inhibitors of cytokines, peptide growth and
differentiation
factors. Methods which are well known to those skilled in the art can be used
to
construct expression vectors containing a nucleic acid encoding the protein
coding
region of interest operatively linked to appropriate
transcriptional/translational control
signals. See, for example, the techniques described in Sambrook, et al., 1992,
Molecular Cloning, A Laboratory Manuel, Cold Spring Harbor Laboratory, N.Y.,
and
Ausebel et al., 1989, Current Protocols in Molecular Biology, Greene
Publishing
Associates & Wiley Interscience, N.Y.
In addition, stem cells may be attached in vitro to a natural or synthetic
matrix that provides support for the transplanted cells prior to
transplantation. The
type of matrix that may be used in the practice of the invention is virtually
limitlessness. The matrix will have all the features commonly associated with
being
"biocompatible", in that it is in a form that does not produce an adverse, or
allergic
reaction when administered to the recipient host. Growth factors capable of
stimulating the growth and regeneration of liver tissue may also be
incorporated into
the matrices. Such matrices may be formed from both natural or synthetic
materials
and may be designed to allow for sustained release of growth factors over
prolonged
periods of time. Thus, appropriate matrices will both provide growth factors
and also
act as an in situ scaffolding in which the transplanted cells differentiate
and proliferate
to form new liver tissue. In preferred embodiments, it is contemplated that a
biodegradable matrix that is capable of being reabsorbed into the body will
likely be
most useful.
To improve stem cell adhesion to the matrix, and survival and function
of the stem cell, the matrix may optionally be coated on its external surface
with
factors known in the art to promote cell adhesion, growth or survival. Such
factors
include cell adhesion molecules, extra cellular matrix molecules and/or growth
factors.
The present invention also relates to the use of bone marrow cells in
three dimensional cell and tissue culture systems to form structures analogous
to liver
tissue counterparts in vivo. Cells cultured on a three-dimensional culture
system will
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grow in multiple layers, forming new liver tissue. The resulting liver tissue
will
survive for prolonged periods of time, and perform liver-specific functions
following
transplantation into the recipient host. Methods for producing such structures
is
described, e.g., in US Patent No. 5,624,840, which is incorporated herein in
its
entirety.
5.2 METHODS FOR ENRICHMENT OF HEPATIC OVAL CELLS
The present invention also provides methods for deriving an enriched
population of hepatic oval cells from liver tissue using a Thy-1 specific
antibody.
This aspect of the invention is based on the observation that hepatic oval
cells express
high levels of Thy-1 on their cell surface.
Hepatic oval cells may be obtained from a variety of different donor
sources. Depending on the degree of liver damage, enriched populations of
autologous hepatic oval cells may be derived from the tissue of the subject
who is to
receive the transplanted hepatic oval cells. This approach avoids the
immunological
rejection of foreign tissue. In yet another preferred embodiment of the
invention,
allogenic liver tissue for use in purifying hepatic oval cells may be obtained
from
donors who are genetically related to the recipient and share the same
transplantation
antigens on the surface of their blood cells. Alternatively, if a sibling is
unavailable,
tissue may be derived from antigenically matched (identified through a
national
registry) donors.
In an embodiment of the invention, hepatic oval cells are isolated from
a disaggregated liver tissue biopsy. This may be readily accomplished using
techniques known to those skilled in the art. For example, the liver tissue
can be
disaggregated mechanically and/or treated with digestive enzymes and/or
chelating
agents that weaken the connections between neighboring cells, making it
possible to
disperse the tissue suspension of individual cells. Enzymatic dissociation can
be
carried out by mincing the liver tissue and treating the minced tissue with
any of a
number of digestive enzymes . Such enzymes include, but are not limited to,
trypsin,
chymotrpsin, collagenase, elastase and/or hylauronidase. A review of tissue
disaggregation technique is provided in, e.g., Freshney, Culture of Animal
Cells, A
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14
Manual of Basic Technique, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 9,
pp.107-
126.
Following preparation of a single cell suspension, Thy 1.1-positive
cells, which represent the hepatic oval cell population of cells may be
purified from
the Thyl-1-negative population of cells using a variety of different methods.
Such
procedures involve a positive selection, such as passage of sample cells over
a column
containing anti-Thy-1 antibodies or binding of cells to magnetic bead
conjugated anti-
Thyl antibodies or by panning on anti-Thy-1 antibody coated plates and
collecting the
bound cells. Alternatively, the single cell suspension may be exposed to a
labeled
antibody that immuno-specifically binds to the Thyl-1 cell surface antigen.
Following
incubation, with the Thy 1.1 antibody, the cells are rinsed in buffer to
remove any
unbound antibody. Hepatic oval cells expressing the Thyl-1 cell surface
antigen can
then be cell sorted by fluorescence-activated cell sorting using, for example,
a Becton
Dickinson FACStar flow cytometer.
Prior to transplantation into the recipient host, the hepatic oval cells
may be contacted with a number of different growth factors that can regulate
tissue
regeneration by affecting cell proliferation, and gene expression. Such growth
factors
include those capable of stimulating the proliferation and/or differentiation
of hepatic
progenitor cells. For example, epidermal growth factor (EGF), transforming
growth
factor a (TGF-a) or hepatocyte growth factor/scatter factor (HGF/SF) may be
utilized.
The hepatic oval cells may be stimulated in vitro prior to transplantation
into the
recipient subject, or alternatively, by injecting the recipient with growth
factors
following transplantation.
The present methods and compositions may employ hepatic oval cells
genetically engineered to enable them to produce a wide range of functionally
active
biologically active proteins including, but not limited to, growth factors,
cytokines,
hormones, inhibitors of cytokines, peptide growth and differentiation factors.
Methods which are well known to those skilled in the art can be used to
construct
expression vectors containing a nucleic acid encoding the protein of interest
linked to
appropriate transcriptional/translational control signals. See, for example,
the
techniques described in Sambrook, et al., 1992, Molecular Cloning, A
Laboratory
"WO 00/50048 PCT/US00/04744
Manuel, Cold Spring Harbor Laboratory, N.Y., and Ausebel et al., 1989, Current
Protocols in Molecular Biology, Greene Publishing Associates & Wiley
Interscience,
N.Y.
In addition, hepatic oval cells may be attached in vitro to a natural or
5 synthetic matrix that provides support for the transplanted hepatic oval
cells prior to
transplantation. The matrix will have all the features commonly associated
with being
"biocompatible", in that it is in a form that does not produce an adverse, or
allergic
reaction when administered to the recipient host. Growth factors capable of
stimulating the growth and regeneration of liver tissue may also be
incorporated into
10 matrices. Such matrices may be formed from both natural or synthetic
materials and
may be designed to allow for sustained release of growth factors over
prolonged
periods of time. Thus, appropriate matrices will both provide growth factors
and also
act as an in situ scaffolding in which the hepatic oval cells differentiate
and proliferate
to form new liver tissue. In preferred embodiments, it is contemplated that a
1 S biodegradable matrix that is capable of being reabsorbed into the body
will likely be
most useful.
To improve oval cell adhesion to the matrix, and survival and function
of the stem cell, the matrix may optionally be coated in its external surface
with
factors known in the art to promote cell adhesion, growth or survival. Such
factors
include cell adhesion molecules, extra cellular matrix molecules or growth
factors.
The present invention also relates to the use of hepatic oval cells in
three dimensional cell and tissue culture systems to form structures analogous
to liver
tissue counterparts in vivo. The resulting liver tissue will survive for
prolonged
periods of time, and perform liver-specific functions following
transplantation into the
recipient host. Methods for producing such structures is described in US
Patent No.
5624,840, which is incorporated herein in its entirety.
5.3. ADMINISTRATION OF BONE MARROW
STEM CELLS OR HEPATIC OVAL CELLS
The bone marrow cells and/or enriched oval cells can be administered
to the recipient in an effective amount to achieve its intended purpose. More
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16
specifically, an effective amount means an amount sufficient to lead to the
development of new liver tissue and restoration of liver function, thereby
alleviating
the symptoms associated with liver disorders.
The number of cells needed to achieve the purposes of the present
invention will vary depending on the degree of liver damage and the size, age
and
weight of the host. For example, the cells are administered in an amount
effective to
restore liver function. The dose range of cells to be used in the practice of
the
invention may vary between 105 - 10'° cells, although the preferable
dose of
administered cells will be between 106 - 108. It may be necessary to use
dosages
outside these ranges in some cases,
as will be apparent to those of skill in the art.
Determination of effective amounts is well within the capability of
those skilled in the art. The effective dose may be determined by using a
variety of
different assays designed to detect restoration of liver function. The
progress of the
transplant recipient can be determined using assays that include blood tests
known as
liver function tests. Such liver function tests include assays for alkaline
phosphates,
alanine transaminase, aspartate transaminase and bilirubin. In addition,
recipients can
be examined for presence or disappearance of features normally associated with
liver
disease such as, for example, jaundice, anemia, leukopenia, thrombocytopenia,
increased heart rate, and high levels of insulin. Further, imaging tests such
as
ultrasound, computer assisted tomography (CAT) and magnetic resonance (MR) may
be used to assay for liver function.
The bone marrow cells and/or enriched oval cells can be administered
to the recipient in one or more physiologically acceptable carriers. Carriers
for these
cells may include, but are not limited to, solutions of phosphate buffered
saline (PBS)
containing a mixture of salts in physiologic concentrations. In addition, the
cells may
be associated with a matrix prior to administration into the recipient host.
In an embodiment of the invention, the bone marrow cells andlor
hepatic oval cells can be administered by intravenous infusion. The cells to
be
injected, are drawn up into a syringe and injected into the recipient host. In
such
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17
instances the cells would be expected to migrate to the recipient's liver
where they
will differentiate and proliferate to form new liver tissue.
Alternatively, the methods of the present invention encompass
administration of the bone marrow cells and/or hepatic oval cells into the
recipient so
as to become located in the liver. The administration of the stem cells and/or
hepatic
oval cells, is accomplished by conventional techniques such as injection of
cells into
the recipient host liver, injection into the portal vein, or surgical
transplantation of
cells into the recipient host liver. In some instances it may be necessary to
administer
the stem cells and/or hepatic oval more than once to restore liver function.
In
addition, growth factors, such as G-CSF, or hormones, may be administered to
the
recipient prior to and following transplantation for the purpose of priming
the
recipients liver and blood to accept the transplanted cells and/or to generate
an
environment supportive of hepatic cell proliferation.
5.4. USE OF BONE MARROW CELLS FOR REGENERATION
OF TISSUE, OTHER THAN LIVER TISSUE
In yet another embodiment of the invention, bone marrow cells may be
used for regeneration of tissues other than liver tissue. Specifically, the
methods and
compositions of the invention provide for the transplantation of bone-marrow
stem
cells into a recipient host in amounts sufficient to result in tissue
regeneration other
than liver regeneration. The invention is based on the observation that bone-
marrow
derived cells can participate in the production of not only liver cells, but
pancreatic
cells as well.
The methods of the present invention encompass administration of the
bone marrow cells into the recipient host so as to become located in the organ
or
tissue in which regeneration is desired. The administration of the stem cells
into the
desired region is accomplished by conventional techniques such as injection of
cells
within the recipient host or surgical transplantation of cells within the
recipient host.
In some instances it may be necessary to administer the stem cells more than
once to
restore the desired tissue function.
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18
In a specific embodiment of the invention, bone marrow cells may be
transplanted into the pancreas of a recipient subject in need of new
pancreatic tissue.
Such subjects include those having pancreatic disorders such as acute or
chronic
pancreatitis or carcinomas of the pancreas. The bone marrow cells can be
administered to the recipient in one or more physiologically acceptable
carriers, in an
effective amount to achieve its intended purpose. More specifically, an
effective
amount means an amount sufficient to lead to the development of new pancreatic
tissue and restoration of pancreatic function, thereby alleviating the
symptoms
associated with the pancreatic disorders.
6. EXAMPLE: BONE MARROW CELLS PARTICIPATE IN THE
PRODUCTION OF HEPATOCYTES, BILE DUCTAL
CELLS AND OVAL CELLS DURING LIVER
REGENERATION
The purpose of the present study was to test the hypothesis that oval
cells and other liver cells may arise from a cell population originating in,
or associated
with, the bone marrow. This hypothesis was tested by three approaches: i) bone
marrow transplantation (BMTx) from male rats into lethally irradiated
syngeneic
females, and detection of donor cells in the recipients by means of DNA probes
to the
Y chromosome sry region; ii) BMTx from Dipeptidyl peptidase-IV positive
(DPPIV+)
male rats into DPPIV- syngeneic females, and detection of DPPIV-expressing
cells in
the recipient animals; and iii) whole liver transplantation (WLTx) using Lewis
rats
that express the L21-6 antigen as recipients, and Brown-Norway (Brown-Norway)
rats
that do not express this antigen as allogenic donors, in order to confirm that
an extra-
hepatic source (L21-6+ cells) could repopulate the transplanted (L21-6- cells)
liver. In
conjunction with these approaches, the 2-AAF/CC14 protocol was used to induce
oval
cell activation and proliferation. Hepatocyte proliferation is generally
suppressed by
exposure of an animal to 2-acetylaminoflourane (2-AAF) and subsequently
hepatic
injury is induced by administration of carbon tetrachloride. In situ
hybridization, PCR,
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19
and immunohisto- and cytochemical techniques were used to distinguish donor
cells
from recipient cells.
6.1. MATERIALS AND METHODS
6.1.1. BONE MARROW AND WHOLE LIVER TRANSPLANTATION
Bone marrow transplantation (BMTx) was performed as previously
described (Murase, N. et al., 1996, Transplantation 61:1 ) with minor
modifications.
In one set of experiments (Set l, two separate experiments), bone marrow from
male
F-344 rats was transplanted into lethally irradiated syngeneic female F-344
rats
(n = 10 animals). The females were given 10.5 Gy from a'3'Cesium source (JL
Shepherd Mark I), and then rescued by injecting about 60 x 106 male bone
marrow
cells via the tail vein. After allowing establishment of a chimeric system
(about 30-45
days), the animals were tested to see if the donor cells had engrafted. PCR
analysis
was performed on DNA extracted from the huffy coat of nucleated cells obtained
from
retinal orbital blood (ROB). All animals survived the transplant procedure,
but
varying degrees of intensity for chimerism, was evident. Only those animals
that
expressed a strong signal for the Y chromosome PCR product were placed on the
2-
AAF/CC14 protocol for oval cell induction. The animals were sacrificed 9-13
days
after CC14 administration. In a second set of BMTx experiments (Set 2), bone
marrow from male F-344 rats expressing the DPPIV enzyme was transplanted into
lethally irradiated DPPIV- syngeneic female F-344 rats, which were then
further
treated as above. Ten female rats were successfully transplanted in Set 1, and
fourteen
female rats in Set 2.
Whole liver transplantation (WLTx) was performed as previously
described (Murase, N et al., 1995, Transplantation 60:158). Brown-Norway rats
were
used as donors of whole liver tissue, and Lewis rats as recipients. The L21-6
monoclonal antibody specific for Lewis rats was used to distinguish
immunohistochemically donor cells from recipient cells (Yagihashi et al.,
1995,
Transplantation Proceedings 27:1519). Once the rats recovered from the WLTx
(about two months), the animals were placed on the 2-AAF/CC 14 protocol for
induction of oval cells as described below. In this experiment six Lewis rats
were
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successfully transplanted; of these, three animals survived until completion
of the
study.
6.1.2. ANIMALS
F-344 male and female rats were obtained from Frederick Laboratories
5 (Frederick, MD) as marrow donors and recipients. DPPIV- female F-344 rats
were a
gift from Dr. Sanjeev Gupta, Albert Einstein College of Medicine, Bronx, NY.
All
procedures involving animals were conducted according to institutionally
approved
protocols.
6.1.3. INDUCTION OF OVAL CELLS
10 2-AAF time-release pellets were prepared (70 mg, 2.5 mg/day release
for 28 days) as described by Hixson et al. (Hixson D.C. et al., 1990,
Pathobiology
58:65) and inserted subcutaneously in the rats 7 days prior to administration
of CCl4.
The animals were sacrificed, and tissue samples were obtained at various time
points
thereafter. CC14 was injected intraperitoneally (i.p) as a single dose of 1.9
ml/kg
15 (1500 mg/kg) b.w. of a 1: 1 (vol/vol) solution in corn oil; this dose was
calculated on
the basis of the LDSO dose (RJ. Lewis, Sr., Ed., SAX Dangerous Properties of
Industrial Material (Van Nostrand Reinhold, New York, NY. 1993), pp. 52 and
1149,
eighth edition).
6.1.4. PCR ANALYSIS
20 PCR analysis for the Y chromosome was performed on DNA
extracted from transplanted and non-transplanted female animals using primers
for the
sry gene of the Y chromosome (An, J. et al., 1997, J Andrology 18:289). The
primer
sequences are as follows:
5'-CATCGAAGGGTTAAAGTGCCA-3' and
5'-ATAGTGTGTAGGTTGTTGTCC-3'. These primers amplify a 549-by nucleic acid
product that has been shown to be very specific for the Y chromosome with no
reactivity to female DNA. PCR was performed as previously described (An, J. et
al.,
1996, Experimental Hematology 24:768). Human (3-actin primers from Clonetec
Laboratories (Palo Alto, CA), included to ensure that each sample of DNA was
intact,
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21
produce an expected product of approximately 1000 base pairs in length
(DeFrances,
M.C., 1992, Development 116:387).
6.1.5. CELL ISOLATION
Hepatocytes and nonparenchymal cells (NPC) were isolated after a 2-
step collagenase digestion of the liver according to an established protocol
(1996,
Methods Cell Biol 13:29) Prior to the isolation procedure, the caudate lobe
was
surgically removed and divided into halves, one being fixed in 10% buffered
formalin,
and the other placed in OTC. compound and frozen in cold 2-Methylbutane. The
samples were stored at -80°C until paraffin or frozen sections were
prepared for
routine examination after hematoxylin and eosin (H&E) staining. This tissue
served
as an internal control for light microscopy and hybridization in situ.
Digestion began by blanching the liver to remove the majority of blood
cells from the liver, by perfusion with a buffered saline solution (S&M) for
10 min,
10 ml/min at 37°C. Digestion of the liver was accomplished by
collagenase digestion
(Worthington Biochemical Co., Freehold, NJ; 100 mg/250 ml S&M supplemented
with CaCI) for 20 min, at 10 ml/min at 37°C. On completion of the
digestion, the
liver was removed from the animal, placed into cold S&M, and repeatedly shaken
to
disrupt the individual cells from the tissue. The resulting cell suspension
was passed
through nylon mesh and centrifuged 3 times at 50 g for 5 min to separate the
hepatocytes from the NPCs. The supernatants on top of the hepatocyte pellets
were
collected after each spin and combined. The hepatocytes were resuspended in
cold
media (MEM Neaa, Gibco, Gaithersburg, MD) and placed on ice until further use.
The collected supernatant was diluted 1:3 with cold S&M to remove the
collagenase
from the cells, and centrifuged at 1100 rpms (400 g) for 10 min to pellet the
NPCs.
The NPC fraction was then suspended in 1X phosphate buffered saline (PBS) and
stored at 4 °C until oval cell isolation. The presence of oval cells in
the NPC fraction
has been previously determined (Yaswen, P., 1984, Cancer Research 44:324). The
oval cells were then isolated from the NPC fraction using flow cytometric
techniques
as previously described (Petersen, B.E. et al., 1998, Hepatology 27:433).
Briefly,
approximately 200 x 106 NPCs were incubated for 20 min at 4°C with
fluorescein
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22
isothiocyanate (FITC)-conjugated anti-rat Thy 1.1 (1 ~g/million cells) and
then rinsed
twice in 1X PBS + 1%FBS, 5 min each time. FITC isotype mouse IgGI was used as
control. The cells were then kept on ice in the dark until sorted with a
Becton
Dickinson FACStar flow cytometer into Thyl.l+ (oval cells) and Thyl.l- sub-
s populations of cells. The purity of the different sorted cell populations
were as
follow: 95-97% pure for the Thy-1+ cells and 99% pure for the Thy-1- cells
(Petersen,
B.E. et al., 1998, Hepatology 27:433).
6.1.7. IN SITU HYBRIDIZATION OF THE SRY
REGION OF THE Y CHROMOSOME
Digoxigenin labeled DNA probes, prepared by random priming using
the Genius System instructions (Boehringer Mannheim, Indianapolis, IN), were
hybridized to paraffin or frozen liver sections as per manufacturer's
instructions.
Briefly, frozen sections were placed on Superfrost Plus microscope slides
(Fisher
Scientific, Pittsburgh, PA) and were pre-hybridized in 10 mM Tris-HCI, 50%
formamide, 0.6 M NaCI, 1 mM EDTA, lx Denhardts, 0.5 mg/ml carrier RNA and
10% dextran sulfate for 1 hr at 37°C. The digoxigenin-labeled probe of
the sry gene
of the Y chromosome was applied to the sections and allowed to hybridize
overnight
at 37°C. Following post-hybridization washes, detection of the probe
was
accomplished by incubation with alkaline phosphatase-conjugated anti-
digoxigenin
antibody (1:500) for 2 hr at RT. Alkaline phosphatase activity was visualized
by
incubation with NBT and BICP (Boehringer Mannheim, Indianapolis, IN) in the
dark.
The color development was monitored and the enzymatic reaction stopped by
immersing the slides in 10 mM Tris, 1 mM EDTA.
6.1.8. IMMUNOHISTOCHEMISTRY AND CYTOCHEMISTRY
All tissue samples were divided into half. One was immediately
placed in liquid nitrogen for later extraction of RNA or DNA. The other half
was
split, with one portion being fixed in 10% buffered formalin and processed for
preparation of 4-~m thick paraffin embedded sections, and the other portion
placed in
OTC freezing compound and cold 2-Methylbutane and processed for preparation of
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23
6 ,um-thick frozen sections which were stored at -80°C until staining.
Routine
histological examinations were performed on both paraffin and frozen sections
stained
with hematoxylin and.eosin (H&E). In addition, spleen and pancreas tissue were
treated the same way and frozen sections and paraffin sections were DPPIV and
hematoxylin and eosin stained.
6.2. RESULTS
Female rats were lethally irradiated and rescued with a bone marrow
transplant from a male animal. Nucleated blood cells of the transplanted
animals were
tested by PCR to establish that the BMTx was successful. Once the female rats
were
determined to have been engrafted with male bone marrow, they were placed on
the
oval cell protocol as stated above. On Day 9 and Day 13 (post hepaic injury)
rats were
anesthetized and their livers were perfused in order to obtain single cell
suspensions of
non-parenchyma) cells (NPC) (cells that are not hepatocytes) and parenchyma)
cells
(primarily hepatocytes).
1 S Flow cytometry was used to isolate the oval cells from the NPC
fraction using anti-Thy-1-FITC. The Thy-1 positive, Thy-1 negative and
hepatocyte
populations were subjected to PCR analysis. Figure 1 shows that both the day 9
and
day 13 Thy-1+ and Thy-1- cell populations of NPCs were positive for the
Y chromosome PCR product. The Thy-1- fraction showed a strong signal, probably
due to the presence of donor hematopoietic cells such as Kupffer cells which
are in the
NPC population and Thy-1 negative, but positive for the Y chromosome. In the
day 9
hepatocyte fraction there was no visible signal. At this time point in oval
cell
activation, the oval cells have not yet begun to differentiate into either
transitional
cells or hepatocytes. By day 13 there were cells in the hepatocyte fraction
expressing
the Y chromosome PCR (549 bp) product. At this time the oval cells are
beginning to
differentiate into hepatocytes (Grishman, J.W., 1997, in Stem Cells, C.S.
Potten, Ed.
(Academic Press, San Diego, CA p233-282). If all oval cells that differentiate
into
hepatocytes were derived from the liver, then one would expect that none of
the
hepatocytes tested would be positive for the Y chromosome. The fording that
some
hepatocytes were Y chromosome positive indicates that they were derived from
the
WO 00/50048 PCT/US00/04744 --
24
bone marrow donor cells. The combined data indicates that at day 9 the oval
cells
(Thy-1+) in the recipient female were derived from the donor male and that
they
continued to differentiate into mature hepatocytes by day 13.
To confirm the PCR results seen in Figure 1, one of the smaller lobes
from every liver that was perfused was ligated, removed and split in half. One
portion
was fixed in 10% buffered formalin and processed into 4-,um thick paraffin
embedded
sections; the other portion was placed in OTC freezing compound and processed
into
6 ,um-thick frozen sections. In situ hybridization for the Y chromosome sry
gene was
performed on both types of fixed tissue. Hepatocytes carrying a positive
reaction
product (blue staining) in their nuclei were readily seen in untreated control
male rats
(Figures 2A). In agreement with the results obtained by PCR analysis of the
isolated
hepatocyte fraction, cells with positive blue staining (Y chromosome positive)
were
detected in females subjected to BMTx and the 2-AAF/CC14 protocol at day
13(Figure
2B). Figure 2C shows no reaction product in the liver of an untreated control
female.
In the second set of bone marrow transplantation experiments, bone
marrow cells from DPPIV+ F-344 male rats were injected into lethally
irradiated
DPPIV- F-344 females. This constituted a system in which the presence of cells
originating from donor cells in the recipient liver could be easily detected,
by
revealing cytochemically the activity of the enzyme DPPIV (Figure 3). As
previously
reported with studies of this enzyme, a diffuse red to brownish-red staining
of the bile
canalicular site between hepatocytes was observed. This type of staining was
seen in
the DPPIV+ F-344 male rats (Figure 3A). The control untreated DPPIV- females
showed no staining (Figure 3B). To determine whether DPPIV would be expressed
in
transplanted animals, staining was performed on liver sections prepared from
transplanted female rats treated with 2-AAF/CC14. As seen in Figure 3C-3F,
DPPIV
expression was observed in several bile canalicular sites between hepatocytes
from
four different transplanted animals. DPPIV expression was also observed on a
few
oval cells/transitional (small hepatocyte) cells in the liver from these rats
(Figure 3D
and 3E).
Four animals showing the strongest evidence of the donor marrow
engraftment, based upon examination of the recipient rat's spleens, were
chosen
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(Figure 3C-3F) for an estimation of the number of hepatocytes originating from
the
donor bone marrow. From these four animals, five different lobes were used to
cut
approximately 10 sections per lobe (50 sections total). All of the sections
were
stained for DPPIV and examined for DPPIV expression. Roughly, 25 random fields
5 (200x) per section were examined, and the numbers indicate the total number
of
hepatocytes positive for DPPIV staining per section.
Table 1 represents the number of DPPIV positive hepatocytes observed
in the transplanted DPPIV- rats. By dividing the total positive cells by the
total
hepatocytes observed, approximately 0.16% of the total number of hepatocytes
were
10 positive for DPPIV expression. The rat liver has approximately 700 x 106
hepatocytes, indicating that, at day 13, approximately 1.0 x 106 hepatocytes
originated
from transplanted bone marrow cells using the oval cell protocol.
TABLE
1.
Percent
of
DPPIV
positive
hepatocytes
in
BMTx
DPPIV
deficient
females
SECTIONSFIELDS DPPIV+
15 AnimalLOBES # of Total # Total # of PERCENT
Number# of Differentof positive HepatocytesTOTAL
Differentnon-serialRandom
Fields
6028 5 10 250 55 0.144
6034 4 10 200 18 0.059
6036 5 10 250 78 0.204
6037 5 10 250 93 0.243
20 The mean value of 0.163% represents that approximately 1x106 hepatocytes
would be
positive for DPPIV expression within the transplanted female rats.
In order to confirm that extra-hepatic cells can repopulate the liver,
whole liver transplantation was used as a final approach. In this series of
experiments,
Lewis rats that express the MHC class II L21-6 isozyme were used as recipients
of
25 liver from Brown-Norway rats which do not express L21-6 (Yagihushi, A. et
al.,
1995, Transplantation Proceedings 27:1 S 19). An anti L21-6 monoclonal
antibody was
used to differentiate donor from recipient cells. In this model, oval cells
that
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26
originated from an extra-hepatic source would be L21-6 positive, while oval
cells
originating in situ would be negative. The results obtained in these
experiments are
illustrated in Figures 4 and 5. Figure 4A and 4B shows stained sections of two
Brown-Norway rat livers transplanted into Lewis rats prior to being placed on
the 2-
AAF/CC14 protocol. A widespread staining of L21-6 was present in the
transplanted
Brown-Norway livers, presumably due to influx of cells of the Lewis host
immune
system reacting to the allogenic Brown-Norway liver. Most notable, though, was
the
presence of ductal structures containing L21-6 positive cells. These
structures
represent a pattern often seen in the organization and differentiation of
actively
proliferating oval cells (Thorgeirsson, S.S. et al., 1993, Proc. Soc. Exp Biol
Med
204:253; N. Fausto, in The Liver: Biology and Pathobiology LM. Arias et al.
Eds
(Raven Press, New York 1994) p. 1501-1518 (third edition)). The presence
therein of
positive cells supports the concept that these oval cells were derived from an
extra-
hepatic source. The structures also contained cells which were clearly L21-6
negative,
suggesting that some oval cells were derived in situ from the donor liver
(Figure 4B).
To better characterize the positive cells seen in the ductal structures
and distinguish them from inflammatory cells invading the Brown-Norway
transplanted livers, double immunofluorence staining was performed using an
antibody against OC.2, a specific oval cell/ductal cell marker (Faris, R.A. et
al., 1956
Cancer Research 16:142), in conjunction with the L21-6 antibody. As can be
seen in
Figure S, individual cells expressing both OC.2 and L21-6 are evident as a
yellow
stain (combination of red and green). The cells, therefore, were identified as
oval
cells or derivatives thereof, and not immunocytes or inflammatory cells.
In addition, when tissues other than the liver were analyzed, such as the
pancreas and spleen, and cell staining was observed, indicating that
transplanted bone
marrow cells were capable of infiltrating and incorporating into these
tissues. As
indicated in Figure 17A-F, when experimental DPPIV deficient rats were
transplanted
with DPPIV positive bone marrow, a large number of cells were positive for
DPPIV
(Figure 17 E-F).
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7. EXAMPLE: PURIFICATION OF HEPATIC OVAL
CELLS USING ANTIBODIES THAT RECOGNIZE THE
THY1.1 CELL SURFACE ANTIGEN
The following subsection discloses experimental data indicating that
hepatic oval cells express the hematopoietic stem cell marker Thy 1.1. The
data
provides a novel cell marker for identification of oval cells. Using Thy 1.1
antibody, a
highly enriched population of oval cells was obtained.
7.1 MATERIALS AND METHODS
CC14, 99% pure high-performance liquid chromatography grade, and 2-
AAF were purchased from Aldrich Chemical Co. (St. Louis, MO). 2-AAF crystals
were incorporated into time-released pellets (70 mg/pellet over a 28-day
release, 2.5
mg/d) supplied by Innovative Research Inc. (Sarasota, FL). Male Fisher 344
rats
(150-170 g) were obtained from Fredericks Laboratories (Frederick, MD).
Microscope Superfrost Plus slides, buffered Formalin-Fresh, and dextran
sulfate were
obtained from Fisher Scientific (Pittsburg, PA). Hematoxylin was purchased
from
Anatech, Ltd. (Battle Creek, MI). Anti-a-fetoprotein (AFP) antibody was
purchased
from Nordic Immunology (Tilburgh, the Netherlands). OV-6 and BD-1 antibodies
were gifts from Dr. Doug Hixson (Brown University, Providence, RI). OC.2
antibody
was a gift from Dr. Ron Faris (Brown University, Providence, RI). Rat anti-GGT
was
a gift from Dr. Benito Lombardi (University of Pitssburgh, Pittsburgh, PA).
Thyl.l
was purchased from PharMingen Inc. (San Diego, CA). Proliferating cell nuclear
antigen (PCNA) was purchased from Signet Laboratory Inc. (Dedham, MA). 5-
Bromodeoxy- uridine (BrdU) was obtained from Boehringer Mannheim
(Indianapolis,
IN). Desmin was obtained from Dako Corp. (Carpinteria, CA). Eosin, propidium
iodide (PI), and all other chemicals used were obtained from Sigma Chemical
Company (St. Louis, MO).
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7.1.2 COMPOUND DELIVERY
An LDS° dose of CC14 as determined by Lewis was used (SAX
Dangerous Properties of industrial Material, In: Lewis, R.J., Sr. Edition 8,
1993
update and vol 3. New York: Van Nostrand Reinhold, 1992 52:1149). A single
dose
of 1.9 mL/kg (1,500 mg/kg) of body weight, in a 1:1 vol/vol dilution in corn
oil, was
administered by intraperitoneal injection. Two hours before they were
sacrificed, the
animals received an intra peritoneal injection of BrdU (50 mg/kg body weight)
to
identify cells in S-phase of the cell cycle, as described by Lindrose et al
(1991,
Hepatology 13:743-750).
7.1.3 OVAL CELL COMPARTMENT PROLIFERATION/ACTIVATION
2-AAF pellets were inserted 7 days before hepatic injury following a
protocol similar to Novikoff et al. (1996, Am J Pathol 148:1473-1491) and
Hixson et
al. (1990, Pathobiology 58:65-73) The time points for this study were counted
from
when the hepatic injury (CC14, Phx) was induced. The dose and delivery for
CC14 was
discussed earlier in the compound delivery section and performed in the same
manner.
For the Phx procedure, rats were hepatectomized under general anaesthesia
according
to the methods described by Higgins and Anderson (1931, Arch. Pathol. 12:186-
202).
The tissue obtained was processed in the same manner described in the
immunohistochemistry methods.
7.1.4 ANIMAL EUTHANIZATION
All procedures involving animals were conducted according to
institutionally approved protocols. Rats were anesthetized by injection with
sodium
pentobarbital (0.1 mL/100 g body weight) before being sacrificed.
7.1.5 IMMUNOHISTOCHEMISTRY
A basic immunohistochemical protocol previously described by Wolf
et al. (1991, Hepatology 12:186-202) was used with slight modification to
conform to
each particular antibody. Liver tissue was divided and fixed in either 10%
buffered
formalin or placed in OTC compound, frozen in cold 2-methylbutane (Fisher
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29
Scientific), and stored at -80°C. All staining procedures for light
microscopy were
performed on 4-~m thick, paraffin-embedded sections or 6-m thick, frozen
sections.
Routine histological examination were made for all liver tissue samples on
sections
(paraffin and frozen) stained with hematoxylin-eosin. Single cell suspensions
were
collected on glass slides by cytocentrifugation and air-dried.
Cytocentrifugation was
performed using a Cytospin 3 Cytocentrifuge (Shandon Inc. Pittsburgh, PA) for
6
minutes at 600 rpm. Immunohistochemistry on cytospin preparations (100,000
cells/slide) was performed using the techniques described above. The cytospin
preps
were then analyzed by confocal microscopy (Multiprobe 2001 Inverted Confocal
Laser Scanning Microscope, CLSM, Molecular Dynamics, Sunnyvale, CA). BrdU
staining was performed on 4-~m thick, paraffin-embedded tissue as described by
Lindroos et al. (1991, Hepatology 12:186-202). For each, antibody-negative
controls
were performed by either blocking with the appropriate nonimmune serum or by
omitting the primary antibody from the protocol.
7.1.6. FLOW CYTOMETRY
Hepatocyte and nonparenchymal cell isolation was performed by a
two-step collagenase digestion according to the protocol established by Seglen
(1976,
Methods in Cell Biol 13:29-83). Oval cell isolation was performed using flow
cytometry. Briefly, the nonparenchymal cell (NPC) fraction has been determined
to
contain the hepatic oval cell population as described by Yaswen et al. (1984,
Cancer
Res 44:324-331). Immunohistochemistry was performed on the parenchyma) and
NPC fractions to ensure that the cells of interest were in the NPC fraction.
The NPC
fraction was found to contain the highest percentage of oval cells. A portion
(approximately 60 to 80 x 106 of the total 200 x 106 cells) of the NPC
fraction was
further purified using flow cytometry. Fluorescein isothiocyanate (FITC)-
conjugated
anti-rat Thyl.l (1 mg/million cells) was used to label the target cells. The
cell
fraction was incubated with the antibody for 20 minutes at 4°C, rinsed
twice in lx
phosphate-buffered saline + 1 % fetal bovine serum for 5 minutes each, and
stored in
the dark on ice until sorting. A FITC mouse G, IgG, was used as an isotype
control.
Cells were sorted using a Becton Dickinson FACStar flow cytometer into two
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populations: Thyl.l-positive (Thyl.l+) and Thyl.l-negative (Thyl.l-). Those
animals
whose livers were perfused had one of the smaller lobes surgically removed
before the
cell isolation procedure. This tissue was used as an internal control for
light
microscopy, as well as for in situ hybridization. The excised liver tissue was
divided
5 in half and fixed in either 10% buffered formalin or placed in OTC compound,
frozen
in cold-2-methylbutane (Fisher Scientific), and stored at -80 ° C.
Cell Cycle Analysis. One x 106 Thyl.l+ cells were fixed in ice-cold
70% ethanol at 4°C overnight. Following centrifugation (5 minutes at
3,000 rpm), the
ethanol was removed and 1 mL of propidium iodide staining solution consisting
of 50
10 mg/mL PI, 100 U/mL RNase A in Ca- and Mg-free phosphate-buffered saline +
glucose was added and incubated for 30 minutes at room temperature. Flow
cytometric cell cycle analysis was then performed.
7.2 RESULTS
7.2.1 TIME LINE OF EVENTS FOR ACTIVATION
15 OF OVAL CELL PROLIFERATION
To activate oval cell proliferation in the liver, certain events must
occur. Figure 6 is an outline that represents the events and times involved in
the
process of the activation of oval cell proliferation. In control animals, a
placebo was
inserted in place of 2-AAF. Oval cells can be seen as early as day 5
posthepatic injury
20 and can still be detected as late as day 42 postinjury. Figure 7A-B shows
the
histological changes in liver sections from rats exposed to 2-AAF for 7 days,
followed
by CC14 exposure and sacrificed 11 days posthepatic injury. With the 2-
AAF/CC14
protocol, massive oval cell proliferation was seen after day 9. The sections
were also
stained with antibodies for specific oval cell markers (e.g., CK-19 and GGT),
and the
25 oval cells generated from the 2-AAF/CC14 protocol were positive for these
markers.
The major peak of BrdU incorporation for oval cells occurred at day 9
posthepatic
injury, with a drastic drop-off in BrdU incorporation on subsequent days.
These
results can be seen in Figure 8A-D, which shows livers sections from rats
sacrificed
on day 9 (Figure 8A), day 11 (Figure 8B), and day 13 (Figure 8C) post-CCl4
exposure.
30 The same type of proliferation pattern was also seen in the 2-AAF/Phx
model.
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7.2.2. IMMUNOHISTOCHEMISTRY FOR THY-1 ON
ACTIVATED HEPATIC OVAL CELLS
To determine whether Thy-l and OC.2 antigens are expressed only by
oval cells, normal rat liver was first examined. Figure 4 represents frozen
sections
obtained from normal rat liver. These sections were stained with Thy-1.1
antibody
(Figure 9A) or OC.2 antibody (Figure 9B). There appears to be no portion of
the liver
expressing the Thy-1 antigen. Figure 9B represents OC.2 staining. The ductular
cells
appear to be positive with little to no staining elsewhere in the liver. Our
results are in
agreement with Faris et al. (1991, Cancer Res 51:1308-1317), who previously
reported this pattern of staining for OC.2 in normal liver.
To test for Thy-1 expression on oval cells, frozen sections of livers
with proliferating oval cells were used. In addition to Thy-1 expression, CK-
19 and
OC.2 expression was also examined. Figure 10 represents frozen liver sections
obtained from rats on the 2-AAF/CCl4 protocol at day 21 after liver injury.
These
sections were stained for CK-19 (Figure l0A and lOD), OC.2 (Figure l OB and
l0E),
and Thy-1 (Figure l OC and lOF). Staining by all three antibodies on serial
sections
showed similar patterns, with all staining located in the periportal region
and
spreading outward; the pericentral region is devoid of staining. At higher
magnification (Figure 10 D-F), the reactivity of the antibodies was primarily
to the
oval cells (arrows), or what has been termed in the art as transitional cells.
Thy-1
antibody stained the ductular epithelium, which showed negative staining. This
is
also evident in Figure 11.
The possibility that cells staining positive for Thy-1 may be
inflammatory cells in response to the CC14 exposure was ruled out. In the 2-
AAF/PHx
model, however, there is presumably no influx of inflammatory cells. Figure 11
shows sections from livers with oval cells, in which the hepatic injury was a
PHx of
two thirds (day 13 2-AAF/PHx). Again, as shown in Figure 10, the pattern of
staining
in the periportal region of the lobule and expanding outward was similar to
the
staining pattern seen in livers from a 2-AAF/PHx model (Figure 11 A-C). At a
higher
magnification (Figure 11D-F), the staining appeared localized to the oval cell
population. These cells were positive for all three markers (CK-19 (Figure 11A
and
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11 D); OC.2 (Figure 11 B and 11 E); Thy-1 (Figure 11 C-11 F)). Clearly, with
both
models of hepatic injury (2-AAF/CCl4, 2-AAF/PHx), the pattern of staining for
CK-
19, OC.2, and Thy-1 was the same within the same population of cells. It
should also
be noted that frozen sections from both 2-AAF/CCl4 and 2-AAF/PHx were double-
labeled with Thy-1 FITC and OC.2-Texas Red, and the same staining pattern was
seen.
7.2.3. FACS ANALYSIS AND SORTING
FOR HOC USING THE THY-1 MARKER
As shown above, using immunohistochemistry, oval cells express
Thyl .1 antigen. The possibility of obtaining a distinct Thy-1+ population by
cell
sorting was next tested. Figure 12 represents FACS analysis of Thy-1+ and Thy-
1'
sorted oval cells obtained from day 12 liver on the 2-AAF/CC14 protocol. Both
the
forward-/side-scatter plots and histograms are shown. Figure 12A (right gate)
is that
of Thy-1+ cells, and Figure 12B (left gate) is Thy-1- cells. The Thyl.l+
population
was sorted to 97% purity and the Thy 1.1 ~ cells to a 99% purity. In each
experiment,
only a portion of the NPC fraction was sorted (60 to 80 x 106 cells) and
typical yields
of Thy-1+ cells obtained were 15 to 20 x 106 cells. These two populations of
sorted
cells were 95% viable by Trypan blue exclusion. The Thyl.l+ sorted cells were
further examined to determine what stage of the cell cycle they were in. Oval
cells
were stained with propidium iodide and analyzed. Cell cycle analysis in Figure
12C
shows a majority (>90%) of the Thyl.l+ cells were in the GO/G1 stage of the
cell
cycle. Figure 12D shows that ~90% of those cells in the GO/Gl stage of the
cell cycle
were Thy-1+.
Immunohistochemical staining for BrdU and PCNA (Figure 13A and
13B, respectively) also revealed that the oval cells were not in the cell
cycle at the
stage of the model used for this study. Only a few cells were positively
stained for
either marker, representing the second peak in the propidium iodide analysis
(Figure
13C and 13D). In a separate study, it was shown that there was only one peak
(day 9)
of BrdU incorporation for oval cells (2-AAF/CC14 protocol), and the days
following
the peak showed very little incorporation. Others have shown that oval cell
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proliferation lasts much longer, and perhaps the differences can be explained
by the
differences in the protocols. The data shown here were from experiments using
the 2-
AAF/CC14 protocol.
7.2.4. IMMLTNOHISTOCHEMISTRY ON THY-1+ SORTED HOC
Having obtained Thyl.l+ and Thy-1- populations, the sorted
populations were further characterized to determine if the traditional oval
cell markers
were expressed. Thy-1+ and Thy-1- cells were cytocentrifugated onto slides.
Figures
14 and 15 represent Thy-1+ and Thy-1- cells, respectively. Figure 14A is a
hematoxylin-eosin stain, and Figure 14B is a representative of a negative
control in
which the primary antibody was omitted. For all antibodies, the appropriate
negative
controls were performed, either by omitting the primary antibody or by
blocking with
an appropriate nonimmune serum. Figure 14C-14F is of sorted Thy-1+ cells
stained
with a-fetoprotein (AFP), CK-19, GGT, and OV6, respectively. To show that the
cells of interest (Thy-1+ cells) are not Ito cells, staining for desmin, an
Ito cell-specific
marker, was performed. These data are shown in Fig. 14G. The Thy-1+ cells were
negative for desmin, which indicated that these cells were Ito cells. Also,
desmin
staining was performed on liver sections obtained from both 2-AAF/CCl4- and 2-
AAF/PHx-treated animals at various time points, and the oval cells were also
negative. The results shown in Fig. 14H are of double-stained oval cells
showing
Thy-1-FITC (green) and AFP-Texas Red (red). In regions of the cell in which
the
antibody binding is in close proximity to each other, the two fluorochrome
wavelengths mix, and the resulting fluorescence is yellow. The presence of
dual
markers (yellow) was evident in most cells shown. In addition, both Thy 1
(green) and
AFP (red) were seen as distinct colors in separate cellular domains on the
same cell.
To show that the Thy-1+ staining was specific for oval cells, Thy-1-
cells were also subjected to the same immunostaining described above. Figure
15
represents these Thy-1- cells; Figure 15A is a representative hematoxylin-
eosin stain
of Thy-1- cells. Figure 15B and 15D are cells stained for AFP and GGT,
respectively.
Their corresponding negative controls are shown in Fig. 15C and 15D,
respectively.
In both cases, the Thy-1- populations were devoid of any staining for these
two
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traditional oval cell markers. The specificity for Thy-1 binding appeared to
be only
specific to oval cells, as seen in Figures 14 and 15.
The present invention is not to be limited in scope by the specific
embodiments described herein which are intended as single illustrations of
individual
aspects of the invention, and functionally equivalent methods and components
are
within the scope of the invention. Indeed, various modifications of the
invention, in
addition to those shown and described herein will become apparent to those
skilled in
the art from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the claims. Various
publications
are cited herein, the contents of which are hereby incorporated, by reference,
in their
entireties.
